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Interaction between two tomato ringspot virus replication proteins and endoplasmic reticulum membranes Zhang, Guangzhi 2007

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INTERACTION BETWEEN TWO TOMATO RINGSPOT VIRUS REPLICATION PROTEINS AND ENDOPLASMIC RETICULUM MEMBRANES by G U A N G Z H I Z H A N G B . S c , Beijing Agricultural University, Beijing, 1989 M . S c , Fujian Agricultural University, Fuzhou, 1995 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF D O C T O R OF P H I L O S O P H Y in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Botany) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A March 2007 © Guangzhi Zhang, 2007 Abstract Genome replication of Tomato ringspot virus (ToRSV) occurs in association with the endoplasmic reticulum (ER) and/or ER-derived membranes. To identify the viral membrane anchor proteins for the replication complex, two hydrophobic proteins encoded by T o R S V R N A 1 , termed N T B - V P g and X 2 , were studied for their ability to associate with intracellular membranes in planta and in vitro. N T B - V P g and X 2 were fused to the green fluorescent protein (GFP) and the fusion proteins were transiently expressed in Nicotiana benthamiana (N. benthamiana) epidermal cells. Subcellular localization of the fusion proteins was examined by confocal microscopy and subcellular fractionation and immunobloting. Mutagenesis was conducted to identify the membrane targeting domains within each protein. In addition, in vitro membrane association assays and glycosylation site mapping were used to investigate the membrane topology of the two proteins. Confocal images showed that both N T B - V P g and X 2 directed G F P to the E R membranes, and both proteins co-fractionated with an E R marker (Bip) when expressed in planta. In the N T B - V P g protein, two distinct ER-binding domains were identified: an N-terminal amphipathic helix and a C-terminal transmembrane domain. The C-terminal transmembrane domain was sufficient to direct G F P to the E R and translocate the downstream V P g domain into the E R lumen, resulting in ER-specific glycosylation at the naturally occurring glycosylation site in the V P g domain; the N -terminal amphipathic helix was also necessary and sufficient to direct G F P to intracellular membranes in planta and translocate the N-terminus of N T B into the E R lumen at least in vitro. In X 2 , three ER-targeting domains were identified by mutagenesis in planta: two C-terminal transmembrane domains and a less-well-defined domain further 11 upstream. In vitro glycosylation mapping studies indicated that all three domains within X 2 were able to traverse the membranes, resulting in an overall Niumen/CCytosoi topology. Taken together, the results indicate N T B - V P g and X 2 are polytopic E R membrane proteins. ER-association of both proteins in the absence of other viral components suggests they are the membrane anchor proteins for the viral replication complex. i n Table of Contents Abstract ii Table of Contents iv List of Tables vii List of Figures viii List of Abbreviations x Acknowledgments xiii Co-Authorship Statement . xv Chapter 1: Literature review: membrane association of the replication complex of positive sense single stranded RNA viruses I 1.1 INTRODUCTION I 1.2 I N T R A C E L L U L A R M E M B R A N E S Y S T E M A N D M E M B R A N E TRAFFIC 4 1.2.1 Membrane organelles 5 1.2.1.1 The endoplasmic reticulum (ER) 5 1.2.1.2 The Golgi apparatus 6 1.2.1.3 Enclosomes 8 1.2.1.4 Lysosome-like organelles 8 1.2.2 Membrane traffic 9 1.2.3 Proteins soiling and localization in the endomembrane system 11 1.3 BIOGENESIS OF M E M B R A N E PROTEINS 13 1.3.1 Co-lranslalional integral membrane protein biogenesis 14 1.3.2 Post-translational membrane protein biogenesis 16 1.4 TOPOGENES1SOF INTEGRAL M E M B R A N E PROTEINS 17 1.5 M E M B R A N E ASSOCIATION OF REPLICATION C O M P L E X E S OF (+) ss RNA VIRUSES 19 1.5.1 Picomavims superfamily 20 1.5.1.1 Poliovirus 20 1.5.1.2 Plant viruses in the picornavirus superfamily 24 1.5.2 Alphavirus superfamily 25 1.5.2.1 Brome mosaic virus (BMV) .' 25 1.5.2.2 Other alpha-like viruses 27 1.5.3 Flavivirus superfamily 29 1.5.3.1 Hepatitis C vims (HCV) 29 1.5.3.2 Other viruses in the flavivirus superfamily 31 1.5.4 Host components involved in the formation of viral replication complexes 32 1.5.4.1 Membranes and proteins involved in lipid biosynthesis and membrane traffic 32 1.5.4.2 Translation factors 34 1.6 OVERVIEW OF ToRSV.. . 36 1.6.1 Molecular biology of ToRSV 37 IV 1.6.1.1 Genomic organization and gene expression 37 1.6.1.2. Genome replication and movement 41 1.7 R E S E A R C H OBJECTIVES 44 1.8 BIBLIOGRAPHY 45 Chapter 2: Evidence that insertion of Tomato ringspot nepovirus NTB-VPg protein in endoplasmic reticulum membranes is directed by two domains: a C-terminal transmembrane helix and an N-terminal amphipathic helix1 : 61 2.1 INTRODUCTION 61 2.2 M A T E R I A L S AND M E T H O D S 64 2.2.1 Plasmid constructions 64 2.2.2 Biolistic delivery of plasmids into N. benthamiana planls and confocal microscopy 68 2.2.3 Agroinfiltralion of N. benthamiana plants 69 2.2.4 Sub-cellular fractionation and deglycosylalionassays 69 2.2.5 In vitro translation assays 71 2.2.6 Computer-assisted prediction of putative transmembrane helices and amphipathic helices 71 2.3 RESULTS •, ?2 2.3.1 ER-largeting of GFP fusion proteins containing the entire NTB-VPg domain 72 2.3.2 Absence of membrane-targeting domains in the central region of NTB 76 2.3.3 ER-specific glycosyiation of GFP fusion proteins containing the hydrophobic domain in the C-terminal regioii of NTB-VPg 76 2.3.4 Partial ER-associalion of GFP fusion proteins containing the hydrophobic • domain in the C-terminal region of NTB-VPg 80 2.3.5 Membrane-association of GFP fusion proteins containing a putative amphipathic helix at the N- terminus of NTB 81 2.3.6 In vitro analysis of the topology of the N-terminal region of NTB-VPg in the membranes 86 2.3.7 Sub-cellular fractionation of truncated proteins containing the C-terminal or N-terminal regions of NTB-VPg fused lo the H A epitope tag 89 2.4 DISCUSSION 92 2.5 BILIOGRAPHY 99 Chapter 3: Characterization of membrane-association domains within the Tomato ringspot nepovirus X2 protein, an endoplasmic reticulum-targeted polytopic membrane protein2 105 3.1 INTRODUCTION 105 ' 3.2 M A T E R I A L S A N D M E T H O D S 108 3.2.1 Plasmid construction 108 3.2.2 Agroinfiltration of N. benthamiana plants and confocal microscopy 111 3.2.3 Subcellular fractionation and membrane flotation assays 1 111 3.2.4 In vitro translation assays and deglycosylalion assays 112 v 3.2.5 Computer-assisted multiple sequence alignemenls and prediction of putative transmembrane helices and amphipathic helices • 112 3.3 RESULTS 113 3.3.1 Computer-assisted prediction of hydrophobic regions within X 2 113 .3.3.2 Subcellular localization of GFP-tagged X 2 proteins 114 3.3.3 X 2 contains multiple ER-largeting domains 120 3.3.4 Topology of X 2 in ER membranes inferred from the pattern of glycosylation in vitro ;I23 3.4 DISCUSSION 126 • 3.5 BIBLIOGRAPHY 133 C h a p t e r 4: G e n e r a l d i scuss ion a n d future projects 138 4.1 G E N E R A L DISCUSSION .' 138 4.2 F U T U R E PROJECTS 148 4.3 BIBLIOGRAPHY 150 vt List of Tables Table 2.1 Primers used in plasmid constructions 66 Table 3.1 Primers used in this study for plasmid constructions 109 List of Figures Figure 1.1 Infection cycle of (+)ss R N A viruses 2 Figure 1.2 A schematic representation of the viral replication complex ( V R C ) 3 Figure 1.3 A simplified scheme of the endomembrane system and membrane traffic in eukaryotic cells 5 Figure 1.4 The membrane topology of integral membrane proteins 18 Figure 1.5 Genomic organization and expression of non-structural proteins encoded by representative viruses of the three superfamilies of (+)ss R N A virus .... 21 Figure 1.6 Morphology of purified T o R S V particles and symptoms and cytopathological effects induced by T o R S V infection 37 Figure 1.7 Schematic representation of the genomic organization of T o R S V and modular arrangement of replication proteins in picornaviruses 39 Figure 1.8 Processing of PI polyprotein encoded by T o R S V R N A I 41 Figure 2.1 Immunodetection of G F P fusion proteins containing the entire N T B - V P g or the central region of N T B 73 Figure 2.2 Subcellular localization of G F P fusion proteins containing the entire N T B - V P g protein or the central region of N T B 75 Figure 2.3 Immunodetection of G F P fusion proteins containing the C-terminal region of N T B - V P g 8 77 Figure 2.4 Sub-cellular localization of G F P fusion proteins containing the C-terminal region of N T B - V P g 81 Figure 2.5 Computer-assisted analysis of putative membrane-association domains in the N-tenninal region of N I B 82 Figure 2.6 Immunodetection of G F P fusion proteins containing the N-terminal domain of N I B : 83 Figure 2.7 Sub-cellular localization of G F P fusion proteins containing the N-terminal portion of N T B in N. benthamiana cells 85 Figure 2.8 Topological analysis of the N-terminus of N T B using an introduced N -glycosylation site . 87 Figure 2.9 Immunodetection of the N-terminal and C-terminal regions of N T B - V P g fused to epitope tags -. 90 Figure 2.10 Updated model for the insertion of the N T B - V P g protein into E R membranes 98 Figure 3.1 Computer-assisted prediction of transmembrane helices (TM) in the T o R S V X 2 protein 114 Figure 3.2 Multiple protein sequence comparison of the X 2 protein domains of . nepoviruses and the 32 kDa protein of comoviruses 115 Figure 3.3 Subcellular localization of G F P - X 2 and X 2 - G F P . : 1 17 Figure 3.4 Subcellular fractionation o X 2 fusion proteins 1 18 Figure 3.5 Subcellular fractionation of X 2 - G F P mutant derivatives 121 Figure 3.6 Subcellular localization of X 2 - G F P derivatives in epidermal cells of . .<V. benthamiana 122 Figure 3.7 In vitro glycosylation assays of wild-type or mutated X 2 124 Figure 3.8 Topological model of X 2 in E R membranes 129 Figure 4.1 Comparison of membrane-binding domains.of proteins from 'Poliovirus 2BC3AJB region, the C P M V 32k and 60K proteins and the T o R S V X 2 and N T B - V P g proteins 142 ix List of Abbreviations 3' Three prime 5' Five prime A Adenosine A r M V Arabis mosaic virus A. thaliana Arabidopsis thaliana A T G Start codon l a B M V helicase-like protein 2a B M V polymerase-like protein 3A Poliovirus 3A protein 3 A B Poliovirus 3 A B protein, precursor for 3 A and 3B Bip Binding protein, an E R chaperone protein B M V Brome mosaic virus BP-80 proteins Vacuole sorting receptor B R S V Beet ringspot nepovirus B R V Blackcurrant reversion nepovirus 2B Poliovirus 2B protein 2 B C Poliovirus 2 B C protein, precursor for 2B and 2C CP Virus coat protein C P M V Cowpea mosaic virus C-terminal Carboxy-terminal C-termini Carboxy-termini C-terminus Carboxy-terminus 2C Poliovirus 2C protein 3C Poliovirus 3C protein proteinase 3 C D Poliovirus 3 C D protein COPI Coat protein for vesicle moving from the Golgi to the E R COPII Coat protein for vesicle moving from the E R to the Golgi D Aspartic acid D N A Deoxyribonucleic acid E Glutamic acid Endo H Endoglycosidase H e E F I A Eukaryotic translation elongation factor 1 alpha eIF3 Eukaryotic translation initiation factor 3 eIF4E Eukaryotic translation initiation factor 4E eIF(wo)4E Eukaryotic translation initiation factor 4E plant isoform E R Endoplasmic reticulum ER-dsred2 A red fluorescent protein targeted into the E R lumen Fig Figure G Glycine in the context of amino acid sequence G D P Guanosine diphosphate G F L V Grapevine fanleaf virus G F P Green fluorescent protein x G T P Guanosine triphosphate GTPase Small proteins that bind to G T P K Lysine kDa Kilodalton 140K Turnip yellow mosaic virus replication protein 66K Turnip yellow mosaic virus polymerase 6-kDa Tobacco etch virus membrane anchor protein H C V Hepatitis C Virus H I V Human immunodeficiency virus I Isoleucine L Leucine L A M P lysosomal-associated membrane proteins L L / I L Endosome/lysosome sorting motifs M M Microsomal membrane M P Movement protein N Asparagine N. benthamiana Nicotiana benthamiana N P I R Plant vacuolar sorting motif on many proteins N S P Non-structural protein nsP 1 Semliki forest virus membrane anchor protein NS3 H C V NS3 proteinase N S 4 A H C V N S 4 A protein, NS3 proteinase cofactor N S 4 B H C V N S 4 B protein, a polytopic membrane protein N S 5 A H C V N S 5 A protein N S 5 B H C V polymerase N T B Nucleotide triphosphate-binding motif, T o R S V protein N T B - V P g T o R S V precursor protein of N T B and V P g N - X - S / T Consensus amino acid sequence of glycosylation, X i s any amino acid except proline P Proline P C R Polymerase chain reaction PNGase F Peptide TV-glycosidase F Pol T o R S V polymerase Poly A Polyadenylate Pro T o R S V proteinase P1 Poliovirus P1 region containing the structural proteins P1 Polyprotein encoded by T o R S V R N A 1 P123 Polyprotein of Semliki forest virus P1234 Polyprotein of Semliki forest virus P2 Poliovirus P2 region containing the 2 A B C proteins P3 Poliovirus P3 region containing the 3 A B C D proteins P30 Membrane-enriched fraction R Arginine S Serine S D S - P A G E Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Sec61 Translocon complex for translocation of protein across the E R xi S N A R E Soluble N-ethylmaleimide sensitive fusion attachment receptor SRP Signal recognition particle (+)ss R N A viruses Positive single strand R N A virus S3 Post-nuclear fraction S30 Soluble protein-enriched fraction T A protein Tail-anchored protein T Threonine T B S V Tomato bushy stunt virus T E V Tobacco etch virus T G N trans- Golgi network TGN38 A Golgi resident membrane protein T M V Tobacco mosaic virus T O M 1 Arabidopsis thaliana protein required for T M V replication T O M 2 A Arabidopsis thaliana protein required for T M V replication T o R S V Tomato ringspot virus T Y M V Turnip yellow mosaic virus U F A Unsaturated fatty acid V P g Vi ra l protein genome-linked VPg-Pro T o R S V precursor protein of V P g and Pro VPg-Pro-Pol T o R S V precursor protein of V P g , Pro, Pol V R C Vi ra l replication complex X 2 - N T B - V P g T o R S V precursor protein of X 2 , N T B and V P g wt/vol Weight per volume Y Tyrosine Acknowledgments I would like to express my gratitude to my supervisor, Dr. Helene Sanfacon, for her continual scientific guidance, encouragement, financial support and other countless help during the course of my Ph. D . study. She gave me the freedom to choose research projects, and spent a lot of time discussing them; she also put great effort in helping me with proposal writing and my comprehensive examination at the beginning of my Ph.D career, and preparation of presentations and manuscripts later on. In addition, she gave me several chances to attend scientific conferences, which greatly enriched my knowledge and opened my mind. Without her help, it would be much more difficult or impossible for me to get to this point. I would also like to thank other members of my supervisory committee, Dr. David Theilmann, Dr. George Haughn and Dr. X i n L i , for attending my annual progress meetings and providing valuable suggestions and advices to my research. I am particularly grateful to Dr. David Theilmann, who had to get up at 5am to catch the airplane from Penticton and to return at 11pm in order to attend my progress meetings. M y thanks must also go to former and current members of the Sanfacon lab for their cooperation, help and friendship. I would like to thank Dr. Shuocheng Zhang for collaboration on the project in Chapter 2. I would like to give my special thanks to Joan Chisholm for her excellent technical help, quick response to order and deliver lab supplies; and for her help with my English by reading manuscripts, thesis, presentation and correcting both written and spoken English. I want to thank the rest of the lab members, Rui Wen, Bita Jafarpour, Juan Jovel, Melanie Walker and Lianying Yang for x i i i scientific discussions, sharing new ideas, daily conversation, and fun times we had together. In particular, I am grateful to them for putting up with me taking extra work bench space. I would like to thank Michael Weis for help with confocal microscopy. I would like to extend my thanks to many people in the Pacific Agri-Food Research Center for their help. Thanks P A R C for generous permission to use the facilities. I was lucky to spend the last three and half years in the beautiful Okanagan Valley. There are so many things to say about this area: the breath-taking views, the climate and delicious fruits and especially the people. I have made many friends here, including John Laurie and his families, Juan and his family, Minggang and Yingchao Nie , Col in Hu, Junhuan X u and Xiaojiang Dai . We had so many fun times traveling together, camping and fishing together and plus numerous parties. Changwen L u and his family, Xiang Y u and his family and our neighbors in Trout Creek including Judy Fox and her family and so many more people gave us so much help during our stay in this area. The last but by no means the least, I would like to thank my wife for her unconditional support during my Ph.D. study. She always stands firmly behind me whatever I choose. She deserved an award for giving birth to our baby boy, Gordon X u Zhang and for taking care of him over the last 10 months. The tears, smiles and laughs of Gordon have brought us lots of happiness and have made the lonely research life more enjoyable. Co-Authorship Statement The studies described in Chapter 2 were initiated by Dr. Shuocheng Zhang and me. Dr. Zhang conducted most of the cloning and some of the in vivo and in vitro protein expression analysis. I conducted some of the cloning and most of the confocal analysis. I was actively involved in the preparation of the manuscript under the guidance of Dr. Helene Sanfacon. Joan Chisholm and Lanying Yang also contributed to this project with their technical assistance. xv C H A P T E R 1 Literature review: membrane association of the replication complex of positive sense single stranded RNA viruses 1.1 INTRODUCTION Viruses are submicroscopic, obligate intracellular parasites. They are structurally simple organisms consisting of a small nucleic acid genome surrounded by a protein shell. Some of them also have a membranous envelope. The genome of a virus, which can be D N A or R N A , single stranded or double stranded, is replicated inside host cells using host translational and metabolic machinery. Many viruses have a single stranded R N A genome of positive polarity [(+)ss R N A virus]. They constitute taxonomically diverse viral groups and species that differ in morphology, genomic organization and terminal structures in their genomes. The only commom feature of (+)ss R N A viruses is the requirement for nonstructural proteins for genome replication, in particular an R N A dependent R N A polymerase (RdRp). Phylogenetic analysis of RdRps of all groups of (+)ss R N A virus suggests that they can be classified into three superfamilies. Superfamily I, the picornavirus superfamily, includes picorna-like, poty-like, sobemo-like and arteri-like viral groups. Superfamily II, the flavivirus superfamily, includes carmo-like viruses and flavi-like viruses. Superfamily III, the alphavirus superfamily, includes tymo-like, rubi-like and tobamo-like viruses (Koonin, 1991; Koonin and Dolja, 1993). In addition to the conserved RdRp, viruses in each superfamily may also have similar helicase and accessory proteins, modular arrangement of replication proteins and conserved 3' and 5' structures of viral genomes. These features suggest that viruses within each superfamily have similar replication strategies. 1 A s an obligate intracellular organism, a virus begins its infection cycle by entering a host cell (Fig. 1.1). Once inside the cell, the virus disassembles and releases its genome. Fig. 1.1 Infection cycle of (+)ss RNA viruses. The infection cycle can be divided into six steps: 1, Entry: a virus enters a host cell; 2, Uncoating: the viral particle dissembles to release its genome; 3, Translation: the viral RNA is translated to produce replicase and capsid proteins; 4, RNA replication: the viral RNA is used as a template to produce more progeny RNAs; 5. Assembly: the progeny RNA and capsid proteins form new viral particles; 6, Exit: the newly formed viral particles exit the cell and are ready for infection of other cells For (+)ss R N A viruses, the viral genomic R N A can serve directly as m R N A to synthesize viral proteins required for R N A replication, and then switches from translation to act as a template for R N A replication. R N A replication takes place in two steps: first, the viral genomic R N A is used as a template to synthesize negative strand R N A ; second, the negative strand R N A is used as a template to produce progeny viral R N A s . The progeny R N A s and capsid proteins, which usually are synthesized late in the infection cycle, assemble to form new viral particles. The progeny viral particles then exit the cell and are ready to infect next cell. Entry o TO 2 The replication of R N A occurs in a multi-component complex, referred to as the viral replication complex ( V R C ) (Fig. 1.2), which consists of the viral replicase proteins, viral Fig. 1.2. A schematic representation of the viral replication complex (VRC). The V C R contains viral RNA, viral replicase proteins and probably host proteins and the V C R is assembled on the inner surface of invaginated intracellular membranes. R N A s and probably host proteins. With no exception, replication complexes of all well-characterized (+)ss R N A viruses are associated with intracellular membranes which can be derived from various organelles including the endoplasmic reticulum (ER), Golgi , tonoplast, mitochondria, chloroplast and endo-lysomal compartment (Salonen et al., 2005a). Targeting of viral replication complexes to specific membrane organelles is often mediated by virus-encoded integral membrane proteins. The viral integral membrane proteins act as membrane anchors for the V R C and other viral proteins or viral R N A are brought to the membranes via protein-protein and protein-RNA interactions. 3 In this review, I w i l l begin with a brief discussion of the intracellular membrane system, membrane traffic and biogenesis of membrane proteins. Then, I w i l l provide a comprehensive overview of what is known about the formation of replication complexes at the specific intracellular membranes by (+)ss R N A viruses. Since Tomato ringspot virus (ToRSV) is used as a model in this study, I w i l l conclude with a general overview of its molecular biology. 1.2 I N T R A C E L L U L A R M E M B R A N E S Y S T E M A N D M E M B R A N E T R A F F I C A l l eukaryotic cells contain extensive intracellular membranes that divide the cell into different membrane-enclosed compartments, called organelles. Each organelle contains distinct membrane lipids and enzymes, and has different functions. The organelles common to both plant and animal cells are the E R , the Golgi apparatus, mitochondria, endosome, and peroxisome. Plant and animal cells also contain special organelles such as chloroplasts and vacuoles (in plant) and lysosomes (in animal). Most of the organelles are involved in either endocytic or biosynthetic/secretory pathways (Fig. 1.3). The organelles in these pathways are interconnected by transport vesicles, which bud from the donor membranes and fuse with acceptor membranes thereby passing membranes and molecules associated with the membranes from one organelle to another. 4 Fig. 1.3 A simplified scheme of endomembrane system and membrane traffic in eukaryotic cells. In the biosynthetic/secretory pathway, cargo synthesized in the ER is transport to the Golgi in COPII coated vesicles; ER resident proteins are recycled in COPI coated vesicles. Secretory cargo as well as cargo destined for downstream organelles are further transferred from the Golgi in clathrin coated vesicles. In endocytic pathways, proteins are internalized at the plasma membrane and transferred to the early endosome, from where the proteins can be recycled to the plasma membrane or transported to the lysosome/vacuole through the late endosome/multiple vesicular body (MVB) or prevacuole compartmemt (PVC) in plants. 1.2.1 Membrane organelles 1.2.1.1 The endoplasmic reticulum (ER) The E R is the most dynamic endomembrane system and typically constitutes over half of the total membrane of a cell (Alberts, 2002). It reaches from the perinuclear areas to the cortical regions and forms a net-like structure throughout the cytosol. A single internal space, called the E R lumen, is enclosed by the E R membrane. Ultrastructually, the E R can be subdivided into many distinct functional domains (Staehelin, 1997). 5 The E R plays central roles in l ipid and protein biosynthesis. It produces almost all of the lipids for cellular organelles and a major portion of proteins. In addition, the E R is the gateway for newly synthesized proteins to enter into the biosynthetic/secretory pathway. Secretory proteins or proteins destined to reside in later compartments start their journey by crossing the E R membranes (Vitale and Denecke, 1999). The E R is also involved in protein folding and modification which usually occurs in the E R lumen. Folding of nascent proteins is assisted by a number of chaperones, e.g. B ip which is commonly used as an E R marker (Gething, 1999). Modification includes removal of the N-terminal signal sequence by a signal peptidase and glycosylation. Both N - and O-linked glycosylation can occur in the E R . N-glycosylation is initially catalyzed by an oligosaccharyl-transferase which transfers a 14 residue oligosaccharide to an asparagine in the consensus sequence - N - X - S / T (where X is any amino acid except proline). The oligosaccharide is further trimmed by other enzymes to produce the final glycan structure. O-glycosylation is the result of addition of a G a l N A c to a serine or threonine by N-acetygalactosaminyl-transferase (Hounsell et al., 1996; Kornfeld and Kornfeld, 1985). 1.2.1.2 The Golg i apparatus The Golgi apparatus consists of a stack of flattened membrane-enclosed cisternae which are further divided into cis-, medial and trans-Golgi. Both cis- and trans- faces are closely associated with two tubular and cisternal structures called cis Golgi network and trans-Go\g\ network (TGN) . Animal cells tend to have fewer and larger Golgi 6 apparatuses that are relatively static. In contrast, plant cells contain as many as hundreds of Golgi stacks. Golgi stacks in plant cells are highly mobile on the tubules of the cortical E R network (Boevink et al., 1998). The Golgi is a major modification and sorting station for proteins destined for the secretory pathway or later compartments (Warren and Malhotra, 1998). The cis- face of the Golgi receives cargo proteins from the E R , which are then transported through the Golgi stack to the trans-Golgi network, where the proteins are sorted and sent to different destinations. Proteins en route to the Golgi are further processed by glycosylation enzymes residing within the Golgi . These enzymes modify the N-l inked glycan on glycoproteins synthesized in the E R . The final structures of the N-l inked glycan can be described as high-mannose, complex or both (Kornfeld and Kornfeld, 1985). The glycosylation enzymes reside in different sub-compartments of the Golg i and thereby are frequently used as Golgi markers (Neumann et al., 2003). The N-l inked glycosylation that occurs in the E R and Golgi can be distinguished using two different deglycosylation enzymes, Endoglycosidase H (EndoH) and Peptide iV-glycosidase F (PNGaseF). EndoH only recognizes the non-modified N-glycosylation from the E R while PNGaseF cleaves all N-l inked glycan (Tarentino and Maley, 1974; Tarentino and Plummer, 1994). Modification in the Golgi is different in plants and animals as some plant modifications are not recongnized by the PNGase F. The Golgi also participates in lipid biosynthesis which increases the concentration of cholesterol and sphingolipids in membranes (van Meer, 1998). In plant cells, the Golgi 7 has one additional function, which is synthesis of cell wall polysaccharides (Dhugga, 2005). 1.2.1.3 E n d o s o m e s Endosomes are fairly well defined in mammalian cells. Three morphologically and functionally distinct compartments have been identified, which include early endosomes, late endosomes and recycling endosomes. Each compartment has different luminal p H and varied protein and lipid composition (Geldner, 2004; Sachse et al., 2002). Early endosomes receive cargo internalized from the plasma membranes. The internalized molecules are sorted at this compartment to either late endosomes or recycling endosomes. The molecules to be degraded are transported to late endosomes while the molecules to be recycled back to the plasma membrane are transported through the recycling endosomes. In addition, early and late endosomes also send to and receive cargo from the T G N (Abazeed et a l , 2005; Cook et al., 2004). Endosome compartments are not well understood in plants, althrough putative early endosomes and late endosomes (referred as prevacuolar compartment) have been identified (Tse et a l , 2004; Ueda et al., 2001). 1.2.1.4 L y s o s o m e - I i k e organe l les In contrast to uniform structures of most other cellular organelles, lysosomes constitute morphologically heterogeneous membrane-bound compartments whose common feature is a high content of hydrolytic enzymes in the lumen (Alberts, 2002). More than 50 acid-8 dependent hydrolases residing in the lumen which perform the the primary digestive function of this compartment have been identified. Macromolecules can be delivered to lysosomes through the biosynthetic or endocytic pathways (Fig. 1.1) (see section 1.1.2 for a discussion of these pathways). In addition to the soluble hydrolases, lysosomes contain a set of highly glycosylated membrane proteins referred as lysosomal-associated membrane proteins ( L A M P ) (Eskelinen et al., 2003). The functions of these L A M P s are not clear. Nevertheless, these proteins, e.g. L A M P 1 and L A M P 2 , are useful as lysosome marker proteins. Plant cells do not possess lysosomes. The degradation function is performed by vacuoles which are related to the lysosomes of animal cells. The vacuoles are one of the hallmarks of plant cells. In plant vegetative tissues, vacuoles usually take up more than 90% of the cell volume. Plant vacuoles are multifunctional organelles. In addition to its digestive function, this organelle also plays important roles in turgor maintenance, protoplasmic homeostasis, storage of metabolic products and sequestration of xenobiotics. Plant reserve tissues, such as seed and fruit, contain a second type of vacuoles, called protein-storage vacuoles. These vacuoles accumulate proteins that are utilized as nutrition during seed germination (Marty, 1999). 1.2.2 Membrane traffic Except for mitochondria and chloroplasts, all other organelles are interconnected by a continuous stream of membranous vesicles which bud from one organelle, travel through the cytosol and fuse with another organelle. In biosynthetic and secretory pathways, 9 membranes and proteins are transported from the E R via the Golgi to the plasma membrane or to lysosomes/vacuoles (via late endosome). In endocytic pathways, molecules are internalized at the plasma membrane and delivered to lysosomes or vacuoles via early and late endosomes (Fig. 1.3). The basic molecular mechanisms underlying the vesicle-mediated membrane traffic appear to be the same, irrespective of organelles involved. The initial step of formation of vesicles involves recruiting coat proteins (COPs) to a donor membrane to induce proteinaceous membrane pits. This process requires a GTPase which is activated by its G D P / G T P exchange factor. There are three well characterized types of coat protein: COPI , COPII and clathrin. COPI - and COPII- coated vesicles mediate transport between E R and Golgi . Clathrin-coated vesicles act in trafficking from the T G N to endosomes and from the plasma membrane to endosomes (Fig. 1.1) (Bonifacino and Lippincott-Schwartz, 2003; Robinson et al., 1998). Following budding from the donor membrane, the coated vesicle pinches off from the membrane via the activity of GTPase dynamin-like proteins, which form a helix around the neck of the vesicle and scissor it off the membrane by G T P hydrolysis (Praefcke and McMahon , 2004). Fusion of the vesicle with its target membrane occurs after the vesicle has shed its coat. This process takes place in two steps. First, the vesicle is attached to the membrane by a tethering complex, which brings the vesicle to the target membrane. Second, the soluble N-ethylmaleimide sensitive fusion attachment receptor proteins, known as the S N A R E proteins, which reside in both the vesicle and target membrane, interact and form a trans-complex, bringing the membranes together and resulting in membrane fusion (Jahn et al., 2003). 10 1.2.3 Proteins sorting and localization in the endomembrane system Despite extensive membrane traffic, cellular organelles in the endomembrane system maintain their own resident proteins while allowing transit of others. Therefore, specific protein sorting is required to separate passenger proteins from residents. The E R is the first sorting station in the biosynthetic-secretory pathway. Newly-synthesized proteins destined for secretion or to downstream organelles leave the E R in COPII vesicles. The cargo proteins can be packaged in the COPII vesicles by selective uptake i f signals present in the cargo are recognized, or through bulk flow in which signals in the proteins are necessary for retrieval and retention, and absence of these signals leads to secretion (Warren and Mellman, 1999). Thus, E R localization is achieved by two complementary mechanisms: retention and retrieval (Teasdale and Jackson, 1996). Proteins could be retained in the E R through interaction with chaperones or by forming large aggregates. Escaped ER-resident proteins are returned from the Golgi in retrograde COPI vesicles. K D E L and dilysine motifs which mediate the retrieval of E R resident proteins are well characterized (Letourneur et al., 1994; Lewis et al., 1990). The Golgi is the major sorting station and proteins are directed to different destinations including retrograde transport to the E R , secretion, the plasma membrane and the endosomes/lysosomes (vacuoles). Soluble secretory cargo proteins follow the default bulk flow route. The proteins for other cellular destinations require specific sorting signals for correct targeting. As mentioned above, the K D E L and dilysine signals mediate retrograde transport from the Golgi to the E R . Three major endosome/lysosome 11 sorting motifs, dileucine (LL/ IL) and tyrosine ( G Y X X ) and mannose-6-phosphate, direct proteins from the T G N to endosomes/lysosomes (vacuoles) (Gu et al., 2001). Plasma membrane targeting uses more diverse mechanisms. In animal polarized cells, a tyrosine based sorting motif and l ipid rafts have been shown to mediate the basolateral targeting and apical plasma membrane from the T G N , respectively (Keller and Simons, 1997). There is also evidence that the length of the transmembrane domain can act as a sorting signal to the plasma membrane (Brandizzi et al., 2002). The sorting signals are recognized by different C O P proteins. A s a result, the cargo proteins are sorted into different coated vesicles and transported to various locations (Gu et al., 2001). A s for E R proteins, both retention and retrieval probably play a role in the localization of proteins to the Golgi . For example, a tyrosine based motif ( S D Y Q R L ) of TGN38 is necessary for retrieval of this protein from the plasma membrane to the T G N while the transmembrane domain of this protein may act as a Golgi retention signal (Humphrey et al., 1993; Ponnambalam et al., 1994). In the case of glycosyltransferase, which is the most extensively studied membrane protein of the Golgi , the transmembrane domain contains a dominant localization signal. Two models are proposed to explain the transmembrane domain-mediated localization. One is lipid bilayer sorting in which the length of the transmembrane domain is the sole determinant for the Golgi localization. Another is oligomerisation in which the transmembrane domain drives the aggregation of the protein which is then excluded from the transport vesicles (van Vlie t et al., 2003). 12 Another major sorting station is the endosome. The sorting begins in the early endosome where the endocytic and biosynthetic pathways converge (Press et al., 1998). In receptor-mediated endocytosis, the receptors are sorted based on the presence or absence of bound ligand in early endosomes. In the presence of ligand, the receptor is transported to late endosomes/lysosomes for degradation which is mediated by mono-ubiquitination of receptors (Raiborg et al., 2003). In the absence of ligand, the receptors return through recycling endosomes to the plasma membrane. For cargo proteins derived from the biosynthetic pathway, sorting to late endosome/lysosome is based on the dileucine, tyrosine and other motifs while targeting to the plasma membrane probably uses the same route as receptor recycling. L ip id rafts may also play a role in this process. Plant cells have two types of vacuoles. Sorting of proteins to lytic vacuoles is mediated by specific vacuole targeting signals, e.g. N P I R motif on many vacuole proteins, which are recognized by members of the BP-80 family of vacuole sorting receptors. The proteins are then transported in clathrin-coated vesicles from the T G N (Paris and Neuhaus, 2002). Transport to storage vacuoles is mediated by dense vesicles which often form at the cis-Golgi cisternae, suggesting the storage route may bypass the T G N (Robinson et al., 2005). 1.3 B I O G E N E S I S O F M E M B R A N E P R O T E I N S Most membrane proteins can be divided into three categories: peripheral membrane proteins, luminal membrane proteins and integral membrane proteins. Peripheral membrane proteins are synthesized in the cytosol and are recruited to the membranes 13 through interaction with other membrane proteins or membrane lipids. Both luminal membrane proteins and integral membrane proteins are initially synthesized in the cytosol and then targeted to the membranes. Luminal proteins are first targeted to the E R by the signal recognition particle (SRP)-dependent pathway and co-translationally translocated into the E R lumen from where the proteins are transported to their final destinations using the sorting signals discussed above (Walter and Johnson, 1994). Biogenesis of integral membrane proteins is much more complicated than that of luminal proteins and involves several interrelated and sometimes overlapping steps: targeting of the nascent chains to the E R , translocation of E R luminal domains, insertion and proper orientation of transmembrane domains into the lipid bilayer and correct folding of the nascent protein. 1.3.1 Co-translational integral membrane protein biogenesis Most eukaryotic integral membrane proteins are targeted to the E R co-translationally. The SRP plays an important role in this process. Initially, the SRP binds to a signal sequence or transmembrane domain emerging from the ribosome and arrests the translation. The SRP-nascent chain-ribosome complex is subsequently targeted to the E R membrane through interaction with SRP receptors on the E R membrane. The ribosome-nascent chain complex is then transferred from the SRP to the translocation site. Translation of the nascent chain resumes, and co-translational translocation initiates (Walter and Johnson, 1994). 14 The translocation machinery in the E R is termed the Sec61 translocon complex. It is a heterotrimer consisting of a-, p1-, and y-subunits (Johnson and van Waes, 1999). The a-subunit contains ten transmembrane helices which adopt a compact helix bundle structure in the membrane. A potential hydrophilic pore formed in the middle of the helix buddle is thought to be the protein translocation channel. A lateral exit side formed between the helices 2 and 7 of the a-subunit might allow the transmembrane segment of the nascent chain to move from the translocon into the lipid bilayer (Van den Berg et al., 2004). A number of proteins including the translocating-chain-associated membrane protein, signal peptidase, oligosaccharyltransferase and chaperones are associated with the translocon. These proteins are required for the biogenesis of some but not all membrane or secretory proteins (Gorlich and Rapoport, 1993; Johnson and van Waes, 1999). Once the nascent chain-ribosome complex is transferred to the translocation site, the signal sequence is recognized by components of the translocon. The recognition is important for tight binding of the ribosome to the translocon, gating of the translocation channel and initiation of translocation (Crowley et al., 1994; Jungnickel and Rapoport, 1995). Secretory and soluble luminal proteins are completely translocated into the E R lumen and the signal sequence in these proteins is cleaved by a signal peptidase following translocation, while integral membrane proteins are only partially translocated into the E R lumen. For the integral membrane proteins with a cleavable N-terminal signal sequence followed by a single transmembrane domain, translocation of the proteins stops when the transmembrane segment enters the translocon (also referred as a stop transfer signal). The transmembrane segment is recognized by the translocon, which in turn 15 mediates the l ipid partitioning of this hydrophobic sequence (Heinrich et al., 2000). Some single-spanning membrane proteins are targeted to the E R by signal-anchor sequences which are located internally in the polypeptide and do not have a signal peptidase cleavage site. The signal anchor sequences are likely integrated into the l ipid bilayer in the same manner as the cleavable signal sequence (McCormick et al., 2003). In the case of polytopic membrane proteins, it is generally believed that the first transmembrane domain targets and initiates the translocation across the membrane. Membrane integration and translocation of E R luminal domains are mediated by the same Sec61 translocon as single-spanning membrane proteins. In contrast to single-spanning membrane proteins, biogenesis of polytopic membrane proteins requires multiple rounds of translocation of the luminal domain and membrane integration of transmembrane segments by the translocon. Two theoretical models for describing this process have been proposed. The first one is the 'en bloc' model, in which all the transmembrane domains accumulate inside the translocon before being simultaneously released into the membrane. The second model is 'sequential transfer', in which transmembrane domains enter the lipid bilayer individually (Lecomte et al., 2003). Both models are probably valid to a certain extent as experimental evidence favouring each of them has been provided (Sadlish et a l , 2005). 1.3.2 Post-translational membrane protein biogenesis Membrane integration of proteins can also occur post-translationally. Tail-anchored (TA) proteins are such examples. T A proteins have a cytosolic N-terminal domain that is 16 anchored to the membranes by a single transmembrane domain located close to the C-terminus. The membrane integration of these proteins is obligatorily post-translational since the signal anchor in these proteins is not synthesized until translation is almost finished. T A proteins have been found in association with many types of intracellular membranes including the E R . Information for targeting to the different membranes is contained in the C-terminal tail (Borgese et al., 2003). However, the cellular machinery involved in T A protein targeting and insertion is not well characterized. The E R integration of some synaptobrevins can occur in an SRP-dependent manner (Abell et al., 2004; Kutay et al., 1995). Proteins with a transmembrane amphipathic helix are likely also integrated into the membranes post-translationally. It has been proposed that membrane integration of an amphipathic helix involves two steps: the amphipathic helix initially lays parallel to the membrane with the hydrophobic side facing the membrane and the hydrophilic side to the cytosol, then the amphipathic helix oligomerizes to obtain enough hydrophobicity to traverse the membrane (Bechinger, 1999; Shai, 1999). 1.4 T O P O G E N E S I S O F I N T E G R A L M E M B R A N E P R O T E I N S Integral membrane proteins can orient their transmembrane domains in many different ways. Single-spanning membrane proteins with a cleavable signal sequence always translocate their N-terminus into the E R lumen, whereas single-spanning membrane proteins with a signal-anchor sequence as well as multi-spanning membrane proteins can translocate either N-or C-termini into the luminal side (Fig. 1. 4). 17 A number of factors can influence the orientation of a transmembrane domain in the membranes. The best characterized topological determinant is the distribution of charged A ) B i t o p i c m e m b r a n e p r o t e i n s C y t o s o l E R - l u m e n T y p e I T y p e d T y p e I I I C - t e r m i n a l a n c h o r S i g n a l a n c h o r B ) P o l y t o p i c m e m b r a n e p r o t e i n s C y t o s o l E R - l u m e n 0 C l e a v a b l e s i g n a l s e q u e n c e Q T r a n s m e m b r a n e d o m a i n Fig.1.4. The membrane topology of integral membrane proteins. (A) Bitopic membrane proteins are classified into type 1 (with a cleavable N-terminal signal sequence, membrane topology: N|umen/CCyt0soi), type II (with an internal signal-anchor sequence, membrane topology: Ncytosoi/Qumen), type III (with a reverse signal-anchor sequence, membrane topology: N|umen/Ccyt0S0i) and type IV (with a C-terminal signal). (B) Polytopic membrane proteins can adopt many different topologies, and the overall topology of a polytopic protein is dictated in most cases by the orientation of the first transmembrane domain and the number of transmembrane segments. amino acids flanking the transmembrane domain: more positively charged segments usually stay in the cytoplasmic side, thereby referred to as 'positive inside rules'(Hartmann et al., 1989; Heijne, 1986). For proteins with a signal-anchor sequence, sequences N-terminal to the signal sequence are exposed to the cytosol before the 18 targeting sequence emerges from the ribosome. The folding state but not simply the size of the N-terminus is also an important factor in determining the orientation of the transmembrane domain in addition to the charged amino acid flanking it (Denzer et al., 1995). Another well characterized topological determinant is the hydrophobicity of the signal or signal-anchor sequence. Increasing the total hydrophobicity produces an increased fraction of Niumen/CCytosoi proteins to 80% while a signal sequence with lower hydrophobicity yields exclusively Ncytosoi/Ciumen topology (Wahlberg and Spiess, 1997). For multi-spanning membrane proteins, the first transmembrane domain usually dictates the topology of the whole protein and the orientation of this domain is determined by the above factors (Wessels and Spiess, 1988). However, there are also examples in which the downstream transmembrane domain can determine orientation of an upstream one (Ota et al., 2000). Other determinants, e.g. glycosylation can also influence the topology of membrane protein (Goder et al., 1999). 1.5 M E M B R A N E A S S O C I A T I O N O F R E P L I C A T I O N C O M P L E X E S O F (+)ss R N A V I R U S E S Infection with (+)ss R N A viruses often induces massive proliferation and rearrangement of intracellular membranes which can be derived from various cellular organelles. Studies from different virus superfamilies demonstrate that these modified membranes carry the viral replication complex (Salonen et al., 2005b; Sanfacon, 2005). Generation of the membrane-bound replication complex depends on viral nonstructural proteins, in particular viral integral membrane proteins. These proteins induce the viral specific membrane structures and promote the interaction of other replication components with the membrane structures to assemble the replication complex. Membrane association of 19 the viral replication complex probably increases local concentration of the replication components by targeting them to a common structure and thereby facilitates efficient R N A replication (Lyle et al., 2002). Membrane association also protects viral R N A s from degradation (Janda and Ahlquist, 1998). In this part, I w i l l discuss the formation of membrane-bound replication complexes using well-studied viruses in each superfamily as examples. Emphasis w i l l be given to viral membrane anchor proteins. 1.5.1 Picornavirus superfamily 1.5.1.1 Poliovirus Poliovirus is one of the best characterized viruses in this supergroup. The poliovirus genome is a single R N A that has a V P g (Viral Protein-Genome-linked, which serves as a primer for R N A replication) covalently linked to its 5' end and a poly A tail at its 3' end. The entire R N A genome is first translated into a single polyprotein which has been divided into three regions called P I , P2 and P3 based on the initial cleavage events. PI contains the virion structural proteins, while P2 and P3 are precursors for nonstructural proteins functioning in viral replication. The polyprotein is processed by two viral-encoded proteinases to produce intermediates and mature proteins (for details see F ig 1.5 A ) . Both mature and intermediate proteins produced from P2 and P3 may participate in R N A replication (Knipe and Howley, 2001). Upon entering into host cells, the viral R N A s apparently migrate to the perinuclear E R 20 A ) P o l i o v i r u s g>@ |1A| 1B I  1C I 1D I 2 A M 2C |3AN 3C I 3D I poly (A) 3' I Translation [ T A O B II 1C I 1D l-2A-|za 2C I3AW 3C I 3D ~ 1 | Polyprotein process ing H p t >i h — P 2 —H h P3 ——»1 |1A| 1B II 1C l " lD~l 12A Eg 2C I |3AH-"3C I 3D - I [ H H ^ M HAI h3C I B) HCV 3 B = V P g g> JJIB—rcrTT II E2 I^S2J NS3 l»lNS4Bi NS5A I NS5B l-UIB-3' * Translation \<— S P — • < NSP • ! Id E1 I  E2 I MS2I NS3 '•' I '1% |NS4B|| NS5A 1 NS5B 1 I Polyprotein process ing to2J|:-:NS3^TOlNS4ilI NS5A if^^sassa C) BMV 5' , , 3' 5' , , 3' 5' . . 3' rap-1 RNA1 I g= n a p - | RNA2 H c a p - | RNA3 [ £ I \ * I -2a- j C P and M P — UTR l - " - l Proteinase I I Putative Helicase [ \ Methyltransferase tRNA like I I VPg I I RNA-dependent RNA polymerase Fig.1.5 Genomic organization and expression of non-structural proteins encoded by representative viruses of the three superfamilies of (+) ss R N A virus. (A) Poliovirus, (B) Hepatitis C Virus (HCV) and (C) Brome mosaic virus (BMV) . The genome of each virus is shown at the top of each diagram with 5' and 3' structures, untranslated regions (UTR, horizontal lines) and open reading frames (boxes). Vertical lines within the open reading frames of poliovirus and H C V represent proteolyic cleavage sites with the names of the mature proteins released by these cleavage events indicated. Expression of non-structural proteins of each virus is shown under its genome. Proteins with known or predicted function(s) are colored. (A) The open reading frame of poliovirus is first translated into a polyprotein which is divided into three regions, PI , P2 and P3 via nascent cleavage by the viral proteases. Only processing of P2 and P3, which contain the replication proteins, is shown. Note that stable processing precursors such as 2BC, 3AB and 3CD also accumulate. (B) The non-structural proteins (NSPs) of H C V are first translated as part of a polyprotein, which also contains structural proteins (SP), and are released by polyprotein processing via tha activities of the viral proteinases. (C) The two replication proteins of B M V , l a and 2a, are expressed from RNA1 and RNA2, respectively, while the coat protein (CP) and movement protein (MP) are produced from RNA3. 2 1 where translation and processing take place (Egger and Bienz, 2005). Translation of the viral R N A is soon followed by the appearance of numerous membrane vesicles in the perinuclear area, a phenomenon typical in early poliovirus infection. With the progress of infection, some vesicles acquire the characteristics of autophagic vacuoles which are double membrane-bound vacuoles originated from the E R (Suhy et al., 2000). P2 and P3 proteins and plus- and minus-strand R N A synthesis are associated with those membrane vesicles. Therefore the vesicles carry the poliovirus replication complexes (Bienz et al., 1994). Formation of the poliovirus replication complex is coupled with translation and vesicle induction (Egger et al., 2000). This suggests that the translation-competent E R membranes are involved in the biogenesis of the poliovirus vesicles. The E R origin of poliovirus vesicles is confirmed by the observation that poliovirus appears to use the cellular COPII proteins to bud from the E R membrane. Interestingly, E R resident proteins are excluded from the budded vesicles. The vesicles accumulate in the cytosol instead of being transported to the Golgi (Rust et al., 2001). The replication complex of poliovirus also contains the Golgi marker galT and the lysosome marker L A M P - 1 , suggesting that membranes other than the E R may also be a source for the poliovirus-induced membrane structures (Schlegel et al., 1996). To identify the viral factors involved in the biogenesis of the poliovirus replication complex, viral proteins have been expressed individually or in combination in the absence of viral R N A and replication. Three proteins, 2B, 2C and 3A, and the precursors 22 containing these domains have been shown to play roles in membrane association and membrane induction observed in poliovirus-infected cells. The 3A protein binds to the E R membrane in a manner consistent with that of an integral membrane protein and the membrane binding region has been mapped to a C-terminal hydrophobic sequence (Towner et al., 1996). The N-terminal portion of 3 A is exposed to the cytosol either when expressed alone or in viral infection (Choe and Kirkegaard, 2004) and the C-terminal hydrophobic segment may form a transmembrane domain (Lee et al., 2006, unpublished observation). 3 A in isolation causes dilation of the E R membrane, inhibits E R to Golgi anterograde traffic but does not induce vesicle accumulation (Doedens et al., 1997). Expression of 2C induces vesicles similar to these observed in infected cells and the 2C protein is localized to the modified E R membranes. Binding of 2C to the cellular membrane resembles that of an integral membrane protein. Both the N - and C-termini are involved in membrane association, whereas only the N-terminal region is responsible for membrane rearrangement (Teterina et al., 1997). 2B is a hydrophobic protein of about 100 amino acids. It contains two membrane-binding elements: an N-terminal putative amphipathic helix and a C-terminal transmembrane domain. 2B in isolation targets to the E R and to Golgi complexes. It inhibits E R to Golgi transport and disassembles the Golgi complexes (Doedens and Kirkegaard, 1995; Sandoval and Carrasco, 1997). 2B can also increase membrane permeability by forming an aqueous pore through oligomerization of the N-terminal amphipathic helix (Gonzalez and Carrasco, 2003). The protein 2 B C retains the ability of both 2B and 2C in inducing membrane proliferation, vesicle formation and inhibiting the exocytic pathway (Barco and Carrasco, 1998). Coexpression of 2 B C and 3A induces membranous vesicles similar to those found in infected cells 23 (Suhy et al., 2000). However, none of these preformed membrane structures can be used by poliovirus to assemble the replication machinery. To form the replication complexes, the vesicle induction must be coupled to viral translation and R N A synthesis. The viral R N A is stably associated with the vesicles only when R N A replication could occur (Egger et al., 2000; Teterina et al., 2001). 1.5.1.2 Plant viruses in the picornavirus superfamily Plant picorna-like viruses, e.g., comoviruses and nepoviruses, share replication modules with poliovirus. Potyviruses have a similar genomic organization and mode of gene expression to that of picornaviruses. These viruses seem to replicate in a way similar to that of poliovirus. Tobacco etch virus (TEV) , a potyvirus, induces aggregates of E R membranes and viral R N A replication is associated with these modified E R membranes (Schaad et al., 1997). The 6-kDa protein (see Fig . 1.7) in isolation can direct G F P to the E R membrane and induces large vesicular compartments derived from the E R . It behaves like an integral membrane protein and membrane binding is mediated by a central hydrophobic domain. A s the 6-kDa protein is the only integral membrane protein encoded by the T E V genome; the replication complexes of T E V are proposed to be directed to the E R membrane by this protein (Schaad et al., 1997). Comoviruses also replicate in association with ER-derived membranes. Cowpea mosaic virus ( C P M V ) infection induces production of vesicles and massive proliferation of the E R membranes but not the Golgi complex (Carette et al., 2000). C P M V replication proteins and R N A synthesis are colocalized with the modified E R membranous structures (Carette et al., 2002a; Carette et al., 2000). In the absence of other viral proteins, the 32K- and 60K-24 target to the E R and cause morphological changes in the membranes, suggesting these two proteins might target and anchor the C P M V replication complexes to the E R (Carette et al., 2002b). In the case of nepoviruses, both Grapevine fanleaf virus ( G F L V ) and T o R S V replicate in association with ER-derived membranes (Han and Sanfacon, 2003; Ritzenthaler et al., 2002). G F L V induces proliferation and a redistribution of the E R but not the Golgi to form the perinuclear viral replication compartments. Interestingly, immunotrapped vesicles from crude extracts of GFLV-infected protoplasts were occasionally aggregated into well-organized "rosette-like structures similar to those observed in poliovirus-infected cells (Ritzenthaler et al., 2002). It is not known which G F L V protein encoded by R N A 1 targets the replication complexes to the E R . However, there is evidence that the 2 A protein directs R N A 2 to the juxta-nuclear location where R N A replication takes place, possibly through interaction with R N A 1-encoded proteins ( G a i r e e t a l , 1999). 1.5.2 A l p h a v i r u s s u p e r f a m i l y 1.5.2.1 B r o m e m o s a i c v i r u s ( B M V ) B M V is a representative member of the alphavirus superfamily. The helicase-like l a and polymerase-like 2a proteins are expressed from two different R N A s and are both required for replication (Fig . l .5-B) . In addition to its natural plant hosts, replication of B M V R N A can occur in yeast, which provides a useful tool to study the formation of replication complexes and to identify host factors involved in B M V replication. 25 In both plants and yeast, B M V infection induces membrane aggregates in the perinuclear E R . Colocalization of l a , 2a and R N A synthesis at these sites indicates the modified E R structures carry B M V replication complexes (Restrepo-Hartwig and Ahlquist, 1999; Restrepo-Hartwig and Ahlquist, 1996). E M images in infected yeast cells show the perinuclear membrane aggregates consist of numerous spherules which invaginate into the E R membranes with open necks connected to the cytosol. Multiple molecules of l a and 2a proteins (at the ratio of 25:1) are associated with the inner surface of the spherules and B M V R N A synthesis occurs within the spherules (Schwartz et al., 2002). The l a protein is the key organizer of B M V replication complexes, l a targets to E R membranes independently of other viral components and induces the formation of membranous spherules similar to those observed during viral infection (Restrepo-Hartwig and Ahlquist, 1999; Schwartz et al., 2002). E R association of l a is mediated by its N -terminal domain. Although 1 a apparently does not traverse the membrane and thus has a peripheral topology, it binds to the E R as an integral membrane protein (den Boon et al., 2001). The peripheral topology is consistent with the role of l a in recruiting 2a and viral R N A replication templates to the replication complexes, and indeed l a can redirect both 2a and viral R N A templates to the membrane spherules from the cytosol through protein-protein or protein-RNA interaction (Chen and Ahlquist, 2000; Chen et al., 2001; Chen et al., 2003). l a dramatically increases viral R N A stability by transferring it to a membrane-associated, nuclease-resistant state. This event does not increase the translation of B M V R N A s (Chen et al., 2001) (Janda and Ahlquist, 1998). Thus, l a induced stability of B M V R N A s may reflect an interaction involved in recruiting viral R N A templates for R N A 26 replication from the competing cellular processes of translation and degradation. Assembly of active RdRp requires not only viral proteins but also viral R N A which may either directly contribute some non-template function or recruit essential host factors into the RdRp complex (Quadt et al., 1995). 1.5.2.2 Other alpha-like viruses Many other alphaviruses also induce membranous spherules in which viral R N A replication takes place but the cellular origin of the spherules appears to be very diverse. Semliki Forest virus, an animal alphavirus, replicates in spherules lining the inner membranes of cytoplasmic vacuoles. The vacuoles are derived from endosomes since they contain the endosome markers L A M P - 1 and L A M P - 2 (Kujala et al., 2001). nsPl (equivalent to B M V 1 a) acts as the membrane anchor for the replication complexes since it is the only viral protein with membrane targeting and binding activity. nsPl alone targets the plasma membrane but does not induce membrane modification. The presence of polyprotein precursors containing the nsPl domain including P123 or P1234 are essential for the induction of the cytoplasmic vacuoles and the assembly of the polymerase complexes (Salonen et al., 2003). The plant-infecting virus, Alfalfa mosaic virus, also appears to assemble the replication complexes on vacuole membranes, however, it is not clear whether spherules structures are induced (Van Der Heijden et al., 2001). Virally-induced spherules have also been observed in Turnip yellow mosaic virus ( T Y M V ) infection. In this case, the spherules are formed on the chloroplast envelope. T Y M V encodes two nonstructural replication proteins: the 140K protein (equivalent to B M V l a , containing domains indicative of methyltransferase, proteinase, and 27 NTPase/helicase), and the 66K putative RdRp. Similar to B M V l a , the 140K protein is the key organizer of T Y M V replication complexes. It is targeted to the chloroplast envelopes in the absence of other viral factors and induces the clumping of the chloroplasts, one of the typical cytological effects of T Y M V infection. It can also redirect the 66K polymerase from the cytosol to chloroplast membranes (Prod'homme et al., 2003). Direct interaction between the 140K and 66K proteins has been demonstrated. In contrast to other alphaviruses, the interaction domain of the 140K protein has been mapped to the proteinase domain instead of the helicase domain, suggesting T Y M V uses a different pathway to assemble the replication complexes (Jakubiec et al., 2004). In most characterized alphaviruses, membrane anchor proteins are encoded by the viruses themselves. A t least one case has been found in which the membrane anchor is provided by the host. Efficient Tobacco mosaic virus ( T M V ) replication in Arabidopsis thaliana requires the host proteins T O M 1 and T O M 2 A (Ishikawa et al., 1993; Ohshima et al., 1998). T O M 1 and T O M 2 A are polytopic membrane proteins (Tsujimoto et al., 2003; Yamanaka et al., 2000). Both proteins are predominantly targeted to vacuole membranes although T O M 1 is also localized to other membranes. The subcellular fractionation pattern of the viral RdRp activity coincides with both the membrane-bound T M V replication proteins and T O M 1 (Hagiwara et al., 2003). Moreover, T O M 1 interacts with the helicase domain of T M V (Yamanaka et al., 2000). Therefore, T O M 1 is likely an essential component of T M V replication complexes and probably tethers the viral replication complexes to membranes. There are two sub-populations of polymerase in TMV-infected cells but only the membrane-bound one is active in replication, probably 28 through interaction with T O M 1 (Nishikiori et al., 2006). T O M 2 A is not an absolute requirement for the assembly of T M V replication complexes. The presence of T O M 2 A may facilitate the formation of T M V replication complexes on distinct intracellular membranes probably through interaction with T O M 1 (Tsujimoto et al., 2003). 1.5.3 F l a v i v i r u s s u p e r f a m i l y 1.5.3.1 H e p a t i t i s C v i r u s ( H C V ) H C V represents the most characterized virus in this superfamily with regard to replication complexes, and the interaction between viral replicases and host membranes. The H C V R N A genome encodes a single polyprotein, which is processed by viral and host proteases into structural and nonstructural proteins (Fig. l .5-C) With the establishment of the subgenomic replicon of H C V , it became clear that proteins N S 3 , N A 4 A , N S 4 B , N S 5 A and N S 5 B are necessary and sufficient for H C V replication (Bartenschlager, 2006). Similar to other (+)ss R N A viruses, these proteins form a complex in a membrane-bound compartment to perform the R N A synthesis activity. In infected cells, H C V non-structural proteins and R N A synthesis activity colocalize to cytoplasmic dot-like structures, which may represent modified E R membranes harboring active H C V replication complexes (Gosert et al., 2003). Immuno-electron microscopy further demonstrates that H C V replication complexes locate on the altered membranes, termed a membranous web, which correspond very well to the dot-like structures observed under light microscopy (Moradpour et al., 2003). 29 H C V is unique in that all the nonstructural proteins except NS3 are membrane proteins which display very diverse modes of membrane interaction. N S 4 A associates with the E R or ER-derived membranes and mitochondria when expressed either alone or in the context of viral infection. The membrane association is mediated by the N-terminal transmembrane a-helix. N S 4 A relocates NS3 from the cytosol to the membranes when they are co-expressed (Nomura-Takigawa et al., 2006; Wolk et al., 2000). N S 4 B is a polytopic membrane protein. It contains five transmembrane segments with both the N -and C-termini translocated inside the membrane (Lundin et al., 2003). In contrast to the downstream four transmembrane a-helices, the N-terminal transmembrane segment is an amphipathic helix. This segment can mediate membrane association and is required for the correct localization of viral replication complexes and R N A replication (Elazar et al., 2004). When expressed individually, N S 4 B is localized to the E R , but also induces a pattern of cytoplasmic foci positive for E R markers (Hugle et al., 2001; Lundin et al., 2003). These foci may correspond to the membrane alteration, designated as the membranous web on which H C V replication complexes assemble, suggesting N S 4 B is a key organizer of the viral replication complexes (Egger et al., 2002). N S 5 A is termed a tip-anchored protein in analogy to tail-anchored proteins. N S 5 A associates with E R or ER-derived membranes either alone or in the context of the H C V polyprotein. The membrane-binding determinant of N S 5 A has been mapped to the N-terminal 31 amino acids, which forms an amphipathic helix. The amphipathic helix binds to the membrane using the hydrophobic side and yields a peripheral membrane topology. Genetically disrupting the amphipathic helix impairs H C V replication (Brass et al., 2002; Elazar et al., 2003). N S 5 B is also associated with E R membranes and the C-terminal 21 amino acids, 30 which form a transmembrane a-helix, are necessary and sufficient for the membrane integration. N S 5 B is targeted to the membrane in a post-translational, ATP-independent manner and therefore is a tail-anchored membrane protein (Schmidt-Mende et al., 2001). The C-terminal transmembrane domain not only has membrane anchor function but also is required for H C V R N A synthesis (Lee et al., 2004). 1.5.3.2 Other viruses in the flavivirus superfamily Flaviviruses such as Dengue virus and West Nile virus are distantly related to H C V and share a similar gene order and common non-structural proteins. The replication complexes of flaviviruses are associated with viral-induced membranes, termed vesicle packets (Mackenzie et al., 1996). Studies on Kunjin virus (Australia strain of West Ni le virus) indicate that the vesicle packets contain the Golgi marker GalT. The Golgi origin of the vesicle packets is further demonstrated by the fact that the Golgi apparatus-disrupting agent brefeldin A prevents the development of immunofluorescent foci of induced membranes (Mackenzie et al., 1999). Two hydrophobic proteins encoded by Kunjin virus, namely N S 2 A and N S 4 A , probably target and anchor the viral replication complexes onto the cytoplasmic surface of the membranes (Mackenzie et al., 1998; Roosendaal et al., 2006). N S 4 A and N S 4 A - 4 B in isolation are localized to the E R and induce membrane rearrangements. However, redistribution of N S 4 A to the Golgi from which the membranes of viral replication complexes are derived requires the 'cleavage of the C-terminal transmembrane 2 K domain (Roosendaal et al., 2006). 31 The membrane association of the V R C s of Tombusviruses, which have been classified in this superfamily, are also characterized. Two different types of membranes are used by the viruses. The first one is peroxisomal membranes (Cymbidium ringspot virus and Cucumber necrosis virus); the second is mitochondrial membranes (Carnation Italian ringspot virus) (Sanfacon, 2005). Tombusviruses encode two replication proteins, a small hydrophobic protein and a readthrough protein which includes the entire small protein at its N-terminus and an RdRp motif at its C-terminus. Both proteins are associated with membranes and are required for viral R N A replication. The small proteins are primary determinants for peroxisome or mitochondria targeting since the proteins, when expressed independently, target to these membranes and induce membrane alterations. Membrane association is mediated by two transmembrane helices interspaced by a small hydrophilic loop (Navarro et al., 2004; Weber-Lotfi et al., 2002). The viral RdRp is also required to produce viral specific membranous structures. Studies on Tomato bushy stunt virus ( T B S V ) indicate that peroxisome localization of the RdRp is dependent on its interaction with the small protein (Panavas et al., 2005a). 1.5.4 Host components involved in the formation of viral replication complexes 1.5.4.1 Membranes and proteins involved in lipid biosynthesis and membrane traffic Membranes are likely playing more roles in assembling viral replication complexes than just providing an anchor for the complexes as discussed above. R N A replication of many (+)ss R N A viruses requires de novo synthesis of membrane since inhibitors of lipid synthesis also inhibit viral R N A replication (Carette et al., 2000; Guinea and Carrasco, 1990; Perez et al., 1991). Continuous lipid synthesis and membrane formation is likely 32 required for proliferation of the specific membranous structures harbouring the viral replication complexes. Formation of a functional replication complex also requires a specific l ipid composition. Addit ion of oleic acid increases membrane fluidity making these membranes non-functional for poliovirus R N A synthesis in HeLa cells (Guinea and Carrasco, 1991). In contrast, unsaturated fatty acids ( U F A ) are required for the R N A replication of B M V in yeast. Mutation in delta9 fatty acid desaturase which reduces the unsaturated fatty acids blocks initiation of negative-strand R N A synthesis although l a still becomes membrane associated, retains its ability to recruit 2a to the membrane, induces spherules formation and recognizes and stabilizes viral R N A templates normally. Biochemical analysis indicates that the spherule-associated membranes are locally depleted in U F A s , suggesting that B M V replication complexes preferentially associate with one or more types of membrane lipid (Aizaki et a l , 2004; Lee et al., 2001). Recently, the replication complexes of H C V have also been shown to be associated with lipid rafts, a specific membrane lipid enriched in cholesterol, glycolipids and sphingolipids (Shi et al., 2003). Nevertheless, there is also an example in which the replication complex of Flock house virus was engineered to be retargeted to an alternative intracellular membrane (Miller et al., 2003), suggesting that a specific intracellular membrane is not required for replication complex formation and function of this virus. In addition to specific membrane lipid requirements, membrane traffic is probably involved in the biogenesis of viral-specific membrane structures. This idea originates from the observation that R N A replication of many viruses is sensitive to brefeldin A , an 33 inhibitor of membrane traffic between the E R and Golgi (Cuconati et al., 1998; Mackenzie et al., 1998; Maynel l et a l , 1992; Ritzenthaler et al., 2002). In recent years, host protein factors involved in membrane traffic have been identified that contribute to this process. Poliovirus probably hijacks the COPII machinery to produce the vesicles while Kunjin virus may use the COPI traffic system (Mackenzie et al., 1999; Rust et al., 2001). Similarly, a cellular vesicle membrane transport protein named hVAP-33 is indispensable for the formation of H C V replication complexes on l ipid rafts, presumably through transporting viral proteins to lipid rafts (Gao et al., 2004). Interestingly, two host proteins (termed VAP27-1 and VAP27-2) that have high homology to the V A P 3 3 family of S N A R E - l i k e proteins from animals interact specifically with the C-terminal domain of the 60K protein of C P M V in yeast-two hybrid screens and colocalize with 60K in CPMV-infected cowpea protoplasts, suggesting a possible role of the two proteins in formation of replication complexes of a plant virus (Carette et al., 2002c). In a genetic screening of host genes affecting T B S V R N A replication, a set of yeast genes involved in vacuolar targeting of proteins and vesicle-mediated transport has been identified, but the exact roles of these proteins remain to be determined (Panavas et al., 2005b). 1.5.4.2 Translation factors The genome of (+)ss R N A viruses can act as m R N A for translation of viral gene products. Efficient translation of the viral R N A is mediated by the interaction between viral translational elements and the host translational machinery including translation factors (Dreher and Mil le r , 2006; Edgi l and Harris, 2006). There is increasing evidence that host translation factors also are indispensable components of V R C s . They interact with either 34 the viral RdRp or cis-acting signals on the viral R N A required for viral R N A replication, suggesting their roles in viral R N A replication. The isolated replication complexes of B M V and T M V contain several host proteins. Interestingly, subunits of eIF3 have been found in both of the complexes. In the case of B M V , a 41kDa subunit of eIF3 interacts with B M V 2a polymerase and stimulates negative-strand synthesis, arguing that the 41 kDa subunit is an integral part of the B M V replication complex (Quadt et al., 1993). With T M V , a distinct subunit of eIF3, the 56 kDa RNA-binding protein, copurifies with the T M V replicases, but it is not clear how they form the complex (Osman and Buck, 1997; Quadt et al., 1993). A second translation factor, e E F I A , has also been found to bind to T M V RdRp in vivo and in yeast and to the 3'-untranslated region of T M V R N A as well , suggesting e E F I A is also a component of T M V replication complex (Yamaji et al., 2006). Host elongation factors have been found to interact with the RdRp of other R N A viruses including poliovirus and Bovine viral diarrhea virus (Harris et al., 1994; Johnson et al., 2001). Although these studies indicate that translation factors are integral parts of V R C s and required for viral R N A synthesis, their exact roles in viral R N A replication are not clear. Many translation factors have helicase activity which may directly participate in viral R N A replication by unwinding secondary structures formed during replication. Alternatively, the translation factor may have functions related to R N A synthesis. Since the positive-sense viral R N A has dual functions in serving as the template for both translation and replication, the requirement for translation factors in both processes suggests a link between translation and replication. These translation factors may be involved in regulating the switch from translation to replication. In fact, coupled translation and replication has been observed in many viruses in the picorna-like 35 superfamily (Gamarnik and Andino, 1998; Mahajan et al., 1996). For other viruses, e.g. B M V , a host protein other than translation factors has also been suggested to play a role in this process. Genetic screening in yeast has revealed that B M V la-recruitment of viral R N A s from translation to replication requires a yeast gene LSM1, which encodes a protein involved in cellular m R N A turnover (Diez et al., 2000). Both processes depend on clearing ribosomes from the R N A . 1.6 O V E R V I E W O F T O R S V T o R S V is a member of the genus Nepovirus, Family Comoviridae. T o R S V has a wide host range including both woody and herbaceous species. In nature, this virus mainly infects small fruits and fruit trees in North America, around the Great Lakes and along the Pacific coast, and sometimes causes serious diseases in these economically important crops (Stace-Smith, 1984). In herbaceous hosts, T o R S V often causes ringspot symptoms although other symptoms such as leaf mottling, vein necrosis and plant stunting can be seen (Fig. 1.6-A, B) . The T o R S V particles purified from infected herbaceous hosts are isometric and have a diameter of 28nm (Fig. l .6-C) . T o R S V is transmitted by a soil-borne nematode vector (Xiphinema americanum) (Brown et al., 1994; Brown et al., 1996). The control of this virus involves the use of nematicides to k i l l the transmission vector, however, the nematicides have been proven to be inefficient and also pose potential environmental hazards (Brown et al., 1996). A better understanding of the replication cycle of this virus and other related viruses at the molecular level may facilitate the development of new anti-viral strategies. 36 Fig. 1.6 Morphology of purified ToRSV particles and symptoms and cytopathological effects induced by ToRSV infection. A) Cucumis sativus infected by ToRSV showing symptoms of chlorotic and necrotic lesions on leaves and dwarfing of the plants. B) Nicotiana benthamiana infected by ToRSV showing multiple ringspot symptoms on the leaf. C) Electron micrograph of purified viral particles stained with uranyl acetate. The two particles with black center are empty viral particles. Bar=25 nm. D) Electron micrograph of ultrathin section of ToRSV-infected plant cells showing that proliferated membranous vesicles (arrows) accumulate in the proximity of the nucleus (Nc). Bar=200nm. (A and B, picture courtesy of Juan Jovel. C and D, picture courtesy of Andrew Wieczorek) 1.6.1 Molecular biology of ToRSV 1.6.1.1 Genomic organization and gene expression The genome of T o R S V consists of two (+)ss R N A molecules. Each R N A molecule has a V P g covalently linked to its 5' end and a poly A tail at its 3' end. The genome of the raspberry isolate has been sequenced (Rott et al., 1995; Rott et al., 1991b). Sequence analysis has revealed that each R N A contains a single long open reading frame which 37 encodes one polyprotein. The two polyproteins are cleaved by the viral-encoded proteinase (Pro) to release the mature viral proteins. The cleavage sites within each polyprotein have been identified by in vitro processing assays, allowing the definition of the coding regions of the mature proteins in the viral genome (Carrier et a l , 1999; Carrier et a l , 2001; Wang et al., 1999; Wang and Sanfacon, 2000). R N A 1 encodes protein domains for the RNA-dependent R N A polymerase (Pol), the proteinase (Pro), the V P g and the nucleoside triphosphate-binding protein (NTB) and X I and X 2 . R N A 2 encodes protein domains for the coat protein (CP), movement protein (MP), X 3 and X 4 (Fig. 1.7-A ) . Extensive areas of sequence similarity in 5' and 3' untranslated regions between R N A 1 and R N A 2 have been observed, and are suggested to have maintained identity via recombination between the two R N A molecules during R N A replication (Rott et al., 1991a). The RNAl-encoded proteins including N T B , V P g , Pro and Pol share signature motifs with their equivalents in other picornaviruses and have the same arrangement in the genome as well (Fig. 1.7-B). In addition, the X 2 protein shares sequence similarity with the C-terminus of the 32K protein of C P M V which acts as a proteinase-cofactor (Peters et al., 1992). These observations suggest that T o R S V uses strategies for gene expression and genome replication that are similar to other picornaviruses. It is currently not clear how T o R S V genomic R N A s recruit the host translational machinery. Picornaviruses use two different mechanisms for translation initiation to compensate for the lack of the 5'cap structure. Some of them, e.g. poliovirus, use an internal ribosomal entry site, which is a highly structured element located in the 5' 38 A 5' VPg X1 | Pro | PQI f -po ly (A) 3' RNA1 poly (A) 3' RNA2 X3 X4 MP CP B ?EJ 2C [ 3 A M 3C~ I 3D 6K 5 K 1 | § | 2C | ^iA^NlAP f 0 | NIB VPg Co^ NTB | Pro VPg VPg M ^ N T B Pro . Pol Poliovirus Potyviruses C P M V G F L V ToRSV | | Proteinase I I Putative Hel icase j | V P g I | RNA-dependent R N A polymerase Fig. 1.7 Schematic representation of the genomic organization of ToRSV and modular arrangement of replication proteins in picornaviruses. (A) ToRSV genome, with the 5' VPg (grey cirle), 5' non-coding region (horizontal line), the open reading frame (box), 3' non-coding region (horizontal line) and the poly A tail. Coding regions for viral proteins are indicated. (B) Schematic representation of modular arrangement of the core replication protein domains in the polyproteins encoded by viruses related to ToRSV. The proteins upstream of the putative helicase are also shown since some proteins, e.g. X2 and Co p r o , share sequence similarity. untranslated region of its genomic R N A , to promote efficient translation (Jang et al., 1990). For others, translation initiation may depend on the interaction between V P g and eIF4E or its isoforms (Goodfellow et a l , 2005; Leonard et al., 2000; Wittmann et al., 39 1997). The 5' untranslated regions of T o R S V are probably too short to accommodate an internal ribosomal entry site. Therefore, it is possible that translation initiation of T o R S V R N A s requires the V P g . A n interaction between a T o R S V protein containing V P g (VPg-pro) and elF(iso)4E has been detected in vitro (Leonard et al., 2002). However, the significance of this interaction to the translation of T o R S V R N A s remains to be determined. Each T o R S V R N A is first translated into a polyprotein which is subsequently cleaved by Pro into intermediates and final products. The T o R S V Pro is a 3C-like cysteine proteinase. 3C is the picornavirus proteinase related to chymotrypsin proteinase (Mosimann et al., 1997). The proteolytic processing of the R N A 1 - and RNA2-encoded polyproteins by Pro has been extensively studied, and a consensus amino acid sequence for T o R S V cleavage sites has been identified [(C or V ) Q / ( G or S)] (Chisholm et al., 2001; Hans and Sanfacon, 1995). The importance of this consensus sequence for processing efficiency has been confirmed by site-directed mutagenesis of two cleavage sites (Carrier et al., 1999). In vitro, the R N A 1-encoded polyprotein is processed in cis while the R N A 2 -encoded polyprotein is cleaved in trans (Carrier et al., 1999). The cleavage efficiency is influenced by the nature of the cleavage sites and also by processing of the proteinase itself. For example, Pro cleaves RNA2-encoded cleavage sites more efficiently than the VPg-Pro precursor (Carrier et al., 1999; Chisholm et al., 2001). In addition, several stable precursors including N T B - V P g , X 2 - N T B - V P g and VPg-Pro-Pol have been detected in infected plant extracts, suggesting that processing of the R N A 1-encoded polyprotein can 40 5' O - x i fxPro * ? & ' ' / / ' — poly (A) 3' Translation / / >' >- >A/ A P1 polyprotein 1 Polyprotein processing X2-NTB-VPg NTB-VPg 7 / / V A v I VPg-Pro-Pol Fig.1.8 Processing of PI polyprotein encoded by ToRSV R N A 1 . Showing at the top is the genomic organization of RNA1. The single open reading frame is indicated by a rectangle. Cleavage sites are shown by vertical lines. Showing at the bottom are the individual intermediates and mature proteins detected in infected plants using antibodies against different protein regions (represented as horizontal on the top of PI polyprotein). XI and X2 have not been detected due to the lack of antibodies against the two proteins. occur using several alternative pathways (Joan Chisholm, personal communication) (Han and Sanfacon, 2003) (Fig.1.8). The stable precursors may have different functions from that of the mature proteins. For example, the VPg-Pro-Pol , rather than the N T B - V P g , may act as a donor of V P g during R N A replication. 1.6.1.2. G e n o m e r e p l i c a t i o n a n d m o v e m e n t Similar to other piconarviruses, T o R S V induces massive proliferation of membranous vesicles and morphological changes in E R membranes in infected cells (Han and Sanfacon, 2003) (Fig. 1.6-D). The vesicles accumulate mostly in the perinuclear areas 41 and are believed to carry the T o R S V replication complexes by analogy to poliovirus, C P M V and G F L V . These vesicles are most likely produced by proliferated E R membranes induced during viral infection. Requirement of continuous l ipid synthesis for E R proliferation has been confirmed in C P M V and G F L V infections by using cerulenin, an inhibitor of de novo phospholipid synthesis (Carette et al., 2000; Ritzenthaler et al., 2002). It is likely that R N A 1 can replicate independently. This assumption comes from the observation that the R N A 1 of G F L V , another nepovirus, can replicate independently of R N A 2 in protoplasts (Viry et al., 1993). On the other hand, replication of R N A 2 is dependent on R N A 1 . Therefore, T o R S V R N A 1 -encoded proteins including both the mature proteins and stable precursors mentioned above are likely to be the main components of the T o R S V replication complex. It is still an open question which protein(s) induces the vesicle formation and how these proteins are associated with the vesicles. One or several of the proteins may act as membrane anchors and other components including the T o R S V R N A and proteins and host proteins are brought to the E R via macromolecule interactions. Several viral proteins containing the N T B domain including N T B - V P g and N T B have been detected in viral-infected extracts. These proteins behave as integral membrane proteins, co-fractionate with the viral replication complex and localize to the modified E R membranes (Han and Sanfacon, 2003), suggesting that the NTB-containing proteins might act as membrane anchors for the viral replication complex. The C-terminus of N T B is translocated into the E R lumen, suggesting that the C-terminal hydrophobic domain mediates the membrane association 42 of N T B (Han and Sanfacon, 2003). Consistent with the in vivo observation, the hydrobobic domain is required for membrane association of the C-terminal fragment of N T B and for translocation of the V P g into the E R lumen in vitro; the translocated N T B -V P g is processed at a position upstream of the junction between the N T B and V P g by a membrane-associated proteinase, probably a signal peptide peptidase (Wang et al., 2004). The role of a putative amphipathic helix in the N-terminus of N T B in membrane association is not clear. X 2 is also a hydrophobic protein, suggesting that it is a second membrane anchor protein for the replication complex. A s mentioned above, T o R S V R N A 2 encodes four protein domains: X 3 , X 4 , CP and M P (Fig. 1.7-A). X 3 may play a role in targeting the R N A 2 to the replication complex in analogy to the equivalent protein encoded by the R N A 2 of another nepovirus G F L V This G F L V protein is required for the replication of R N A 2 and is localized to replication sites (Belin et al., 1999). The function of X 4 is unknown. The C P and M P are not necessary for replication; instead they are required for encapsidation and cell-to-cell movement (Verver et al., 1998). Both proteins have been detected in infected plants and protoplasts (Sanfacon et al., 1995; Wieczorek and Sanfacon, 1993). In infected protoplasts, it appears that the M P is less stable than the C P (Sanfacon et a l , 1995). In infected plants, M P is associated with tubular structures containing virus-like particles present in or near the cell wall (Wieczorek and Sanfacon, 1993). The tubular structures are probably involved in cell to cell movement of T o R S V as has been suggested for C P M V , G F L V and Caulimoviruses. A current model for cell-to-cell movement of these viruses is that the viral particles move from cell to cell through large tubular structures that extend from the 43 cell wall to adjacent uninfected cells (Lazarowitz and Beachy, 1999). The M P of C P M V and G F L V in isolation can induce the formation of the tubular structures (Laporte et al., 2003) (Carvalho et al., 2003; Pouwels et al., 2004). Studies on the G F L V M P suggest that the intracellular targeting and tubular assembly of the M P may involve the cytoskeleton and a functional plant secretory pathway, respectively (Laporte et al., 2003). In the case of C P M V , the M P also interacts with the larger subunit of the C P , suggesting that the M P facilitates intracellular and/or cell-to-cell movement of the viral particles through the M P - C P interaction (Carvalho et al., 2003). 1.7 R E S E A R C H O B J E C T I V E S T o R S V assembles the replication complexes in association with the E R or ER-derived membranes. However, the molecular mechanism underlying the ER-association of the replication complexes is largely unknown. The goal of this thesis is to provide new information on the assembly of T o R S V replication complexes at the molecular level by characterizing interactions between intracellular membranes and T o R S V replication proteins, in particular the two hydrophobic proteins, N T B and X 2 . The specific objectives of this thesis are as follows: 1. To determine the sub-cellular localization of N T B and X 2 in the absence of other viral-encoded proteins. 2. To identify the membrane-binding domains of the two proteins. 3. To determine the membrane topologies of the two proteins. 44 1.8 B I B L I O G R A P H Y Abazeed, M . E . , Blanchette, J. M . , and Fuller, R. S. (2005). Cell-free transport from the trans-golgi network to late endosome requires factors involved in formation and consumption of clathrin-coated vesicles. J Biol Chem 280(6), 4442-50. Abe l l , B . M . , Pool, M . R., Schlenker, O., Sinning, I., and High, S. (2004). Signal recognition particle mediates post-translational targeting in eukaryotes. Embo J 23(14), 2755-64. Aizak i , H . , Lee, K . J., Sung, V . M . , Ishiko, H . , and La i , M . M . (2004). Characterization of the hepatitis C virus R N A replication complex associated with l ipid rafts. Virology 324(2), 450-61. Alberts, B . (2002). "Molecular biology of the cell." 4 th ed. Barco, A . , and Carrasco, L . (1998). Identification of regions of poliovirus 2 B C protein that are involved in cytotoxicity. J Virol 72(5), 3560-70. Bartenschlager, R. (2006). Hepatitis C virus molecular clones: from c D N A to infectious virus particles in cell culture. Curr Opin Microbiol 9(4), 416-22. Bechinger, B . (1999). The structure, dynamics and orientation of antimicrobial peptides in membranes by multidimensional solid-state N M R spectroscopy. Biochim Biophys Acta 1462(1-2), 157-83. Bel in , C , Schmitt, C , Gaire, F. , Walter, B . , Demangeat, G . , and Pinck, L . (1999). The nine C-terminal residues of the grapevine fanleaf nepovirus movement protein are critical for systemic virus spread. J Gen Virol 80 (6), 1347-56. Bienz, K . , Egger, D . , and Pfister, T. (1994). Characteristics of the poliovirus replication complex. Arch Virol Suppl 9, 147-57. Boevink, P., Oparka, K . , Santa Cruz, S., Martin, B . , Betteridge, A . , and Hawes, C. (1998). Stacks on tracks: the plant Golgi apparatus traffics on an actin/ER network. Plant J15(3), 441-7. Bonifacino, J. S., and Lippincott-Schwartz, J. (2003). Coat proteins: shaping membrane transport. Nat Rev Mol Cell Biol 4(5), 409-14. Borgese, N . , Colombo, S., and Pedrazzini, E . (2003). The tale of tail-anchored proteins: coming from the cytosol and looking for a membrane. J Cell Biol 161(6), 1013-9. Brandizzi, F. , Frangne, N . , Marc-Martin, S., Hawes, C , Neuhaus, J. M . , and Paris, N . (2002). The destination for single-pass membrane proteins is influenced markedly by the length of the hydrophobic domain. Plant Cell 14(5), 1077-92. 45 Brass, V . , Bieck, E . , Montserret, R., Wolk, B . , Hellings, J. A . , B lum, H . E . , Penin, F. , and Moradpour, D . (2002). A n amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis C virus nonstructural protein 5 A . J Biol Chem 277(10), 8130-9. Brown, D . J. F. , Halbrendt, J. M . , Jones, A . T., Vrain, T. C , and Robbins, R. T. (1994). Transmission of three North American nepoviruses by populations of four distinct species of the Xiphinema americanum group. Phytopathology 84(6), 646-649. Brown, D . J. F. , Trudgill , D . L . , and Robertson, W . M . (1996). Nepoviruses: Transmission by nematodes. In "The plant viruses, Volume 5: Polyhedral virions and bipartite R N A genomes" (B. D . Harrison, and A . F. Murant, Eds.), pp. 187-209. Plenum Press, New York. Carette, J. E . , Guhl , K . , Wellink, J., and Van Kammen, A . (2002a). Coalescence of the sites of cowpea mosaic virus R N A replication into a cytopathic structure. J Virol 76(12), 6235-43. Carette, J. E . , Stuiver, M . , Van Lent, J., Wellink, J., and Van Kammen, A . (2000). Cowpea mosaic virus infection induces a massive proliferation of endoplasmic reticulum but not Golgi membranes and is dependent on de novo membrane synthesis. J Virol 74(14), 6556-63. Carette, J. E . , van Lent, J., MacFarlane, S. A . , Wellink, J., and van Kammen, A . (2002b). Cowpea mosaic virus 32- and 60-kilodalton replication proteins target and change the morphology of endoplasmic reticulum membranes. J Virol 76(12), 6293-301. Carette, J. E . , Verver, J., Martens, J., van Kampen, T., Wellink, J., and van Kammen, A . (2002c). Characterization of plant proteins that interact with cowpea mosaic virus '60K' protein in the yeast two-hybrid system. J Gen Virol 83(Pt 4), 885-93. Carrier, K . , Hans, F. , and Sanfacon, H . (1999). Mutagenesis of amino acids at two tomato ringspot nepovirus cleavage sites: effect on proteolytic processing in cis and in trans by the 3C-like protease. Virology 258(1), 161-75. Carrier, K . , Xiang, Y . , and Sanfacon, H . (2001). Genomic organization of R N A 2 of Tomato ringspot virus: processing at a third cleavage site in the N-terminal region of the polyprotein in vitro. J Gen Virol 82(Pt 7), 1785-90. Carvalho, C. M . , Wellink, J., Ribeiro, S. G . , Goldbach, R. W. , and V a n Lent, J. W . (2003). The C-terminal region of the movement protein of Cowpea mosaic virus is involved in binding to the large but not to the small coat protein. J Gen Virol 84(Pt 8), 2271-7. 46 Chen, J., and Ahlquist, P. (2000). Brome mosaic virus polymerase-like protein 2a is directed to the endoplasmic reticulum by helicase-like viral protein 1 a. J Virol 74(9), 4310-8. Chen, J., Noueiry, A . , and Ahlquist, P. (2001). Brome mosaic virus Protein l a recruits viral R N A 2 to R N A replication through a 5' proximal R N A 2 signal. J Virol 75(7), 3207-19. Chen, J., Noueiry, A . , and Ahlquist, P. (2003). A n alternate pathway for recruiting template R N A to the brome mosaic virus R N A replication complex. J Virol 77(4), 2568-77. Chisholm, J., Wieczorek, A . , and Sanfacon, H . (2001). Expression and partial purification of recombinant tomato ringspot nepovirus 3C-like proteinase: comparison of the activity of the mature proteinase and the VPg-proteinase precursor. Virus Res 79(1-2), 153-64. Choe, S. S., and Kirkegaard, K . (2004). Intracellular topology and epitope shielding of poliovirus 3 A protein. J Virol 78(11), 5973-82. Cook, N . R., Row, P. E . , and Davidson, H . W . (2004). Lysosome associated membrane protein 1 (Lampl) traffics directly from the T G N to early endosomes. Traffic 5(9), 685-99. Crowley, K . S., Liao, S., Worrell , V . E . , Reinhart, G . D . , and Johnson, A . E . (1994). Secretory proteins move through the endoplasmic reticulum membrane via an aqueous, gated pore. Cell 78(3), 461-71. Cuconati, A . , Mo l l a , A . , and Wimmer, E . (1998). Brefeldin A inhibits cell-free, de novo synthesis of poliovirus. J Virol 72(8), 6456-64. den Boon, J. A . , Chen, J., and Ahlquist, P. (2001). Identification of sequences in Brome mosaic virus replicase protein l a that mediate association with endoplasmic reticulum membranes. J Virol 75(24), 12370-81. Denzer, A . J., Nabholz, C. E . , and Spiess, M . (1995). Transmembrane orientation of signal-anchor proteins is affected by the folding state but not the size of the N -terminal domain. Embo J 14(24), 6311-7. Dhugga, K . S. (2005). Plant Golgi cell wall synthesis: from genes to enzyme activities. Proc Natl Acad Sci USA 102(6), 1815-6. Diez, J., Ishikawa, M . , Kaido, M . , and Ahlquist, P. (2000). Identification and characterization of a host protein required for efficient template selection in viral R N A replication. Proc Natl Acad Sci USA 97(8), 3913-8. 47 Doedens, J. R., Giddings, T. H . , Jr., and Kirkegaard, K . (1997). Inhibition of endoplasmic reticulum-to-Golgi traffic by poliovirus protein 3A: genetic and ultrastructural analysis. J Virol 7 1 ( 1 2 ) , 9054-64. Doedens, J. R., and Kirkegaard, K . (1995). Inhibition of cellular protein secretion by poliovirus proteins 2B and 3A. Embo J 1 4 ( 5 ) , 894-907. Dreher, T. W. , and Mil le r , W . A . (2006). Translational control in positive strand R N A plant viruses. Virology 3 4 4 ( 1 ) , 185-97. Edgi l , D . , and Harris, E . (2006). End-to-end communication in the modulation of translation by mammalian R N A viruses. Virus Res 1 1 9 ( 1 ) , 43-51. Egger, D . , and Bienz, K . (2005). Intracellular location and translocation of silent and active poliovirus replication complexes. J Gen Virol 8 6 ( P t 3), 707-18. Egger, D . , Teterina, N . , Ehrenfeld, E . , and Bienz, K . (2000). Formation of the poliovirus replication complex requires coupled viral translation, vesicle production, and viral R N A synthesis. J Virol 7 4 ( 1 4 ) , 6570-80. Egger, D . , Wolk, B . , Gosert, R., Bianchi, L . , B lum, H . E . , Moradpour, D . , and Bienz, K . (2002). Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex. J Virol 7 6 ( 1 2 ) , 5974-84. Elazar, M . , Cheong, K . H . , L i u , P., Greenberg, H . B . , Rice, C. M . , and Glenn, J. S. (2003). Amphipathic helix-dependent localization of N S 5 A mediates hepatitis C virus R N A replication. J Virol 7 7 ( 1 0 ) , 6055-61. Elazar, M . , L i u , P., Rice, C. M . , and Glenn, J. S. (2004). A n N-terminal amphipathic helix in hepatitis C virus ( H C V ) N S 4 B mediates membrane association, correct localization of replication complex proteins, and H C V R N A replication. J Virol 7 8 ( 2 0 ) , 11393-400. Eskelinen, E . L . , Tanaka, Y . , and Saftig, P. (2003). A t the acidic edge: emerging functions for lysosomal membrane proteins. Trends Cell Biol 1 3 ( 3 ) , 137-45. Gaire, F. , Schmitt, C , Stussi-Garaud, C , Pinck, L . , and Ritzenthaler, C. (1999). Protein 2 A of grapevine fanleaf nepovirus is implicated in R N A 2 replication and colocalizes to the replication site. Virology 2 6 4 ( 1 ) , 25-36. Gamarnik, A . V . , and Andino, R. (1998). Switch from translation to R N A replication in a positive-stranded R N A virus. Genes Dev 1 2 ( 1 5 ) , 2293-304. 48 Gao, L . , A i zak i , H . , He, J. W. , and L a i , M . M . (2004). Interactions between viral nonstructural proteins and host protein hVAP-33 mediate the formation of hepatitis C virus R N A replication complex on lipid raft. J Virol 7 8 ( 7 ) , 3480-8. Geldner, N . (2004). The plant endosomal system—its structure and role in signal transduction and plant development. Planta 2 1 9 ( 4 ) , 547-60. Gething, M . J. (1999). Role and regulation of the E R chaperone B i P . Semin Cell Dev Biol 1 0 ( 5 ) , 465-72. Goder, V . , Bier i , C. , and Spiess, M . (1999). Glycosylation can influence topogenesis of membrane proteins and reveals dynamic reorientation of nascent polypeptides within the translocon. J Cell Biol 1 4 7 ( 2 ) , 257-66. Gonzalez, M . E . , and Carrasco, L . (2003). Viroporins. FEBS Lett 5 5 2 ( 1 ) , 28-34. Goodfellow, I., Chaudhry, Y . , Gioldasi, I., Gerondopoulos, A . , Natoni, A . , Labrie, L . , Laliberte, J. F. , and Roberts, L . (2005). Calicivirus translation initiation requires an interaction between V P g and eIF4E. EMBO Rep. Gorlich, D . , and Rapoport, T. A . (1993). Protein translocation into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane. Cell 7 5 ( 4 ) , 615-30. Gosert, R., Egger, D . , Lohmann, V . , Bartenschlager, R., B lum, H . E . , Bienz, K . , and Moradpour, D . (2003). Identification of the hepatitis C virus R N A replication complex in Huh-7 cells harboring subgenomic replicons. J Virol 7 7 ( 9 ) , 5487-92. Gu, F., Crump, C. M . , and Thomas, G . (2001). Trans-Golgi network sorting. Cell Mol Life Sci 5 8 ( 8 ) , 1067-84. Guinea, R., and Carrasco, L . (1990). Phospholipid biosynthesis and poliovirus genome replication, two coupled phenomena. Embo J9(6), 2011-6. Guinea, R., and Carrasco, L . (1991). Effects of fatty acids on lipid synthesis and viral R N A replication in poliovirus-infected cells. Virology 1 8 5 ( 1 ) , 473-6. Hagiwara, Y . , Komoda, K . , Yamanaka, T., Tamai, A . , Meshi , T., Funada, R. , Tsuchiya, T., Naito, S., and Ishikawa, M . (2003). Subcellular localization of host and viral proteins associated with tobamovirus R N A replication. Embo . 7 2 2 ( 2 ) , 344-53. Han, S., and Sanfacon, H . (2003). Tomato ringspot virus proteins containing the nucleoside triphosphate binding domain are transmembrane proteins that associate with the endoplasmic reticulum and cofractionate with replication complexes. J Virol 7 7 ( 1 ) , 523-34. 49 Hans, F. , and Sanfacon, H . (1995). Tomato ringspot nepovirus protease: characterization and cleavage site specificity. J Gen Virol 7 6 ( 4 ) , 917-27. Harris, K . S., Xiang, W. , Alexander, L . , Lane, W . S., Paul, A . V . , and Wimmer, E . (1994). Interaction of poliovirus polypeptide 3CDpro with the 5' and 3' termini of the poliovirus genome. Identification of viral and cellular cofactors needed for efficient binding. JBiol Chem 2 6 9 ( 4 3 ) , 27004-14. Hartmann, E . , Rapoport, T. A . , and Lodish, H . F. (1989). Predicting the orientation of eukaryotic membrane-spanning proteins. Proc Natl Acad Sci USA 8 6 ( 1 5 ) , 5786-90. Heijne, G . V . (1986). The distribution of positively charged residues in bacterial inner membrane proteins correlates with the trans-membrane topology. Embo J 5(11), 3021-3027. Heinrich, S. U . , Mothes, W. , Brunner, J., and Rapoport, T. A . (2000). The Sec61p complex mediates the integration of a membrane protein by allowing l ipid partitioning of the transmembrane domain. Cell 1 0 2 ( 2 ) , 233-44. Hounsell, E . F. , Davies, M . J., and Renouf, D . V . (1996). O-linked protein glycosylation structure and function. Glycoconj J 1 3 ( 1 ) , 19-26. Hugle, T., Fehrmann, F., Bieck, E . , Kohara, M . , Krausslich, H . G . , Rice, C. M , Blum, H . E . , and Moradpour, D . (2001). The hepatitis C virus nonstructural protein 4B is an integral endoplasmic reticulum membrane protein. Virology 2 8 4 ( 1 ) , 70-81. Humphrey, J. S., Peters, P. J., Yuan, L . C. , and Bonifacino, J. S. (1993). Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence. J Cell Biol 1 2 0 ( 5 ) , 1123-35. Ishikawa, M . , Naito, S., and Ohno, T. (1993). Effects of the toml mutation of Arabidopsis thaliana on the multiplication of tobacco mosaic virus R N A in protoplasts. J Virol 6 7 ( 9 ) , 5328-38. Jahn, R., Lang, T., and Sudhof, T. C. (2003). Membrane fusion. Cell 1 1 2 ( 4 ) , 519-33. Jakubiec, A . , Notaise, J., Tournier, V . , Hericourt, F. , Block, M . A . , Drugeon, G. , van Aelst, L . , and Jupin, I. (2004). Assembly of turnip yellow mosaic virus replication complexes: interaction between the proteinase and polymerase domains of the replication proteins. J Virol 7 8 ( 1 5 ) , 7945-57. Janda, M . , and Ahlquist, P. (1998). Brome mosaic virus R N A replication protein l a dramatically increases in vivo stability but not translation of viral genomic R N A 3 . Proc Natl Acad Sci USA 9 5 ( 5 ) , 2227-32. 50 Jang, S. K . , Pestova, T. V . , Hellen, C. U . , Witherell, G . W. , and Wimmer, E . (1990). Cap-independent translation of picornavirus R N A s : structure and function of the internal ribosomal entry site. Enzyme 4 4 ( 1 - 4 ) , 292-309. Johnson, A . E . , and van Waes, M . A . (1999). The translocon: a dynamic gateway at the E R membrane. Annu Rev Cell Dev Biol 1 5 , 799-842. Johnson, C. M . , Perez, D . R., French, R., Merrick, W . C , and Donis, R. O. (2001). The N S 5 A protein of bovine viral diarrhoea virus interacts with the alpha subunit of translation elongation factor-1. J Gen Virol 8 2 ( P t 12), 2935-43. Jungnickel, B . , and Rapoport, T. A . (1995). A posttargeting signal sequence recognition event in the endoplasmic reticulum membrane. Cell 8 2 ( 2 ) , 261-70. Keller, P., and Simons, K . (1997). Post-Golgi biosynthetic trafficking. J Cell Sci 1 1 0 ( P t 2 4 ) , 3001-9. Knipe, D . , and Howley, P. (2001). Fields Virology 1 , 685. Koonin, E . V . (1991). The phylogeny of RNA-dependent R N A polymerases of positive-strand R N A viruses. J Gen Virol 7 2 ( P t 9 ) , 2197-206. Koonin, E . V . , and Doha, V . V . (1993). Evolution and taxonomy of positive-strand R N A viruses: implications of comparative analysis of amino acid sequences. Crit Rev Biochem Mol Biol 2 8 ( 5 ) , 375-430. Kornfeld, R., and Kornfeld, S. (1985). Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem 5 4 , 631-64. Kujala, P., Ikaheimonen, A . , Ehsani, N . , Vihinen, H . , Auvinen, P., and Kaariainen, L . (2001). Biogenesis of the Semliki Forest virus R N A replication complex. J Virol 7 5 ( 8 ) , 3873-84. Kutay, U . , Ahnert-Hilger, G . , Hartmann, E . , Wiedenmann, B . , and Rapoport, T. A . (1995). Transport route for synaptobrevin via a novel pathway of insertion into the endoplasmic reticulum membrane. Embo J 1 4 ( 2 ) , 217-23. Laporte, C , Vetter, G. , Loudes, A . M . , Robinson, D . G . , Hillmer, S., Stussi-Garaud, C , and Ritzenthaler, C. (2003). Involvement of the secretory pathway and the cytoskeleton in intracellular targeting and tubule assembly of Grapevine fanleaf virus movement protein in tobacco B Y - 2 cells. Plant Cell 1 5 ( 9 ) , 2058-75. Lazarowitz, S. G . , and Beachy, R. N . (1999). Vi ra l movement proteins as probes for intracellular and intercellular trafficking in plants. Plant Cell 1 1 ( 4 ) , 535-48. 51 Lecomte, F. J., Ismail, N . , and High, S. (2003). Making membrane proteins at the mammalian endoplasmic reticulum. Biochem Soc Trans 31(Pt 6), 1248-52. Lee, H . , L i u , Y . , Mejia, E . , Paul, A . V . , and Wimmer, E . (2006). The C-terminal hydrophobic domain of hepatitis C virus R N A polymerase N S 5 B can be replaced with a heterologous domain of poliovirus protein 3A. J Virol. Lee, K . J., Choi , J., Ou, J. H . , and L a i , M . M . (2004). The C-terminal transmembrane domain of hepatitis C virus ( H C V ) R N A polymerase is essential for H C V replication in vivo. J Virol 78(7), 3797-802. Lee, W . M . , Ishikawa, M . , and Ahlquist, P. (2001). Mutation of host delta9 fatty acid desaturase inhibits brome mosaic virus R N A replication between template recognition and R N A synthesis. J Virol 75(5), 2097-106. Leonard, S., Chisholm, J., Laliberte, J. F., and Sanfacon, H . (2002). Interaction in vitro between the proteinase of Tomato ringspot virus (genus Nepovirus) and the eukaryotic translation initiation factor iso4E from Arabidopsis thaliana. J Gen Virol 83(Pt 8), 2085-9. Leonard, S., Plante, D . , Wittmann, S., Daigneault, N . , Fortin, M . G . , and Laliberte, J. F. (2000). Complex formation between potyvirus V P g and translation eukaryotic initiation factor 4E correlates with virus infectivity. J Virol 74(17), 7730-7. Letourneur, F., Gaynor, E . C , Hennecke, S., Demolliere, C , Duden, R., Emr, S. D . , Riezman, H . , and Cosson, P. (1994). Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum. Cell 79(7), 1199-207. Lewis, M . J., Sweet, D . J., and Pelham, H . R. (1990). The E R D 2 gene determines the specificity of the luminal E R protein retention system. Cell 61(7), 1359-63. Lundin, M . , Monne, M . , Widel l , A . , V o n Heijne, G. , and Persson, M . A . (2003). Topology of the membrane-associated hepatitis C virus protein N S 4 B . J Virol 77(9), 5428-38. Lyle , J. M . , Bullitt, E . , Bienz, K . , and Kirkegaard, K . (2002). Visualization and functional analysis of RNA-dependent R N A polymerase lattices. Science 296(5576), 2218-22. Mackenzie, J. M . , Jones, M . K . , and Westaway, E . G . (1999). Markers for trans-Golgi membranes and the intermediate compartment localize to induced membranes with distinct replication functions in flavivirus-infected cells. J Virol 73(11), 9555-67. 52 Mackenzie, J. M . , Jones, M . K . , and Young, P. R. (1996). Improved membrane preservation o f flavivirus-infected cells with cryosectioning. J Virol Methods 5 6 ( 1 ) , 67-75. Mackenzie, J. M . , Khromykh, A . A . , Jones, M . K . , and Westaway, E . G . (1998). Subcellular localization and some biochemical properties of the flavivirus Kunjin nonstructural proteins N S 2 A and N S 4 A . Virology 2 4 5 ( 2 ) , 203-15. Mahajan, S., Dolja, V . V . , and Carrington, J. C. (1996). Roles of the sequence encoding tobacco etch virus capsid protein in genome amplification: requirements for the translation process and a cis-active element. J Virol 7 0 ( 7 ) , 4370-9. Marty, F. (1999). Plant vacuoles. Plant Cell 1 1 ( 4 ) , 587-600. Maynel l , L . A . , Kirkegaard, K . , and Klymkowsky, M . W . (1992). Inhibition of poliovirus R N A synthesis by brefeldin A . J Virol 66(4), 1985-94. McCormick, P. J., Miao , Y . , Shao, Y . , L i n , J., and Johnson, A . E . (2003). Cotranslational protein integration into the E R membrane is mediated by the binding of nascent chains to translocon proteins. Mol Cell 1 2 ( 2 ) , 329-41. Mil le r , D . J., Schwartz, M . D . , Dye, B . T., and Ahlquist, P. (2003). Engineered retargeting of viral R N A replication complexes to an alternative intracellular membrane. J Virol 77(22), 12193-202. Moradpour, D . , Gosert, R., Egger, D . , Penin, F., B lum, H . E . , and Bienz, K . (2003). Membrane association of hepatitis C virus nonstructural proteins and identification of the membrane alteration that harbors the viral replication complex. Antiviral Res 6 0 ( 2 ) , 103-9. Mosimann, S. C. , Cherney, M . M . , Sia, S., Plotch, S., and James, M . N . (1997). Refined X-ray crystallographic structure of the poliovirus 3C gene product. J Mol Biol 2 7 3 ( 5 ) , 1032-47. Navarro, B . , Rubino, L . , and Russo, M . (2004). Expression of the Cymbidium ringspot virus 33-kilodalton protein in Saccharomyces cerevisiae and molecular dissection of the peroxisomal targeting signal. J Virol 7 8 ( 9 ) , 4744-52. Neumann, U . , Brandizzi, F. , and Hawes, C. (2003). Protein transport in plant cells: in and out of the Golgi . Ann Bot (Lond) 9 2 ( 2 ) , 167-80. Nishikiori , M . , Dohi , K . , M o r i , M . , Meshi , T., Naito, S., and Ishikawa, M . (2006). Membrane-bound tomato mosaic virus replication proteins participate in R N A synthesis and are associated with host proteins in a pattern distinct from those that are not membrane bound. J Virol 8 0 ( 1 7 ) , 8459-68. 53 Nomura-Takigawa, Y . , Nagano-Fujii, M . , Deng, L . , Kitazawa, S., Ishido, S., Sada, K . , and Hotta, H . (2006). Non-structural protein 4 A of Hepatitis C virus accumulates on mitochondria and renders the cells prone to undergoing mitochondria-mediated apoptosis. J Gen Virol 8 7 ( P t 7), 1935-45. Ohshima, K . , Taniyama, T., Yamanaka, T., Ishikawa, M . , andNaito, S. (1998). Isolation of a mutant of Arabidopsis thaliana carrying two simultaneous mutations affecting tobacco mosaic virus multiplication within a single cell. Virology 2 4 3 ( 2 ) , 472-81. Osman, T. A . , and Buck, K . W. (1997). The tobacco mosaic virus R N A polymerase complex contains a plant protein related to the RNA-bind ing subunit of yeast elF-3. J Virol 71(8), 6075-82. Ota, K . , Sakaguchi, M . , Hamasaki, N . , and Mihara, K . (2000). Membrane integration of the second transmembrane segment of band 3 requires a closely apposed preceding signal-anchor sequence. JBiol Chem 2 7 5 ( 3 8 ) , 29743-8. Panavas, T., Hawkins, C. M . , Panaviene, Z . , and Nagy, P. D . (2005a). The role of the p33:p33/p92 interaction domain in R N A replication and intracellular localization of p33 and p92 proteins of Cucumber necrosis tombusvirus. Virology 3 3 8 ( 1 ) , 81-95. Panavas, T., Serviene, E . , Brasher, J., and Nagy, P. D . (2005b). Yeast genome-wide screen reveals dissimilar sets of host genes affecting replication of R N A viruses. Proc Natl Acad Sci USA 1 0 2 ( 2 0 ) , 7326-31. Paris, N . , and Neuhaus, J. M . (2002). BP-80 as a vacuolar sorting receptor. Plant Mol Biol 5 0 ( 6 ) , 903-14. Perez, L . , Guinea, R., and Carrasco, L . (1991). Synthesis of Semliki Forest virus R N A requires continuous lipid synthesis. Virology 1 8 3 ( 1 ) , 74-82. Peters, S. A . , Voorhorst, W. G. , Wery, J., Wellink, J., and van Kammen, A . (1992). A regulatory role for the 32K protein in proteolytic processing of cowpea mosaic virus polyproteins. Virology 1 9 1 ( 1 ) , 81-9. Ponnambalam, S., Rabouille, C , Luzio, J. P., Nilsson, T., and Warren, G . (1994). The TGN38 glycoprotein contains two non-overlapping signals that mediate localization to the trans-Golgi network. J Cell Biol 1 2 5 ( 2 ) , 253-68. Pouwels, J., van der Velden, T., Willemse, J., Borst, J. W. , van Lent, J., Bisseling, T., and Wellink, J. (2004). Studies on the origin and structure of tubules made by the movement protein of Cowpea mosaic virus. J Gen Virol 8 5 ( P t 12), 3787-96. Praefcke, G . J., and McMahon , H . T. (2004). The dynamin superfamily: universal membrane tubulation and fission molecules? Nat Rev Mol Cell Biol 5 ( 2 ) , 133-47. 54 Press, B . , Feng, Y . , Hoflack, B . , and Wandinger-Ness, A . (1998). Mutant Rab7 causes the accumulation of cathepsin D and cation-independent mannose 6-phosphate receptor in an early endocytic compartment. J Cell Biol 140(5), 1075-89. Prod'homme, D. , Jakubiec, A . , Tournier, V . , Drugeon, G . , and Jupin, I. (2003). Targeting of the turnip yellow mosaic virus 66K replication protein to the chloroplast envelope is mediated by the 140K protein. J Virol 77(17), 9124-35. Quadt, R., Ishikawa, M . , Janda, M . , and Ahlquist, P. (1995). Formation of brome mosaic virus RNA-dependent R N A polymerase in yeast requires coexpression of viral proteins and viral R N A . Proc Natl Acad Sci USA92(l\), 4892-6. Quadt, R., Kao, C. C. , Browning, K . S., Hershberger, R. P., and Ahlquist, P. (1993). Characterization of a host protein associated with brome mosaic virus R N A -dependent R N A polymerase. Proc Natl Acad Sci USA 90(4), 1498-502. Raiborg, C , Rusten, T. E . , and Stenmark, H . (2003). Protein sorting into multivesicular endosomes. Curr Opin Cell Biol 15(4), 446-55. Restrepo-Hartwig, M . , and Ahlquist, P. (1999). Brome mosaic virus R N A replication proteins l a and 2a colocalize and l a independently localizes on the yeast endoplasmic reticulum. J Virol 73(12), 10303-9. Restrepo-Hartwig, M . A . , and Ahlquist, P. (1996). Brome mosaic virus helicase- and polymerase-like proteins colocalize on the endoplasmic reticulum at sites of viral R N A synthesis. J Virol 70(12), 8908-16. Ritzenthaler, C , Laporte, C , Gaire, F. , Dunoyer, P., Schmitt, C , Duval , S., Piequet, A . , Loudes, A . M . , Rohfritsch, O., Stussi-Garaud, C , and Pfeiffer, P. (2002). Grapevine fanleaf virus replication occurs on endoplasmic reticulum-derived membranes. J Virol 76(17), 8808-19. Robinson, D . G . , Hinz, G . , and Holstein, S. E . (1998). The molecular characterization of transport vesicles. Plant Mol Biol 38(1-2), 49-76. Robinson, D . G . , Oliviusson, P., and Hinz, G . (2005). Protein sorting to the storage vacuoles of plants: a critical appraisal. Traffic 6(8), 615-25. Roosendaal, J., Westaway, E . G . , Khromykh, A . , and Mackenzie, J. M . (2006). Regulated cleavages at the West Ni le virus N S 4 A - 2 K - N S 4 B junctions play a major role in rearranging cytoplasmic membranes and Golgi trafficking of the N S 4 A protein. J Virol 80(9), 4623-32. Rott, M . E . , Gilchrist, A . , Lee, L . , and Rochon, D . (1995). Nucleotide sequence of tomato ringspot virus R N A 1 . J Gen Virol 76 ( Pt 2), 465-73. 55 Rott, M . E . , Tremaine, J. H . , and Rochon, D . M . (1991a). Comparison of the 5' and 3' termini of tomato ringspot virus R N A 1 and R N A 2 : evidence for R N A recombination. Virology 1 8 5 ( 1 ) , 468-72. Rott, M . E . , Tremaine, J. H . , and Rochon, D . M . (1991b). Nucleotide sequence of tomato ringspot virus R N A - 2 , J Gen Virol 7 2 ( P t 7 ) , 1505-14. Rust, R. C , Landmann, L . , Gosert, R., Tang, B . L . , Hong, W. , Hauri, H . P., Egger, D . , and Bienz, K . (2001). Cellular COPII proteins are involved in production of the vesicles that form the poliovirus replication complex. J Virol 7 5 ( 2 0 ) , 9808-18. Sachse, M . , Ramm, G. , Strous, G . , and Klumperman, J. (2002). Endosomes: multipurpose designs for integrating housekeeping and specialized tasks. Histochem Cell Biol 1 1 7 ( 2 ) , 91-104. Sadlish, H . , Pitonzo, D . , Johnson, A . E . , and Skach, W . R. (2005). Sequential triage of transmembrane segments by Sec61 alpha during biogenesis of a native multispanning membrane protein. Nat Struct Mol Biol 1 2 ( 1 0 ) , 870-8. Salonen, A . , Ahola, T., and Kaariainen, L . (2005a). Vi ra l R N A replication in association with cellular membranes. Curr Top Microbiol Immunol 2 8 5 , 139-73. Salonen, A . , Ahola, T., and Kaariainen, L . (2005b). Vi ra l R N A replication in association with cellular membranes. Curr Top Microbiol Immunol 2 8 5 , 139-173. Salonen, A . , Vasiljeva, L . , Merits, A . , Magden, J., Jokitalo, E . , and Kaariainen, L . (2003). Properly folded nonstructural polyprotein directs the semliki forest virus replication complex to the endosomal compartment. J Virol 7 7 ( 3 ) , 1691-702. Sandoval, I. V . , and Carrasco, L . (1997). Poliovirus infection and expression of the poliovirus protein 2B provoke the disassembly of the Golgi complex, the organelle target for the antipoliovirus drug Ro-090179. J Virol 7 1 ( 6 ) , 4679-93. Sanfacon, H . (2005). Replication of positive-strand R N A viruses in plants: Contact points between plant and virus components. Can JBot 8 3 ( 1 2 ) , 1529-1549. Sanfacon, H . , Wieczorek, A . , and Hans, F. (1995). Expression of the tomato ringsport nepovirus movement and coat proteins in protoplasts. J Gen Virol 7 6 ( P t 9 ) , 2299-303. Schaad, M . C , Jensen, P. E . , and Carrington, J. C. (1997). Formation of plant R N A virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein. Embo J 1 6 ( 1 3 ) , 4049-59. 56 Schlegel, A . , Giddings, T. H . , Jr., Ladinsky, M . S., and Kirkegaard, K . (1996). Cellular origin and ultrastructure of membranes induced during poliovirus infection. J Virol 7 0 ( 1 0 ) , 6576-88. Schmidt-Mende, J., Bieck, E . , Hugle, T., Penin, F. , Rice, C. M . , B lum, H . E . , and Moradpour, D . (2001). Determinants for membrane association of the hepatitis C virus RNA-dependent R N A polymerase. JBiol Chem 2 7 6 ( 4 7 ) , 44052-63. Schwartz, M . , Chen, J., Janda, M . , Sullivan, M . , den Boon, J., and Ahlquist, P. (2002). A positive-strand R N A virus replication complex parallels form and function of retrovirus capsids. Mol Cell 9 ( 3 ) , 505-14. Shai, Y . (1999). Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim Biophys Acta 1 4 6 2 ( 1 - 2 ) , 55-70. Shi, S. T., Lee, K . J., A izak i , PL, Hwang, S. B . , and La i , M . M . (2003). Hepatitis C virus R N A replication occurs on a detergent-resistant membrane that cofractionates with caveolin-2. J Virol 7 7 ( 7 ) , 4160-8. Stace-Smith. (1984). Tomato ringspot virus. CMI/AAB Description of Plant Viruses 2 9 0 . Staehelin, L . A . (1997). The plant E R : a dynamic organelle composed of a large number of discrete functional domains. Plant J11(6), 1151-65. Suhy, D . A . , Giddings, T. H . , Jr., and Kirkegaard, K . (2000). Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles. J Virol 7 4 ( 1 9 ) , 8953-65. Tarentino, A . L . , and Maley, F. (1974). Purification and properties of an endo-beta-N-acetylglucosaminidase from Streptomyces griseus. J Biol Chem 2 4 9 ( 3 ) , 811-7. Tarentino, A . L . , and Plummer, T. H . , Jr. (1994). Enzymatic deglycosylation of asparagine-linked glycans: purification, properties, and specificity of oligosaccharide-cleaving enzymes from Flavobacterium meningosepticum. Methods Enzymol 2 3 0 , 44-57. Teasdale, R. D . , and Jackson, M . R. (1996). Signal-mediated sorting of membrane proteins between the endoplasmic reticulum and the golgi apparatus. Annu Rev Cell Dev Biol 1 2 , 27-54. Teterina, N . L . , Egger, D . , Bienz, K . , Brown, D . M . , Semler, B . L . , and Ehrenfeld, E . (2001). Requirements for assembly of poliovirus replication complexes and negative-strand R N A synthesis. J Virol 7 5 ( 8 ) , 3841-50. 57 Teterina, N . L . , Gorbalenya, A . E . , Egger, D . , Bienz, K . , and Ehrenfeld, E . (1997). Poliovirus 2C protein determinants of membrane binding and rearrangements in mammalian cells. J Virol 7 1 ( 1 2 ) , 8962-72. Towner, J. , Ho, T., and Semler, B . (1996). Determinants o f membrane association for poliovirus protein 3 A B . JBiol Chem 2 7 1 ( 4 3 ) , 26810-8. Tse, Y . C , M o , B . , Hillmer, S., Zhao, M . , L o , S. W. , Robinson, D . G . , and Jiang, L . (2004). Identification of multivesicular bodies as prevacuolar compartments in Nicotiana tabacum B Y - 2 cells. Plant Cell 1 6 ( 3 ) , 672-93. Tsujimoto, Y . , Numaga, T., Ohshima, K . , Yano, M . A . , Ohsawa, R., Goto, D . B . , Naito, S., and Ishikawa, M . (2003). Arabidopsis T O B A M O V I R U S M U L T I P L I C A T I O N (TOM) 2 locus encodes a transmembrane protein that interacts with T O M 1 . Embo . 7 2 2 ( 2 ) , 335-43. Ueda, T., Yamaguchi, M . , Uchimiya, H . , and Nakano, A . (2001). Ara6, a plant-unique novel type Rab GTPase, functions in the endocytic pathway of Arabidopsis thaliana. Embo J 20(17), 4730-41. V a n den Berg, B . , demons, W . M . , Jr., Collinson, I., Modis , Y . , Hartmann, E . , Harrison, S. C , and Rapoport, T. A . (2004). X-ray structure of a protein-conducting channel. Nature 4 2 7 ( 6 9 6 9 ) , 36-44. Van Der Heijden, M . W. , Carette, J. E . , Reinhoud, P. J., Haegi, A . , and B o l , J. F. (2001). Alfalfa mosaic virus replicase proteins PI and P2 interact and colocalize at the vacuolar membrane. J Virol 7 5 ( 4 ) , 1879-87. van Meer, G . (1998). Lipids of the Golgi membrane. Trends Cell Biol 8(1), 29-33. van Vliet , C , Thomas, E . C , Merino-Trigo, A . , Teasdale, R. D . , and Gleeson, P. A . (2003). Intracellular sorting and transport of proteins. Prog Biophys Mol Biol 83(1), 1-45. Verver, J., Wellink, J., Van Lent, J., Gopinath, K . , and Van Kammen, A . (1998). Studies on the movement of cowpea mosaic virus using the jellyfish green fluorescent protein. Virology 2 4 2 ( 1 ) , 22-7. Vi ry , M . , Serghini, M . A . , Hans, F. , Ritzenthaler, C , Pinck, M . , and Pinck, L . (1993). Biologically active transcripts from cloned c D N A of genomic grapevine fanleaf nepovirus R N A s . J Gen Virol 7 4 ( P t 2 ) , 169-74. Vitale, A . , and Denecke, J. (1999). The endoplasmic reticulum-gateway of the secretory pathway. Plant Cell 1 1 ( 4 ) , 615-28. 58 Wahlberg, J. M . , and Spiess, M . (1997). Multiple determinants direct the orientation of signal-anchor proteins: the topogenic role of the hydrophobic signal domain. J Cell Biol 1 3 7 ( 3 ) , 555-62. Walter, P., and Johnson, A . E . (1994). Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane. Annu Rev Cell Biol 1 0 , 87-119. Wang, A . , Carrier, K . , Chisholm, J., Wieczorek, A . , Huguenot, C , and Sanfacon, H . (1999). Proteolytic processing of tomato ringspot nepovirus 3C-like protease precursors: definition of the domains for the V P g , protease and putative R N A -dependent R N A polymerase. J Gen Virol 8 0 ( P t 3 ) , 799-809. Wang, A . , Han, S., and Sanfacon, H . (2004). Topogenesis in membranes of the N T B - V P g protein of Tomato ringspot nepovirus: definition of the C-terminal transmembrane domain. J Gen Virol 8 5 ( P t 2), 535-45. Wang, A . , and Sanfacon, H . (2000). Proteolytic processing at a novel cleavage site in the N-terminal region of the tomato ringspot nepovirus RNA-1-encoded polyprotein in vitro. J Gen Virol 8 1 ( P t 11), 2771-81. Warren, G . , and Malhotra, V . (1998). The organisation of the Golgi apparatus. Curr Opin Cell Biol 1 0 ( 4 ) , 493-8. Warren, G . , and Mellman, I. (1999). Bulk flow redux? Cell 9 8 ( 2 ) , 125-7. Weber-Lotfi, F. , Dietrich, A . , Russo, M . , and Rubino, L . (2002). Mitochondrial targeting and membrane anchoring of a viral replicase in plant and yeast cells. J Virol 7 6 ( 2 0 ) , 10485-96. Wessels, H . P., and Spiess, M . (1988). Insertion of a multispanning membrane protein occurs sequentially and requires only one signal sequence. Cell 5 5 ( 1 ) , 61-70. Wieczorek, A . , and Sanfacon, H . (1993). Characterization and subcellular localization of tomato ringspot nepovirus putative movement protein. Virology 1 9 4 ( 2 ) , 734-42. Wittmann, S., Chatel, H . , Fortin, M . G . , and Laliberte, J. F . (1997). Interaction of the viral protein genome linked of turnip mosaic potyvirus with the translational eukaryotic initiation factor (iso) 4E of Arabidopsis thaliana using the yeast two-hybrid system. Virology 2 3 4 ( 1 ) , 84-92. Wolk, B . , Sansonno, D . , Krausslich, H . G. , Dammacco, F. , Rice, C. M . , B lum, H . E . , and Moradpour, D . (2000). Subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus N S 3 - N S 4 A complex expressed in tetracycline-regulated cell lines. J Virol 7 4 ( 5 ) , 2293-304. 59 Yamaji, Y . , Kobayashi, T., Hamada, K . , Sakurai, K . , Yosh i i , A . , Suzuki, M . , Namba, S., and H i b i , T. (2006). In vivo interaction between Tobacco mosaic virus R N A -dependent R N A polymerase and host translation elongation factor 1 A . Virology 347(1), 100-8. Yamanaka, T., Ohta, T., Takahashi, M . , Meshi , T., Schmidt, R., Dean, C , Naito, S., and Ishikawa, M . (2000). T O M 1 , an Arabidopsis gene required for efficient multiplication of a tobamovirus, encodes a putative transmembrane protein. Proc NatlAcadSci USA 97(18), 10107-12. 60 CHAPTER 2 Evidence that insertion of Tomato ringspot nepovirus NTB-VPg protein in endoplasmic reticulum membranes is directed by two domains: a C-terminal transmembrane helix and an N-terminal amphipathic helix1 2.1 I N T R O D U C T I O N Infection by positive-strand R N A viruses results in massive proliferation and modification of the structure of the host intracellular membranes. Vi ra l replication occurs in association with intracellular membranes and is associated with various modified membrane structures including membranous vesicles and membranous webs (Moradpour et al., 2003; Osman and Buck, 1997; Salonen et al., 2005a; Schwartz et al., 2004). The specific nature of the membranes affected varies from one virus to another (e.g., endoplasmic reticulum (ER), tonoplast, mitochondrial membranes). Animal picornaviruses induce the formation of replication-competent membranous vesicles which are derived from the E R probably using mechanisms similar to those of the secretory pathway (Cuconati et a l , 1998; Gazina et al., 2002; Rust et al., 2001; Schlegel et al., 1996; Suhy et al., 2000). Replication proteins and replication intermediates from plant viruses related to picornaviruses (e.g. potyviruses, comoviruses and nepoviruses) are also found in association with ER-derived membranous vesicles (Carette et al., 2002a; Carette et al., 2000; Ritzenthaler et al., 2002; Schaad et al., 1997). It is thought that these vesicles constitute mini-virus factories which are physically separated from the cytoplasmic content of the cell and offer protective environments for viral R N A replication (Salonen et al., 2005a). 1 A version of this chapter has been published. Zhang, S. C , Zhang, G., Yang, L., Chisholm, J., and Sanfacon, H. (2005) Evidence that insertion of Tomato ringspot nepovirus NTB-VPg protein in endoplasmic reticulum membranes is directed by two domains: a C-terminal transmembrane helix and an N-terminal amphipathic helix. J Virol 79(18), 11752-65. The initial targeting of viral replication proteins to the E R is a key step in the formation of replication-competent vesicles. One or several viral or host proteins act as membrane anchors for the replication complex. These membrane anchors have the ability to associate with the E R in the absence of other viral proteins. Other replication proteins are brought in the replication complex either as polyprotein precursors or through protein-protein interactions with the membrane-anchor proteins (Chen and Ahlquist, 2000; Salonen et al., 2005a; Towner et al., 1998). Vi ra l membrane anchor proteins are usually integral membrane proteins and often have the ability to induce drastic modifications in membrane morphology (Agol et al., 1999; Aldabe and Carrasco, 1995; Brass et al., 2002; Carette et a l , 2002b; Cho et al., 1994; Datta and Dasgupta, 1994; den Boon et a l , 2001; dos Reis Figueira et al., 2002; Schaad et al., 1997; Teterina et al., 1997; Towner et al., 1996). Various membrane-association domains, including amphipathic helices and/or transmembrane helices, are involved in the association of the membrane anchors to the E R (Brass et al., 2002; Carrere-Kremer et al., 2002; Ciervo et al., 1998; Echeverri and Dasgupta, 1995; Lundin et al., 2003; Salonen et al., 2005a; Wang et al., 2004; Yamaga and Ou, 2002). The association of the animal picornavirus membrane anchor proteins has been studied in detail: membrane-association domains have been identified in the 3 A B , 2B and 2 B C proteins, models for the topology of these proteins in the membrane have been established (Agirre et al., 2002; Ciervo et al., 1998; Echeverri and Dasgupta, 1995; Towner et al., 1996) and the ability of these proteins to induce permeabilization of intracellular membranes has been extensively documented (Agirre et al., 2002; Aldabe et al., 1996; de Jong et al., 2003). Much less is known about membrane-anchor proteins from plant viruses related to picornaviruses. Two comovirus and one potyvirus 62 replication proteins have been shown to associate with E R membranes when expressed individually or in the context of a viral infection and to induce drastic modifications of the E R structure (Carette et a l , 2002b; Schaad et al., 1997). However, the mechanism of ER-targeting and the topology of these proteins in the membrane are largely unknown. Several nepovirus proteins co-localize with membrane-bound replication complexes in infected cells (Gaire et a l , 1999; Han and Sanfacon, 2003; Ritzenthaler et a l , 2002). One of these proteins ( N T B - V P g ) is an integral membrane protein and can associate with microsomal membranes in vitro (Han and Sanfacon, 2003; Wang et al., 2004). However, it is not known whether this protein can independently associate with E R membranes in planta and its possible role as a membrane-anchor for the replication complex has not been confirmed. The genome of T o R S V , a nepovirus, consists of two molecules of R N A (Sanfacon, 1995). R N A 1 encodes a polyprotein (PI) containing the domains for the replication proteins including the RNA-dependent R N A polymerase, a 3C-like proteinase, the genome-linked protein (VPg), a putative nucleoside triphosphate-binding protein (NTB) and two additional proteins ( X I and X2) of unknown function (Rott et al., 1995; Wang et al., 1999; Wang and Sanfacon, 2000). The 3C-like proteinase is responsible for cleavage of PI and of the RNA2-encoded polyprotein (Carrier et al., 1999). The mature N T B protein along with several polyprotein precursors, notably N T B - V P g , are associated with E R -derived membranes active in virus replication (Han and Sanfacon, 2003). A hydrophobic domain at the C-terminus of N T B contains a transmembrane helix that traverses the membranes in infected plants and in vitro resulting in a luminal orientation of the V P g 63 domain in at least a portion of the membrane-associated N T B - V P g protein (Han and Sanfacon, 2003; Wang et al., 2004). In vitro, translocation of the V P g in the lumen results in N-l inked glycosylation at a glycosylation site within the V P g domain (Wang et al., 2004). In addition to the C-terminal hydrophobic domain, a putative amphipathic helix was identified at the N-terminus of the protein that may also be involved in the interaction of the protein with the membranes although this has not been confirmed experimentally (Han and Sanfacon, 2003). In this study, we have analyzed membrane-targeting sequences within the N T B - V P g protein in planta using in-frame fusions to the G F P and have examined the topology of the N-terminal region of the N T B - V P g protein. We show that both the C-terminal hydrophobic domain and the N-terminal putative amphipathic helix can direct the G F P fluorescence to intracellular membranes. Our results also suggest that the putative amphipathic helix can promote the translocation of the N-terminus of N T B into the lumen of the membrane at least in vitro. 2.2 M A T E R I A L S A N D M E T H O D S 2.2.1 Plasmid constructions Plasmid pCD-327 (obtained from the Arabidopsis Biological Resource Centre, Ohio State University) contains the coding region for the red-shifted G F P (smRS-GFP) under the control of the Cauliflower mosaic virus 35S promoter (Davis and Vierstra, 1998). To introduce restriction sites allowing in-frame fusions to the C-terminus of G F P , a fragment corresponding to the entire length of pCD-327 was amplified with primers 24 (see Table 64 2.1) and 25 and Pfu polymerase (Stratagene). The P C R product was digested with Kpnl and religated resulting in plasmid psmRS-GFP(S-K) with unique Sstl and Kpnl sites. To construct pER-dsRed2, psmRS-GFP(S-K) was digested with EcoRl and treated with Klenow enzyme prior to religation resulting in psmRS-GFP(S-KARI) . The small Hind lll-Sstl fragment of pmGFP5-ER (Zhang et a l , 2002) was ligated into the corresponding sites of psmRS-GFP(S-KARI) to give psmRS-ER-GFP(S-KARI) . A fragment containing the reading frame for dsRed2 was amplified from pDsRed2 ( B D Biosciences Clontech) using primers 50 and 51, digested with EcoRl and Sstl and ligated into the corresponding sites of psmRS-ER-GFP(S-KARI) to give pER-dsRed2. p G F P - N V and its derivatives were constructed by amplifying the corresponding portion of the coding region of N T B -V P g using p M R l O (Rott et al., 1995) as a template, and the following pairs of primers: 21 and 22 for p G F P - N V , 48 and 49 for p G F P - m N , 36 and 22 for p G F P - c N V 3 , 21 and 35 for pGFP-nN, 21 and 46 for p G F P - n N - T M D and 47 and 35 for p G F P - n N A T M D . The amplified fragments were digested with Sstl and Kpnl and inserted into the corresponding sites of psmRS-GFP(S-K) . To construct p G F P - c N V 3 - T 1 2 2 9 / A and p G F P - c N V 3 A T M l , fragments were amplified using primers 36 and 22 and pT7-cNV- T 1 2 2 9 / A and pT7-c N V A T M l (Wang et al., 2004) as templates, respectively. To obtain a vector allowing fusions to the N-terminus of G F P , a c D N A fragment containing the G F P coding region was amplified from pCD-327 using primers 39 and 2868, digested with Bglll and Sstl and ligated into the BamHI and Sstl sites of psmRS-GFP to give psmRS-GFPn(B-K) containing unique BamHI and Kpnl sites. The internal A U G start codon at the beginning of the G F P open reading frame was then mutated to a G T G codon by site-directed mutagenesis to prevent the possibility of internal initiation at this site. This vector was 65 Table 2.1. Pr imers used in plasmid constructions No. Sequence (5' to 3') ' Comments 21 tatatgagctcggtggcggatcagggctcactgacgtttttgg 22 gcgcgcggtaccttactgtacagattgtgggcgga 24 gatcgaggtacctaagaatttccccgatcgttcaaac 2 5 tataggtacctatgagctctttgtatagttcatccatgccatgtg 30 acgcccatggttcctctgagtatcatga 31 acgcgtcgaccattttcccgacagcagc 35 gcgcgccggtaccaattaacgtggcaagttcacg 3 6 ttatatgagctcggtggcggaggattgttcgttgaagcgtatgactgg 3 9 tataagatctaaggagatataacaggatcccttggtaccggaggtgga ggtatgagtaaaggagaagaact 40 ttatatggatccatggggctcactgacgtttttgg 45 agcggatccatgttgttcgttgaagcgtatgactg 46 gcgcgcggtaccaagagtatcaagggaatggtg 47 ttatatgagctcggtggcggaggaacgttaatggggaaatttgg 48 ttatatgagctcggtggcggaggaaattttgatgttgaaaagtggg 49 gcgcgcggtaccttctgtaagtgagcgccctg 50 ttatatgaattcatggcctcctccgagaacgtc 51 ttattagagctcttaaagctcatcatgcaggaacaggtggtggcggc 53 gcgcgcggtaccctgtacacattgtgggcgga 54 ctagtaccatggaacgagctatacaagg 55 ctagtaggatccttactcgctttctttttcgaag 56 ataacgatgagacgaactcgaatcaagataatcc 57 ggattatcttgattcgagttcgtctcatcgttat 5 8 gacaggacggaagcctcactgcacagagtc 5 9 gactctgtgcagtgaggcttccgtcctgtc 64 ttattatggccaactcgacgtcacaaggatctcaggctccagtagc acagggaggttcacaaggagaagggctcactgacgttttttggcg 65 ttattactcgagaattaacgtggcaagttcacg 71 actcttacgttaatggggaaaactggcaagcgcacttctt 72 aagaagtgcgcttgccagttttccccattaacgtaagagt 73 ttattatggccaactcgacgtcacaaggatctcaggctccagtagc acagggaggttcacaaggagaagatgggctagtgcaccattccc 7 5 tattactcgagatatctcattgtcgttatcaattgaatg 106 ttatatggatccatggccaactcgacgtcacaaggatc 109 gcgcgcggtaccttacgcatagtcaggaacatcgtctgggtaaatta acgtggcaagttcacg 110 cgaccaggatecatggaacaaaaacttatttctgaagaagatctgtt gttcgttgaagcgtatgactgg 111 gcgcgcggtaccttaggcatagtcaggaacatcgtatgggtactgta cagattgtgggcgga 2868 cgcaagaccggcaacagg 5'NTB + 55/1 + GGGS (+) 3'VPg + /:p«I + taa(-) pCD327 + Kpnl (+) pCD327 + Kpnl + Sstl (-) 5'NTB+NcoI (+) 3 'NTB + 5a/I (-) a.a.870 NTB + Kpnl (-) a.a.l 153 NTB + Sstl + G G G G (+) 5'GFP + Bglll +BamHl +Kpnl +GGGG (+) 5'NTB + 5amHI + atg(+) a.a. 1153 NTB+ BamHI + atg (+) a.a.648 NTB + Kpnl (-) a.a.649 NTB + 5s/I + G G G G (+) a.a.702 NTB + 5 M + G G G G (+) a.a. 1 1 5 2 N T B + ^ « I (-) 5 'dsRed2+£coRI(+) 3'dsRed2 + 5 /^1 + H D E L + taa(-) 3 'VPg + Kpnl (-) 5 'TBSVpl9+iVcoI (+) 3 ' T B S V p l 9 + Sa/nHI +taa(-) rnut.l5' £coRl site TBSV pi9 (+) mut . l s t £coRI site T B S V pi9 (-) mut.2n d EcoRl site T B S V pi9 (+) mut.2nd EcoRl site T B S V pi9 (-) TB + Mscl + N-glyc. site + atg (+) a.a.701 NTB +Xhol (-) A a.a.654-686 NTB (+) A a.a.654-686 N T B (-) a.a.649 NTB + Mscl + N-glyc. site(+) a.a.870 N T B + ^ o l (-) N-glyc. site + BamHI + atg (+) a.a. 870 NTB + Kpnl + H A tag + taa (-) a.a. 1153 NTB + BamHI +myc tag + atg (+) 3' VPg + Kpnl + HA tag + taa (-) within nos polyA signal (-) a Introduced restriction sites are underlined. Point mutations or junction points in deletions are shown in bold and underlined characters. Insertion of amino acid spacers (GGGG or GGGS), of a consensus N-glycosylation site (N-glyc. site) and of epitope tags are shown in bold. b For each plasmid the start point of the region homologous to ToRSV sequence or other genes of interest is indicated (e.g. specific amino acid of the ToRSV PI sequence, or 5' end or 3' end of specific protein domains). Additional features are also indicated including: introduced restriction sites, introduced start or stop codons (atg or taa) or linker amino acid sequences (in the one letter code). Finally the strand to which the primer corresponds (+ for coding strand and - for non-coding strand) is also indicated. In the case of primers used to introduce mutations, the nature of the mutations is indicated (i.e. deletion of specific amino acids, or mutation of specific restriction sites). 66 used to insert c D N A fragments produced by amplification with the following pairs of primers: 40 and 53 for p N V - G F P , 40 and 35 for pnN-GFP and 45 and 53 for p c N V 3 - G F P . To construct the agroinfiltration vectors, a Hindlll-EcoRI fragment from p G F P - n N or pnN-GFP was inserted into the corresponding sites of binary vector pBIN19 (Clontech Laboratory Inc.). The resulting plasmids, p B I N - G F P - n N and pBIN-nN-GFP , contained unique Sstl/Kpnl or BamHVKpnl restriction sites bordering the NTB-derived sequence. These restrictions sites were used to insert other fragments encoding regions of N T B - V P g fused to the C- and N-termini of G F P . Plasmid pBIN19-p l9 containing the Tomato bushy stunt virus ( T B S V ) suppressor of gene silencing was constructed by amplifying a c D N A fragment corresponding to the T B S V p l 9 protein using an infectious T B S V c D N A clone as a template. The fragment was amplified using a previously described two-step P C R protocol (Zhang et al., 2001), primers 54 and 55 and mutagenic primers 56, 57, 58 and 59 designed to abolish two internal £coRI sites without affecting the amino acid sequence of the protein. The Ncol-BamKl digested fragment was inserted into the corresponding sites of pBBI525 (Sun et al., 2001). A Hindlll-EcoRI fragment from the resulting plasmid was then transferred into pBIN19. To construct pT7-G-nN2, a fragment containing the N-terminal region of N T B was amplified using p M R l O as a template and primers 64 and 75, digested by Mscl and Xhol and introduced into the corresponding sites of pCITE-4a(+) (Novagen). Other plasmids were constructed in a similar manner using the following pairs of primers: 73 and 75 for p T 7 - G - n N 2 A T M D and 64 and 65 for pT7-G-nN. p T 7 - G - n N 2 A A H 3 was produced using a two-step P C R protocol as above and mutagenic primers 71 and 72 in addition to 67 primers 64 and 75. p T 7 - N V was described previously (Wang et al., 2004). pT7-N was constructed by amplifying a fragment containing the N T B coding region using primers 30 and 31, and inserting the Ncol-Sall digested fragment into the corresponding sites of 1 990 pCITE4a(+). p T 7 - N V - T ' ^ 7 A was constructed by replacing the small Xhol fragment of p T 7 - N V with that of p T 7 - c N V - T 1 2 2 9 / A (Wang et al., 2004). To obtain pT7-G-N, pT7-G-N V and p T 7 - G - N V - T 1 2 2 9 / A , the small Bglll fragment of pT7-G-nN2 was replaced by a large Bgl II fragment from pT7-N, p T 7 - N V or p T 7 - N V - T 1 2 2 9 / A . Plasmid p G - n N - H A was constructed by amplifying a fragment using primers 106 and 109 and pT7-G-nN as a template. The BamHl-Kpnl digested fragment was inserted into the corresponding sites of psmRS-GFP (S-K). The BamHI site in this plasmid is located upstream of the G F P coding region which is therefore deleted from the resulting plasmid. Plasmid p N V - H A , p n N - H A and p c N V 3 - H A was constructed in a similar manner using p G F P - N V as a template and primer pairs 40 and 111, 40 and 109 and 110 and 111, respectively. The BamHl-Kpnl fragments of p G - n N - H A , p N V - H A , p n N - H A and p c N V 3 - H A were ligated with the large BamHl-Kpnl of p B I N - G F P - N V resulting in p B I N - G - n N - H A , p B I N - N V - H A , p B I N - n N - H A and p B I N - c N V 3 - H A . 2 . 2 . 2 Biolistic delivery of plasmids into N. benthamiana plants and confocal microscopy Biolistic delivery of purified plasmids into N. benthamiana plant cells was as described (Zhang et al., 2001). A confocal microscope (Leica) was used to visualize the subcellular distribution patterns of the G F P and dsRed2 fusion proteins. In some cases 68 the fluorescence associated with G F P fusion proteins was weak; therefore the green and red images were collected in a sequential manner to avoid cross-talk between the channels. To minimize organelle movement between the collections of each image, the channels were switched between each line with an average of 4 to 8 data collections for each line. Green and red fluorescence images were merged using the Leica confocal software. 2.2.3 Agroinfiltration of N. benthamiana plants The binary vectors containing the plant expression cassettes of the targeting fusion proteins were transformed into Agrobacterium tumefaciens L B A 4 0 4 4 (Invitrogen) by electroporation. Colonies confirmed to contain the binary vector by P C R were used for agroinfiltration assays as described (Voinnet et a l , 2003). To allow optimal expression of the fusion proteins, the T B S V p l 9 protein was co-expressed in the agroinfiltrated plant cells to prevent induction of post-trancriptional gene silencing (Voinnet et al., 2003). After infiltration, the plants were cultured in the greenhouse for 3 to 5 days. The infiltrated area of the inoculated leaves was collected for extraction. 2.2.4 Sub-cellular fractionation and deglycosylation assays Extraction of plant tissues and production of post-nuclear (S3), soluble (S30) and membrane-enriched (P30) fractions were carried out as described (Han and Sanfacon, 2003; Schaad et al., 1997). The P30 fraction was resuspended in a volume of homogenization buffer corresponding to that of the S30 fraction to allow direct comparison of the protein concentration in each fraction. In some experiments, the P30 69 fraction was further concentrated by centrifugation at 100,000 x g for 1 h followed by resuspension in one tenth of the initial volume (P100 fraction). Membrane flotation assays were conducted essentially as described (Brignati et al., 2003). Briefly, 800 u.1 of S3, P30 or P100 fractions were adjusted to a final volume of 1.9 ml of 71.5% sucrose (wt/vol) in N T E buffer (Brignati et al., 2003) and overlaid with 7 ml of 65% sucrose in N T E and then 3.1 ml of 10% sucrose in N T E . After centrifugation at 100,000 x g for 18 h, twelve 1-ml fractions were collected from the bottom of the tube. To purify HA-fusion proteins, 300 pl of S3, S30 or P30 fractions were adjusted to a final concentration of 1% Triton X-100, 0.1% SDS, 0.05% sodium deoxycholate and 150mM N a C l , supplemented with 50 u.1 of resuspended anti-HA affinity matrix (Roche) and incubated overnight at 4 C with constant agitation. The matrix was pelleted and washed two times with homogenization buffer as described by the supplier. Captured proteins were released from the matrix by addition of 50 |Jl of protein loading buffer (Laemmli, 1970) to the pellet followed by boiling for 5 minutes. For deglycosylation assays, protein loading buffer (Laemmli, 1970) was added to the P30 fractions (0.33 volumes of a 4 X stock solution). The samples were boiled for 5 minutes, centrifuged for 5 minutes and the supernatant was collected. Nine volumes of the appropriate deglycosylation buffer were added to each sample along with either N -glycosidase F (PNGase F, 150 m U per 50 pl of reaction, from Roche) or endoglycosidase H (EndoH, 1 U per 50 u.1 of reaction, from Roche). The PNGase F buffer was 200 m M sodium phosphate buffer, p H 7; 25 m M E D T A and 1% Triton X-100. The EndoH buffer 70 was 100 m M sodium citrate buffer, p H 5; 100 m M (3-mercaptoethanol and 1% Triton X -100. The reactions were incubated at 37 C overnight. Protein loading buffer (0.33 volume of a 4 X stock solution) was added and the samples were boiled for 5 minutes. Separation of proteins by S D S - P A G E and immunodetection were conducted as described (Han and Sanfacon, 2003) using a mouse monoclonal anti-GFP antibody (BD Biosciences), a rabbit polyclonal anti-Bip antibody (donated by M . Chrispeels), a rabbit polyclonal anti-NTB antibody (Wang et al., 1999) or a mouse monoclonal anti-HA antibody (Bio/Can Scientific). The secondary antibodies were goat anti-mouse or goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase (Bio/Can). 2.2.5 In vitro translation assays Coupled in vitro transcription/translation reactions in the presence or the absence of canine microsomal membranes and deglycosylation assays of translation products were conducted as described (Wang et al., 2004). 2.2.6 Computer-assisted prediction of putative transmembrane helices and amphipathic helices Prediction of transmembrane helices in the N-terminal region of N T B was performed using the following programs: PHDhtm (Rost et a l , 1996), Sosui (Hirokawa et al., 1998), Tmpred (Hofmann and Stoffel, 1993), TopPred2 (von Heijne, 1992) , T M H M M (Sonnhammer et al., 1998), H M M T O P (Tusnady and Simon, 1998) and M E M S A T 2 (Jones et al., 1994). Prediction and projection of the putative amphipathic helix was conducted using the Antheprot program (Deleage et al., 2001). 71 2.3 R E S U L T S 2.3.1 E R - t a r g e t i n g o f G F P fus ion p ro te ins c o n t a i n i n g the en t i re N T B - V P g d o m a i n We have previously shown that N T B - V P g is an integral membrane protein associated with ER-derived membranes in ToRSV-infected cells (Han and Sanfacon, 2003). To determine whether the N T B - V P g protein alone has the ability to associate with intracellular membranes, we examined the intracellular localization of N T B - V P g expressed independently of other viral proteins in planta. Both N-terminal and C-terminal fusions of N T B - V P g with G F P ( G F P - N V and N V - G F P , F ig . 2.1 A ) were expressed in N. benthamiana plants (a natural host for T o R S V ) using agroinfiltration. Five days after agroinfiltration, the leaves were collected and sub-cellular fractions enriched in soluble cytoplasmic proteins (S30 fraction) and in membrane-associated proteins (P30 fraction) were produced by differential centrifugation. Proteins were separated by S D S - P A G E and analyzed by immunoblot experiments using either an anti-G F P antibody (to detect G F P fusion proteins) or an antibody that recognize an endogenous ER-luminal protein (Bip) (Han and Sanfacon, 2003). A s expected, B ip was detected predominantly in the membrane-enriched fraction while free G F P was detected predominantly in the S30 fraction (Fig. 2 . IB, lanes 1-2 and 5-6). The full-length 96 kDa G F P - N V and N V - G F P proteins were detected exclusively in the membrane-enriched fractions (P30) (Fig. 2. I B , lanes 11-14). The concentration of these proteins in the plant extracts was much lower than that of the free G F P and extended exposure of the immunoblots was required to detect the proteins. Smaller proteins (of 28 to 32 kDa) were also detected in the S30 and P30 fractions derived from plants expressing G F P - N V and N V - G F P . One possibility is that they represent degradation products of the full-length 72 A GFP (27 kDa) Y////////A GFP-NV (96 kDa, a.a. 621-1236) NV-GFP (96 kDa, a.a. 621-1236) m a ' / / / / / / / / / , GFP-mN (77 kDa, a.a.702-1152) V///////A " Bip A b s GFP A b s ck GFP GFP-mN ck GFP-NV NV-GFP kDa S P S P S P S P S P S P S P 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Fig. 2.1 Immunodetection of GFP fusion proteins containing the entire NTB-VPg or the central region of NTB. (A) Schematic representation of GFP fusion proteins. The hatched areas and the black squares represent the GFP and VPg domains, respectively. The black triangle indicates a putative amphipathic helix at the N-terminus of NTB and the asterisks represent stretches of hydrophobic amino acids. The expected molecular mass of each fusion protein is indicated in parenthesis along with the amino acids of the PI polyprotein included in each fusion protein [numbering according to Rott et al (Rottetal., 1995)]. (B) Immunodetection of GFP fusion proteins. N. benthamiana plants were agroinfiltrated with A. tumefaciens transformed with binary vectors allowing the expression of the various GFP fusion proteins as indicated above each lane. Plant extracts were fractionated into a soluble fraction (S) or membrane-enriched fraction (P) by differential centrifugation as described in Materials and Methods (S30 and P30 fractions). Proteins were separated by SDS-PAGE (11% polyacrylamide) and immunodetected with anti-Bip (lanes 1-2) or anti-GFP (lanes 3-14) antibodies. The following amount of each fraction was loaded in the corresponding lanes: lanes 1-2: 5 ul, lanes 3-8: 2.5 ul, lanes 9-14: 10 ul. The film was exposed for 10 minutes (lanes 1-8) or 18 hrs (lanes 9-14). Migration of molecular mass standards is indicated on the left, ck: negative control, agroinfiltrated with pBIN-pl9 only. The arrow points to the band corresponding to the full-length GFP-NV and NV-GFP proteins. 73 proteins. The presence of the full-length G F P - N V and N V - G F P proteins in the P30 fraction suggested that they are membrane associated although this experiment could not exclude the possibility that protein aggregation was responsible for the recovery of these proteins in the P30 fraction. The concentration of these proteins in the plant extracts was too low to allow membrane flotation experiments that would eliminate this possibility (see below). The sub-cellular localization of the fusion proteins was examined in greater detail using confocal microscopy. To allow dual labelling of E R membranes and the various G F P fusion proteins, we engineered an ER-targeted red fluorescent protein (ER-dsRed2) using E R targeting and retention signals (including an N-terminal signal peptide and a C -terminal H D E L retention signal) previously described for an ER-targeted G F P reporter (Zhang et al., 2002). The plasmids were delivered into N. benthamiana plant cells using biolistic bombardment. Twenty hours after transfection, the red fluorescence associated with the ER-dsRed2 was clearly distinguished from the duller chloroplast autofluorescence (i.e. cells adjacent to those expressing the E R marker were not visible under the conditions used for the detection of the dsRed2 fluorescence; see Fig . 2.2, panel 1, ER-dsRed2). The red fluorescence was concentrated in the perinuclear area and in the cortical network of the E R (Fig. 2.2, panels 1-3, ER-dsRed2), a pattern similar to that previously described for E R - G F P (Carette et al., 2002b). In contrast, fluorescence associated with the free G F P was distributed throughout the cell and diffused through the nuclear pore (Fig. 2.2, panels 1 and 2, GFP) . In some cases, the green fluorescence displayed a diffused network pattern that was in proximity to the E R network but did not 74 coincide with the E R marker when examined at higher magnification (Fig. 2.2, panels 2 and 3). The sub-cellular localizations of free G F P and ER-dsRed2 did not change whether they were expressed individually or in combination (data not shown) suggesting that there was no interaction between the two proteins. Fluorescence associated with G F P - N V and N V - G F P was concentrated around the nucleus or in a sharp web-like structure that coincided Fig. 2.2. Sub-cellular localization of G F P fusion proteins containing the entire N T B - V P g protein or the central region of N T B . TV. benthamiana leaf epidermal cells were transfected by biolistic delivery. Each transfection experiment included one plasmid allowing the expression of the GFP fusion protein as indicated on the left and one plasmid allowing the expression of the ER-dsRed2 marker. Individual transfected cells were examined by confocal microscopy 24 hrs after transfection and a single slice is shown. The green channel shows the fluorescence associated with the GFP fusion proteins and the red channel shows the ER-dsRed2 fluorescence. The digitally superimposed images, where green and red signals that coincide produce a yellow signal are also shown (merge). Panels 2, 5, 7 and 9 are close-up views of the region included in the white square in panels 1, 4, 6 and 8, respectively. A region of the cortical ER network is shown in panels 3 and 10. The bars on the merge panels represent 10 urn for panels 1, 4, 6, 8 and 11 and 2 urn for panels 3 and 10. GFP ER-4sRcd3 merge with the fluorescence associated with ER-dsRed2 (Fig. 2.2, panels 4-10). Aggregates of green and red fluorescence in the perinuclear area were occasionally observed in cells 75 expressing G F P - N V or N V - G F P and ER-dsRed2 (panels 6, 7 and data not shown) but not in cells expressing free G F P and ER-dsRed2. Taken together, these results suggested that G F P - N V and N V - G F P were directed to perinuclear and cortical E R membranes. 2 . 3 . 2 Absence of membrane-targeting domains in the central region of NTB We have previously suggested that sequences at the N - and C-termini of the N T B domain are involved in its association with the E R (Han and Sanfacon, 2003). To determine whether the central region of N T B contains unidentified membrane-targeting domains, a G F P fusion protein including only the central region of N T B (GFP-mN, Fig . 2.1 A ) was expressed in plants. After sub-cellular fractionation of plant extracts, G F P - m N was only detected in the S30 fraction (Fig. 2.1, lanes 7 and 8). The green fluorescence associated with G F P - m N (Fig. 2.2, panel 11) was similar to that observed for the free G F P and did not coincide with the ER-dsRed2 fluorescence. Taken together, these results indicated that G F P - m N does not associate with intracellular membranes and that the middle portion of N T B does not contain elements that could independently promote membrane association. 2 . 3 . 3 ER-specific glycosylation of GFP fusion proteins containing the hydrophobic domain in the C-terminal region of NTB-VPg We have previously shown that a truncated protein, c N V 3 , containing the hydrophobic domain located at the C-terminus of N T B and the V P g domain could associate with canine microsomal membranes in vitro (Wang et al., 2004). Deletion of the hydrophobic region prevented in vitro membrane-association (mutant A T M 1 ) (Wang et al., 2004). To 76 determine i f c N V 3 has the ability to associate with intracellular membranes in vivo, G F P was fused to the wild-type (wt) or A T M 1 derivative of c N V 3 ( G F P - c N V 3 and c N V 3 -G F P - C N V 3 ( 3 6 . 5 k D a , a . a . 1 1 5 3 - 1 2 3 9 ) G F P - c N V 3 A T M l ( 3 2 k D a , A a . a . 1 1 6 9 - 1 2 1 2 ) C N V 3 - G F P ( 3 6 . 5 k D a , a . a . 1 1 5 3 - 1 2 3 9 ) C N V 3 A T M 1 - G F P ( 3 2 k D a . A a . a . l 1 6 9 - 1 2 1 2 ) a kz2 G F P - C N V 3 C N V 3 - G F P A T M 1 w t A T M 1 S P S P S P 1 2 3 4 G F P - c N V 3 'IV A c N V 3 - G F P w t k D P N G a s e F E n d o H 4 5 3 1 2 3 4 Fig. 2.3 Immunodetection of GFP fusion proteins containing the C-terminal region of NTB-VPg. (A) Schematic representation of GFP fusion proteins. The hatched box represents the GFP domain, the open box represents the NTB domain (with the hydrophobic region shown by the asterisk) and the black box represents the VPg domain. A conserved N-glycosylation site in the VPg domain is shown by the letter Y . The predicted molecular mass of each fusion protein is indicated in parenthesis along with the amino acids of the P l polyprotein included in the protein. The amino acids deleted in the ATM1 derivatives of GFP-cNV3 and cNV3-GFP are also indicated. (B) Immunodetection of GFP fusion proteins. Plant extracts from N. benthamiana plants agroinfiltrated with plasmids allowing the expression of wt or mutated versions of GFP-cNV3 and cNV3-GFP were subjected to differential centrifugation as indicated in Fig. 2. I. Proteins present in 5 ul of soluble (S) or membrane-enriched (P) fractions were separated by SDS-PAGE (12% polyacrylamide) and immunodetected with the anti-GFP antibody. (C) Analysis of the glycosylation status of GFP-cNV3 and cNV3-GFP. Proteins present in membrane-enriched fractions (P30) derived from plants expressing the wt or mutated versions (T/A mutant including a mutation of the VPg N-glycosylation site) of the fusion proteins were analyzed. In the case of the wt proteins, the P30 fractions were treated with two deglycosylation enzymes (PNGase F or EndoH) as indicated above each lane. The proteins were separated by SDS-PAGE as above and immunodetected with the anti-GFP antibody. The diamonds point to glycosylated forms of the proteins. Migration of molecular mass standards is indicated on the left. 77 G F P , F ig . 2.3A). Homogenates of leaves expressing these proteins were separated into S30 and P30 fractions. G F P - c N V 3 (with an apparent M r of 36 kDa) was present in both the S30 and P30 fractions (Fig. 2.3B, lanes 1-2). The full-length c N V 3 - G F P protein (with an apparent M r of 38 kDa) was only detected in the P30 fraction, although smaller proteins were also detected in the S30 fraction (lanes 5-6). In contrast, both G F P -C N V 3 A T M 1 and c N V 3 A T M 1-GFP were present predominantly in the S30 fraction (lanes 3-4 and 7-8). This result suggested that P30-fractionation of G F P - c N V 3 and c N V 3 - G F P was dependent on the presence of the hydrophobic region. Multiple forms of G F P - c N V 3 and c N V 3 - G F P were detected in the membrane-enriched fractions raising the possibility that membrane-dependent modification of the proteins, such as glycosylation, occurred in planta. We have previously shown that a naturally occurring N-glycosylation site ( N S T 1 2 2 9 ) in the V P g domain is glycosylated in vitro upon addition of canine microsomal membranes (Wang et al., 2004). To test for the presence of glycosylated forms of G F P - c N V 3 and c N V 3 - G F P in the P30 fraction we conducted two sets of experiments. In one set of experiments, we introduced a mutation in the glycosylation site (mutation T L 2 2 9 / A ) in G F P - c N V 3 . This mutation was previously shown to prevent glycosylation in vitro (Wang et a l , 2004). A protein with an apparent M r of 42 kDa was detected in P30 fractions derived from plants expressing wt G F P - c N V 3 but not in the P30 fraction derived from plants expressing the G F P - c N V 3 - T / A derivative (Fig. 2.3C, lane 1-2) suggesting that this protein was a glycosylated form of G F P - c N V 3 . In a second set of experiments, P30 extracts derived from plants expressing wt G F P -c N V 3 were treated with two deglycosylation enzymes specific for N - l inked 78 oligosaccharides. PNGase F releases all classes of N-l inked oligosaccharides, including complex-type N-l inked oligosaccharides produced in the Golgi , provided that they do not contain a fucose linked a (1-3) to A s n - G L c N A c , a structural motif present in many plant glycoproteins (Tarentino and Maley, 1974). EndoH digests the high mannose carbohydrate side chains added in the E R but does not recognize oligosaccharides further modified in the Golgi (Tarentino and Maley, 1974). After treatment of the P30 extracts with either PNGase F or EndoH, the 42 kDa protein disappeared but was replaced by a new protein with an apparent M r of 38 kDa (Fig. 2.3C, lanes 2-4). Similarly, in P30 fractions derived from plants expressing wt c N V 3 - G F P , a 42 kDa protein was eliminated after treatment with either deglycosylation enzymes (lanes 5-7). Possibly, the deglycosylated protein co-migrated with the 38 kDa c N V 3 - G F P protein. These results confirmed the above suggestion that the 42 kDa protein is a glycosylated form of G F P -c N V 3 and c N V 3 - G F P . The sensitivity of the 42 kDa glycosylated protein to Endo H also suggested that it was retained in the E R and did not translocate into the Golgi apparatus. In addition to the 36 and 42 kDa proteins (detected for G F P - c N V 3 ) and 38 and 42 kDa proteins (detected for c N V 3 - G F P ) , larger forms of the proteins (approximately 66 to 80 kDa) were observed although the relative concentration of these proteins varied from one sample to another (Fig. 2.3C, lanes 2 and 5). Many membrane proteins can maintain their oligomeric structure in the presence of SDS (DeGrado et al., 2003). Therefore, the 66 to 80 kDa proteins may correspond to dimeric forms of the 36 to 42 kDa G F P - c N V 3 and c N V 3 - G F P proteins. Interestingly, an 80 kDa protein present in the P30 fractions from plants expressing G F P - c N V 3 (shown by the diamond in the upper region of the gel, 79 lanes 2-4) was sensitive to the PNGase F and EndoH treatments indicating that it was glycosylated. This result raise the possibility that dimerization of G F P - c N V 3 occurred in the membrane environment with the V P g domain translocated in the lumen. Finally, in P30 extracts from plants expressing c N V 3 - G F P and c N V 3 - G F P A T M l , smaller proteins (with apparent M r of 31-33 kDa) were also detected predominantly in the S30 fractions (Fig. 2.3B, lanes 5-8). One possibility is that they are degradation products of the full-length proteins. 2.3.4 Partial ER-association of GFP fusion proteins containing the hydrophobic domain in the C-terminal region of NTB-VPg In N. benthamiana plant cells, expression of G F P - c N V 3 and c N V 3 - G F P proteins resulted in a fluorescence pattern that was suggestive of a partial association with E R membranes (Fig. 2.4, panels 1-4). Diffuse fluorescence was observed in the nucleus suggesting the presence of soluble protein. However, there were also clear patterns of green fluorescence that coincided with the ER-dsRed2 fluorescence. The proportion of green fluorescence that coincided with the E R marker varied from cell to cell and with time but was in general higher in cells expressing c N V 3 - G F P than in cells expressing G F P - c N V 3 . The fluorescence in cells transfected with the G F P - c N V 3 construct was predominantly diffuse at early time points (6 and 20 hrs post-transfection, data not shown) and it was not possible to conclusively determine whether fluorescence associated with membranes was also present at these time points. After 44 hrs, G F P - c N V 3 was partially co-localized with the E R marker (panels 1-2). Taken together these results suggested a partial association 80 of G F P - c N V 3 and c N V 3 - G F P with E R membranes. This result was consistent with the partial distribution of these proteins in membrane- enriched fraction described above. > i 9 i z I © L i . T 1 1 ( ry Co) t mmsm • $ - -PI /** -7 ... . Fig. 2.4. Sub-cellular localization of GFP fusion proteins containing the C-terminal region of NTB-VPg. N. benthamiana leaf epidermal cells were transfected by biolistic delivery with plasmids allowing the expression of the GFP fusion proteins indicated on the left of the figure and the ER-dsRed2 marker. Each panel represent a single slice of an individual transfected cells examined by confocal microscopy 44 hrs after transfection. As in Fig. 2.2, the green and red channels show the fluorescence associated with the GFP fusion proteins and the ER-dsRed2 protein, respectively. Panels 2, 4, 6 and 8 are close-up views of the region included in the white square in panels 1, 3, 5 and 7, respectively. The bars in the merge panels represent 10 urn. GFP merge Mutated derivatives of G F P - c N V 3 and c N V 3 - G F P with a deletion of the entire hydrophobic region ( A T M 1 mutants, panels 5-8) did not localize to E R membranes confirming the above suggestion that this region is responsible for the partial ER-association of G F P - c N V 3 and c N V 3 - G F P . 2.3.5 M e m b r a n e - a s s o c i a t i o n o f G F P fus ion p ro te ins c o n t a i n i n g a pu t a t i ve a m p h i p a t h i c he l i x at the N - t e r m i n u s o f N T B We have previously identified a putative amphipathic helix in the N-terminal region of N T B and suggested that this helix is involved in membrane association (predicted from a. 81 a. 654 to 689, see Fig. 2.5A) (Han and Sanfacon, 2003). A projection of the predicted amphipathic helix is shown in Fig . 2.5B. Examination of the N-terminal region of N T B with a number of transmembrane helix prediction programs (see Materials and Methods) also revealed the presence of a hydrophobic region located immediately upstream of the putative amphipathic helix which was predicted to form a transmembrane helix by some but not all prediction programs (a. a. 621-643, Fig . 2.5C). The degree of confidence in the TmPred and Sosui predictions was not very strong (see legend of Fig. 2.5C). A P u t a t i v e amphipathic h e l i x (a.a. 654-689) FGAAMDNVRKGITCMRS FVSWLMEHLALALDKITGKRT WT TGKRT AAH3 P u t a t i v e trans-membrane h e l i c e s GLTDVFGVPLSIMNAIGDGLVHH (a.a. 621-643) PHDhtm g l t d v f g v p l s i m n a i g d g l v h h S o s u i g L t d v f g v p l s i m n a i g d g l Tmpred g l t d v f g v p l s i m n a i g d g l v - - TopPred2 TMHMM HMMTOP MEMSAT2 Fig. 2.5. Computer-assisted analysis of putative membrane-association domains in the N -terminal region of N T B . (A) Amino acid sequence of a putative amphipathic helix located in the N-terminal region of NTB. The dashed lines represent amino acids that were deleted by site-directed mutagenesis in mutant GFP-nNAAH3. (B) Edmunson helical wheel representation of the NTB N-terminal amphipathic helix. Hydrophobic and charged amino acids are shown in black and white, respectively. (C) Prediction of a putative transmembrane helix in the N-terminal region of N T B . The deduced amino acid sequence of the first 23 amino acids of the N T B domain are shown at the top of the figure. Below this sequence, the transmembrane helices predicted by each individual program are shown. Only three programs predicted a transmembrane helix at this position. The score for the TMpred prediction for this transmembrane helix was 546, compared to a score of 2698 for the previously identified transmembrane helix at the C-terminus of N T B (only scores above 500 are considered significant). In the Sosui program, the N-terminal transmembrane helix was predicted to be a secondary transmembrane helix, while the transmembrane helix at the C-terminus of N T B was predicted to be a primary transmembrane helix. To evaluate the role of the N-terminal region of N T B in ER-association, G F P was fused to the first 80 amino acids of N T B (GFP-nN and nN-GFP , Fig . 2.6A). In addition, fusion 82 proteins G F P - n N - H R (containing only the upstream hydrophobic region) and G F P -n N A H R (containing only the amphipathic helix) were also tested. Immunodetection of cell extracts derived from plants expressing the various G F P fusion proteins revealed that proteins containing the amphipathic helix (GFP-nN, G F P - n N A H R and nN-GFP) were Fig. 2.6 Immunodetection of G F P fusion proteins containing the N-terminal domain of N T B . (A) Schematic representation of GFP fusion proteins. As in Fig. 2.1, the hatched box represents the GFP domain and the open box represents the NTB domain (with the putative amphipathic helix shown by the triangle and a hydrophobic region shown by the asterisk). The predicted molecular mass of each fusion protein is indicated in parenthesis along with the amino acids of the P l polyprotein included in the proteins. (B) Immunodetection of GFP fusion proteins. Extracts from agroinfiltrated plants were fractionated into a soluble (S) and membrane-enriched (P) fraction as described in Fig. 2.1. Proteins present in 2 pl of each fraction were separated by SDS-PAGE (12% polyacrylamide) and immunodetected with the anti-GFP antibody. Migration of molecular mass standards is shown on the left. (C) Membrane-flotation assays. Sub-cellular fractions were deposited at the bottom of a sucrose step gradient as described in Materials and Methods. Starting material was a membrane-enriched fraction (P30) in the case of GFP-cNV3 and GFP-nN and a post-nuclear fraction (S3) in the case of the soluble GFP. GFP fusion proteins present in 25 ul of fractions 1 to 12 collected from the bottom of the gradient were separated by SDS-P A G E and detected by immublotting using anti-GFP antibodies. For detection of the endogenous Bip protein, fractions collected from the GFP-cNV3 gradient were probed with anti-Bip antibodies. Probing of fractions from the GFP and GFP-nN gradients with the anti-Bip antibodies resulted in similar results (data not shown). Only the portion of each gel displaying the monomeric form of the protein is shown although larger forms of the proteins (probably corresponding to oligomers) were observed for GFP-cNV3 and GFP-nN. In the case of GFP-cNV3, the two bands shown correspond to the unmodified and glycosylated forms of the protein. present predominantly in the membrane-enriched fraction, while a protein that included GFP-nN (36 kDa, a.a. 621-701) GFP-nN-HR (30.5 kDa, a.a. 621 -648) G F P - n N A H R (32.7 kDa, a.a. 649-701) \///S/Ak\ nN-GFP (36 kDa, a.a. 621-701) *LVSSSSS\ B GFP-nN GFP-nN -HR GFP-nN A H R nN-GFP kDa S P S P S P S P 116 mm 97 ^ = 66 — 45 — 31 — —mm 21.5 — 1 2 3 4 5 6 7 8 c GFP-cNV3 " Bip 1 2 3 4 5 6 7 9 10 11 12 8 3 only the upstream hydrophobic region (GFP-nN-HR) was almost exclusively in the soluble fraction (Fig. 2.6B). Larger forms of G F P - n N , G F P - n N A H R and nN-GFP were detected which may correspond to dimers and larger oligomers (lanes 1-2, 5-6 and data not shown). These results suggested that the putative amphipathic helix is involved in membrane-association or is responsible for protein aggregation. To distinguish between these possibilities, we conducted membrane-flotation assays. Membrane-enriched fractions (P30) were adjusted to a concentration of 71.5 % sucrose and deposited at the bottom of a sucrose step-gradient. After high-speed centrifugation, soluble or aggregated proteins remain at the bottom of the gradient while membrane-associated proteins float at the interface between the 65 % and 10 % sucrose layers due to the buoyant density of lipids (Brignati et al., 2003). We have shown that G F P - c N V 3 , present in P30 fractions, is glycosylated in an ER-specific manner in plants (Fig. 2.3C) demonstrating that it is membrane-associated. Thus, this protein was used as a positive control for the membrane-flotation assay. The collected fractions were probed with either anti-GFP antibody to detect the G F P fusion proteins or with anti-Bip antibody to detect the endogenous ER-luminal protein. A s expected, G F P - c N V 3 and Bip partitioned predominantly at the interface between the 65% and 10% sucrose layers (fraction 9, F ig . 2.6C). In contrast, free G F P (present in a post-nuclear fraction, S3) remained at the bottom of the gradient as previously described (Brignati et al., 2003). G F P - n N partitioned into fraction 9 confirming that the protein is membrane-associated. Similar results were obtained with G F P - n N A H R (data not shown). 84 In N. benthamiana cells expressing G F P - n N , the green fluorescence coincided with the perinuclear ER-dsRed2 fluorescence and also partially with the ER-dsRed2 fluorescence associated with the cortical network (Fig. 2.7, panels 1-2). There were also aggregates of green fluorescence in the perinuclear area or throughout the cell, often in close proximity to the E R network (panel 2). In cells expressing n N - G F P , the E R network was drastically altered. Instead of a distinct E R network, large aggregates of red fluorescence were CT 3 5 7 GFP ER-dsRed2 merge Fig. 2. 7. Sub-cellular localization of GFP fusion proteins containing the N-terminal portion of NTB in A7, benthamiana cells. N. benthamiana leaf epidermal cells were transfected by biolistic delivery with plasmids allowing the expression of the GFP fusion proteins indicated on the left and the ER-dsRed2 marker. Each panel represent a single slice of individual transfected cells examined by confocal microscopy 24 hrs after transfection. The green and red channels show the fluorescence associated with the GFP fusion proteins and ER-dsRed2 fluorescence, respectively. The merge panels represent the digitally superimposed images. Panel 2, 4, and 6 are close-up views of the area shown in the white square in panels 1, 3 and 5, respectively. The bars in the merge panels represent 10 urn. present, often in the proximity of the nucleus (panels 3-4). The green fluorescence co-localized with these aggregates. The fluorescence associated with G F P - n N A H R in which the upstream hydrophobic region was deleted clearly coincided with the ER-dsRed2 fluorescence associated with the perinuclear and cortical E R (Fig. 2.7 panels 5-6). In contrast, deletion 85 of the amphipathic helix from G F P - n N (GFP-nN-HR) prevented its association with membranes (Fig. 2.7, panel 7). These results indicated that the putative amphipathic helix is involved in membrane-association. 2.3.6 In vitro analysis of the topology of the N-terminal region of NTB-VPg in the membranes To further analyze the ability of various elements in the N-terminal region of N T B to promote membrane association, we used in vitro membrane association assays (Wang et al., 2004). To examine the topology of the N-terminus of N T B in the membrane, we introduced a glycosylation site immediately upstream of the N T B sequence. This glycosylation site was shown by others to be recognized efficiently by canine microsomal membranes in vitro when placed upstream of a transmembrane domain and when it is translocated in the luminal side of the membrane (Ponnambalam et al., 1994). In construct G-nN2, the first 250 amino acids of N T B were fused in frame with this glycosylation signal (Fig. 2.8A). A protein of the expected size was produced in a coupled in vitro transcription/translation system (Fig. 2.8B, lane 1). Addit ion of canine microsomal membranes to the reaction resulted in the production of an additional protein with slower mobility (lane 2, shown by the diamond). Treatment of the reaction with PNGase F resulted in the disappearance of this additional protein, indicating that it was a glycosylated form of G-nN2 (lane 3). Similar results were obtained with construct G - n N which included only the first 80 amino acids of N T B , corresponding to the region of N T B included in the G F P - n N protein used for the in planta experiments (lanes 10-12). A larger form of G - n N was also observed in the upper region of the gel which probably 86 Fig. 2. 8. Topological analysis of the N-terminus of N T B using an introduced N-glycosylation site. (A) Schematic representation of truncated proteins containing the N-terminal domain of NTB fused to an inserted artificial N-glycosylation site. Amino acids inserted at the N -terminus of the NTB domain are shown with the shaded area (introduced N-glycosylation site represented by the letter Y). The black triangle represents the predicted amphipathic helix and the asterisk represents the upstream hydrophobic region. The sequence of amino acids inserted in frame with the N-terminus of NTB is shown at the bottom with the consensus N-glycosylation site underlined. The name of each protein is shown on the left. Numbers in parenthesis indicate the amino acids at the N - and C-terminus of the region of Pl contained in each fusion protein along with the predicted molecular mass of the proteins. G-nN2AAH3 contained the same region of NTB as G-nN2 with the exception that amino acids 654 to 686 were deleted. (B) In vitro membrane association assays of truncated proteins containing the N-terminal domain of NTB. Proteins were translated in the presence (+) or the absence (-) of canine microsomal membranes (MM). After translation, the in vitro translation products were treated with PNGase F as indicated above each lane. The translation products were separated by SDS-PAGE (12% polyacrylamide for lanes 1-9 and 18% polyacrylamide for lanes 10-12) and detected by autoradiography. Diamonds indicate the glycosylated forms of the protein monomers or dimers. Migration of molecular mass standards is shown on the left for lanes 1-9 and on the right for lanes 10-12. (C) Schematic representation of proteins containing either the NTB-VPg or NTB domains fused to an inserted artificial N-glycosylation site. The NTB domain is shown by the open box and the VPg domain is shown by the black box. N-glycosylation sites in inserted amino acids at the N-terminus of NTB or in the VPg domain are shown by the letter Y above the shaded box and black box, respectively. In the T/A mutant (shown in the G-NV T A) the glycosylation site in the VPg domain is inactivated. (D) In vitro membrane association assays of wt or mutated versions of NTB-VPg (NV) and NTB (N). The G-NV, G-NV™ and G-N proteins contained the inserted artificial N-glycosylation site at their N-terminus as shown in (C). N V T / A and G-NV T / A were mutated at the conserved N-glycosylation site present in the VPg domain. Proteins were translated in the presence (+) or in the absence (-) of canine microsomal membranes (MM). The translation products were separated by SDS-PAGE (10% polyacrylamide) and detected by autoradiography. Diamonds indicate the single or double glycosylated forms of the proteins. Migration of molecular mass standards is shown on the left. G-nN2 (30 kDa, a.a. 621-870) G - n N 2 A H R (28 kDa, a.a. 649-870) G - n N 2 A A H 3 (26 kDa, Aa.a. 654-686) F ^ T ^ J I G - n N (11 kDa, a. a. 621-701) I V A I M A N S T S O G S O A P V A O G G S O G E G-nN2 A H R A A H 3 G-nN 1 2 3 4 7 8 9 10 11 12 G - N V (72 kDa) G - N V T / A G - N (70 kDa) N V N V T / A G - N V G - N V T / A N kDa 85—1 6 0 - 1 G - N - + - + - + - + - + MM • ft . at mm*. • mm ^ m mm* m 87 corresponds to a dimer (its apparent mobility was 28 kDa compared to 13 kDa for the monomeric form of the protein). The 28 kDa protein was glycosylated in the presence of membranes (lanes 10-12, shown by the diamond in the upper region of the gel), raising the possibility that dimerization of G - n N occurred in the membrane environment. Deletion of the upstream hydrophobic region (G-nN2AHR) did not prevent recognition of the glycosylation site (lanes 4-6), while deletion of the putative amphipathic helix (G-n N 2 A A H 3 ) drastically decreased the extent of glycosylation (lanes 7-9). These results suggested that amino acids 654 to 686, containing the putative amphipathic helix, directed the translocation of the N-terminus of N T B to the luminal face of the membranes in vitro. We next wished to confirm this result in the context of the entire N T B - V P g . We have previously shown that the C-terminus of the protein is translocated in the lumen, resulting 1 990 in the recognition of a glycosylation site in the V P g domain. The T / A mutation was introduced into N T B - V P g to produce N V T / A which did not contain a glycosylation site. The glycosylation site described above was then fused in frame with the N-terminus of N V (consisting of the wild-type N T B - V P g ) , N V T / A or N (consisting of the entire N T B domain without the V P g domain), to produce G - N V , G - N V ™ and G - N , respectively (Fig. 2. 8C). A s shown previously, wt N V was glycosylated in the presence of the canine microsomal membranes (Fig. 2.8D, lanes 1-2) (Wang et al., 2004). Glycosylation was prevented after mutation of the V P g glycosylation site ( N V r / A , lanes 3-4) or deletion of the V P g domain (N, lanes 9-10). These results confirmed that the V P g domain is translocated in the lumen in the context of the entire N T B - V P g . Glycosylated forms of 88 the proteins were also observed after translation of G - N V , G - N V 1 / A and G - N in the presence of membranes (lanes 5-8 and 11-12) suggesting that the artificial glycosylation site at the N-terminus of N T B was translocated in the lumen. Interestingly, two forms of glycosylated proteins were observed for G - N V (lane 6). One form co-migrated with the glycosylated form of proteins containing a single glycosylation site, while the other form had a slower mobility and was apparently glycosylated at two sites. This protein contains two glycosylation sites: the N-terminal introduced glycosylation site and the C-terminal V P g glycosylation site. This result suggests that several topologies of the protein co-existed, one of which implied the translocation of both N - and C-termini of the protein in the lumen. 2.3.7 Sub-cellular fractionation of truncated proteins containing the C-terminal or N-terminal regions of NTB-VPg fused to the HA epitope tag The results presented above and previously (Wang et al., 2004) suggest that the C-terminal and N-terminal regions of N T B contain elements that can target G F P fusion proteins to intracellular membranes in planta and can promote membrane association of the native N T B - V P g or smaller truncated proteins in vitro. We next wished to confirm that these membrane association elements were also active in planta when fused to smaller epitope tags. In initial experiments, we tested a fusion protein containing the entire N T B and V P g domains and the H A epitope tag ( Y P Y D V P D Y A ) . Unfortunately, we were not able to detect the fusion protein in extracts from agroinfiltrated plants using anti-HA or anti-NTB antibodies (data not shown). We then tested smaller fusion proteins containing the c N V 3 or n N truncated proteins ( c N V 3 - H A and n N - H A , Fig . 2.9A). G -89 n N - H A was a derivative of G - n N described above and included the H A epitope and the artificial glycosylation site at the N-terminus of the N T B domain. The concentration of the fusion proteins in plant extracts was low and detection of specific proteins by the anti-H A antibody was hindered by the presence of large amounts of a 14 kDa protein from cNV3-HA( l l kDa, a.a.l 153-1239) nN-HA (10 kDa, a.a. 621 -701) G-nN-HA (12 kDa, a.a. 621-701) k B ck CNV3-HA G-nN-HA kDa o c-i c<-) oo oo P30 o oo oo P30 r i 0Q o o c-> cn 00 — 21.5- m • 14.5- • • 1 1 2 4 5 6 7 8 9 Fig. 2. 9. Immunodetection of the N-terminal and C-terminal regions of NTB-VPg fused to epitope tags. (A) Schematic representation of fusion proteins. The boxes with horizontal hatches represent HA epitope tags. The open and black boxes represent the NTB and VPg domains, respectively (with the putative amphipathic helix shown by the triangle and hydrophobic regions shown by the asterisk). The shaded box represents the artificial glycosylation site as described in Fig. 8. The predicted molecular mass of each fusion protein is indicated in parenthesis along with the amino acids of the PI polyprotein included in the proteins. (B) Immunodetection of HA fusion proteins. HA-fusion proteins present in post-nuclear (S3), soluble (S30) and membrane-enriched (P30) fractions derived from agroinfiltrated plants were purified by HA-affinity chromatography as described in Materials and Methods. Proteins were separated by SDS-PAGE (16% polyacrylamide) and immunodetected with anti-HA antibodies. Migration of molecular mass standards is shown on the left. The positions of various forms of the G-nN-HA and cNV3-HA proteins are indicated by the black squares on the left of the gels, ck: negative control, agroinfiltrated with pBIN-pl9 only. (C) Membrane-flotation assays. PI00 fractions were deposited at the bottom of a sucrose step gradient. HA fusion proteins present in 25 |jl of fractions 1 to 12 collected from the bottom of the gradient were separated by SDS-PAGE as above and immunodetected with anti-HA antibodies. plants in post-nuclear (S3) and cytoplasmic (S30) fractions (data not shown). To circumvent this problem, HA-fusion proteins were purified from S3, S30 and P30 fractions using an ant i-HA affinity matrix in the presence of mild detergents. Using this 90 method, we detected specific proteins in S3 fractions derived from plants expressing G -n N - H A and c N V 3 - H A but not in those derived from plants expressing n N - H A (Fig. 2.9B and data not shown). Both G - n N - H A and c N V 3 - H A were found predominantly in the P30 fractions, although a portion of the c N V 3 - H A protein was also present in the S30 fraction. Membrane flotation assays were conducted using a reconcentrated membrane-enriched fraction (PI00 fraction, resuspended in one tenth of the original volume of the P30 fraction). Both proteins separated at the interface between the 65 % and 10 % sucrose layers confirming that they were membrane associated (fraction 9, F ig . 2.9C). The sub-cellular fractionation behaviour of the c N V 3 - H A and G - n N - H A proteins was very similar to that of the corresponding G F P fusion proteins (see G F P - c N V 3 and G F P -nN, Figs. 2.3 and 6). Several forms of the c N V 3 - H A and G - n N - H A proteins were detected. In the case of c N V 3 - H A , these proteins had an apparent molecular mass of 14 and 18 kDa. Both proteins were found in association with intracellular membranes suggesting that the 18 kDa protein is a glycosylated form of the 14 kDa protein (see fraction 9, Fig. 2.9C). In the case of G - n N - H A , a protein of approximately 13 kDa was predominant. L o w amounts of a 17 kDa protein were also occasionally detected (Fig. 2.9B, lane 7), raising the possibility that glycosylation of G - n N - H A also occurred. Unfortunately, the low concentration of this protein in the extracts did not allow us to conduct deglycosylation assays that could have confirmed this suggestion. 91 2 . 4 D I S C U S S I O N The results presented in this study demonstrate that the T o R S V N T B - V P g protein, the putative membrane-anchor for the replication complex, has the ability to associate with intracellular membranes in planta when fused to G F P . Deletion of hydrophobic domains in the N-terminal and C-terminal regions of N T B - V P g prevented the membrane association (see G F P - m N , Fig . 2.1 and 2.2). Although we cannot exclude the possibility that fusion to the G F P alters the natural sub-cellular localization of N T B - V P g , it is noteworthy that similar ER-associated localization was observed for fusion proteins with C-terminal or N-terminal fusions to the GFP . The results presented in this study are also consistent with previous observations that N T B - V P g is an integral membrane protein associated with the E R in ToRSV-infected plants and that it has the ability to independently associate with canine microsomal membranes (which consist predominantly of ER-derived membranes) in vitro (Han and Sanfacon, 2003; Wang et al., 2004). Taken together these results suggest that membrane association elements within N T B - V P g are functional in the context of G F P fusion proteins. In fact, fusion of the truncated c N V 3 protein to G F P (GFP-cNV3 and c N V 3 - G F P fusion proteins) did not prevent the recognition of the naturally occurring V P g glycosylation site, suggesting that these fusion proteins inserted in the proper orientation in the membrane (see below). The n N and c N V 3 truncated proteins also had similar sub-cellular fractionation behaviour when fused to G F P or to the H A epitope tag. The C-terminal region of N T B contains a previously characterized hydrophobic domain that includes a transmembrane helix. This domain directs the translocation of the V P g in 92 the E R lumen in vitro and in ToRSV-infected plants (Han and Sanfacon, 2003; Wang et al., 2004). In this study, we provide several lines of evidence confirming that the hydrophobic domain functions as an ER-targeting domain in vivo. First, wt G F P - c N V 3 and c N V 3 - G F P co-localized at least partially with E R markers and were detected in membrane-enriched fractions. Deletion of the hydrophobic domain ( A T M 1 mutant) prevented the membrane-association. Second, glycosylated forms of the wt proteins were present in the membrane-enriched fractions confirming that they were associated with intracellular membranes and that the V P g domain was translocated in the lumen. Sensitivity of the glycosylation to EndoH indicated that the glycosylated proteins were retained in the E R , suggesting that ER-retention signals are present in the C-terminal region of N T B - V P g . The characterization of these ER-retention signals w i l l be the subject of further studies. Although it is clear that the hydrophobic region within the C-terminal region of N T B can act as an ER-targeting domain, the membrane-association of G F P - c N V 3 and c N V 3 - G F P was only partial as evidenced by the diffusion of a portion of the green fluorescence within the nucleus and the presence of a portion of the proteins immunodetected by the G F P antibodies in the soluble fraction. A similar phenomenon was observed with c N V 3 -H A , although the proportion of protein in the S30 fraction was lower than with the corresponding G F P fusion proteins. One possible explanation is that targeting of these truncated proteins to E R membranes is inefficient at least in the context of the GFP fusions. Another possible explanation is that degradation of the fusion proteins release soluble fragments with G F P activity. A truncated 31 kDa protein, detected in the S30 93 and P30 fractions of plants expressing c N V 3 - G F P , may correspond to such a cleavage product (Fig. 2.3B, lanes 5-6). In the case of G F P - c N V 3 , the predominant 36 kDa protein detected in the S30 and P30 fractions may also correspond to a cleavage fragment for the following reasons. First, it was smaller than the detected c N V 3 - G F P protein even though the predicted molecular masses of the two fusion proteins are identical (Fig. 2.3B, lanes 1-2 and 5-6). Second, the 36 kDa protein did not co-migrate with a 38 kDa protein produced after deglycosylation treatment of the G F P - c N V 3 membrane-enriched fraction (Fig. 2.3C, lanes 2-4). Interestingly, we have previously shown that c N V 3 is cleaved by a membrane-associated proteinase in vitro (Wang et al., 2004). Although it is possible that this cleavage is responsible at least in part for the release of G F P - c N V 3 and c N V 3 -G F P in the cytosolic fraction, further work w i l l be necessary to conclusively determine whether membrane-associated cleavage of c N V 3 (and the full-length N T B - V P g ) occurs in plants. Our results demonstrate that the N-terminal putative amphipathic helix can promote efficient targeting to intracellular membranes in planta and in vitro. The putative role of an upstream hydrophobic region is not clear at this point as it was not able to promote membrane-association in the absence of the amphipathic helix. In addition, deletion of this region did not prevent the membrane-association of proteins that contained the amphipathic helix. However, our results do not exclude the possibility that the upstream hydrophobic region influences the exact positioning of the amphipathic helix in the membrane. 94 The results of the glycosylation site mapping revealed that the N-terminus of N T B can be translocated in the membrane lumen in vitro in the context of truncated proteins containing only the N-terminal amphipathic helix or in the context of the entire N T B -V P g . Amphipathic helices usually orient parallel to the membrane with the hydrophobic face towards the membrane and the hydrophilic face towards the cytoplasm, unless they form membrane-embedded oligomers through the barrel-stave mechanism and assemble into aqueous pores or ion channels (Bechinger, 1999; Shai, 1999). The barrel-stave mechanism is derived from studies of pore-forming cytolytic amphipathic peptides and implies the formation of trans-bilayer oligomers of the amphipathic helices with their hydrophobic sides facing the l ipid bilayer and their hydrophilic sides oriented toward the water-filled pore. Using S D S - P A G E , we have observed larger forms of truncated proteins containing the amphipathic helix, which may correspond to oligomers of the protein (Fig. 2.8 and 2.9). Membrane-dependent glycosylation of one of these putative oligomeric forms was observed in vitro (Fig. 2.9). These observations provide support for the suggestion that oligomerization of the amphipathic helix promote its translocation in the membrane. It is not known whether translocation of the N-terminus of N T B - V P g in the membrane lumen occurs in vivo. Further experimentation w i l l be necessary to confirm that the amphipathic helix has the ability to oligomerize in the membrane environment and to determine whether the N-terminus of N T B - V P g is translocated in the membrane lumen in vivo. The formation of aqueous pores is often associated with permeabilization of the membrane and several viral proteins (viroporins) have been found to destabilize and 95 permeabilize membranes (Gonzalez and Carrasco, 2003). In animal viruses, viroporins have been suggested to play a role in the release of virus particles from infected cells. Many viroporins are located predominantly at the intracellular membranes and have the ability to promote intracellular membrane remodelling which is a prerequisite for viral replication (Gonzalez and Carrasco, 2003). Some viroporins such as the HIV-1 V P u and the influenza virus M 2 proteins induce the formation of intracellular ion channels although the role of these channels in the virus replication cycle is not clear (Gonzalez and Carrasco, 2003). Viroporins have not been identified so far in plant viruses. The Cowpea mosaic comovirus 60K protein (which contains the N T B and V P g domains and is related to the T o R S V N T B - V P g protein) is cytotoxic in plants and induces drastic morphological changes of the E R membranes. It can also induce cell lysis when expressed in insect cells (Carette et al., 2002b). The domains of the 60K protein involved in these cytotoxic effects have not been characterized. It is noteworthy, however, that a putative amphipathic helix has been identified at the N-terminus of the 60K protein (Carette et al., 2002b). In this study, we did not observe severe changes in the structure of the E R in cells expressing G F P fusion proteins containing the entire T o R S V N T B - V P g , although aggregates of the ER-dsRed2 fluorescence were occasionally observed (Fig. 2.2, panels 5-6). One possibility is that the levels of expression of these proteins were not sufficient to significantly alter the E R structure. Interestingly, high levels of expression of a fusion protein containing the N-terminal amphipathic helix (nN-GFP) in plant cells resulted in drastic modification of the morphology of the E R . However, we cannot exclude the possibility that the interaction of the amphipathic helix with the membranes is influenced by the presence of the G F P domain. In fact, modification of the E R 96 structure was not observed to the same extent in cells expressing a fusion protein containing the amphipathic helix fused to the C-terminus of G F P (GFP-nN). The ability of the T o R S V N T B - V P g protein to modify and possibly permeabilize membranes wi l l be the subject of further studies. Multiple interactions between transmembrane helices within the membrane environment have been documented for a large number of polytopic membrane proteins (Curran and Engelman, 2003; Engelman et al., 2003), suggesting that interactions between the T o R S V N T B - V P g amphipathic and transmembrane helices are at least theoretically possible. The current two stage model for membrane insertion and folding of polytopic membrane proteins suggest that individual transmembrane helices initially insert in the membrane independently, followed by a second stage in which transmembrane helices interact with each other in the membrane environment to give higher order folding and oligomerization (Curran and Engelman, 2003; Engelman et al., 2003). A n example of this is provided by the poliovirus 2B protein, a viroporin which forms aqueous pores driven by the tetramerization of an amphipathic helix and further stabilized by intramolecular hydrophobic interactions between the amphipathic helix and an adjacent transmembrane helix and by intermolecular hydrophobic interactions between the transmembrane helices of the four 2B monomers (Agirre et al., 2002). According to the two stage model for membrane protein folding, we propose an updated model for the insertion of N T B - V P g in the membrane (Fig. 2.10). In this model, N T B - V P g initially interacts with the membranes as a monomer with the transmembrane helix traversing the membrane and the amphipathic helix parallel to the membranes (step 1). The ability of each of these 97 elements to oligomerize could result in the formation of an aqueous pore with the hydrophilic side of the amphipathic helix lining the pore (step 2). Possible interactions between the hydrophobic face of the amphipathic helix and the transmembrane helix within a monomer unit and between the transmembrane helices of the various monomers Fig. 2. 10. Updated model for the insertion of the NTB-VPg protein into ER membranes. The protein initially associates as a monomer within the membrane ( 1 ) with the N-terminal amphipathic helix (represented by the light grey cylinder) parallel to the membrane and the C-terminal transmembrane helix (represented by the dark grey cylinder) traversing the membrane. In a second step (2), the protein oligomerizes through the N-terminal amphipathic helix and possibly also the C-terminal transmembrane helix to form an aqueous pore with an a-loop-a conformation. Hydrophobic interactions between the N-terminal amphipathic helix and the C-terminal transmembrane helix may help stabilize the formation of the pore. To simplify the drawing only two monomers are shown interacting in the structure. However, formation of the pore would imply the interaction of at least four NTB-VPg molecules. The VPg domain is represented by the black box. The region containing the conserved motif for the NTB-binding domain is also shown (shaded box). could result in the formation of an a-loop-a motif that spans the bilayer and helps stabilize the structure. In this model, both the N - and C-termini of N T B - V P g are oriented to the luminal face of the membranes, while the central region of N T B is oriented towards the cytoplasm and accessible for protein-protein interactions with other viral or host proteins present in the replication complex. 98 2 . 5 B I L I O G R A P H Y Agirre, A . , Barco, A . , Carrasco, L . , andNieva, J. L . (2002). Viroporin-mediated membrane permeabilization. Pore formation by nonstructural poliovirus 2B protein. JBiol Chem 211 {A3), 40434-41. Ago l , V . I., Paul, A . V . , and Wimmer, E . (1999). Paradoxes of the replication of picornaviral genomes. Virus Res 6 2 ( 2 ) , 129-47. Aldabe, R., Barco, A . , and Carrasco, L . (1996). Membrane permeabilization by poliovirus proteins 2B and 2 B C . JBiol Chem 2 7 1 ( 3 8 ) , 23134-7. Aldabe, R., and Carrasco, L . (1995). Induction of membrane proliferation by poliovirus proteins 2C and 2 B C . Biochem Biophys Res Commun 2 0 6 ( 1 ) , 64-76. Bechinger, B . (1999). The structure, dynamics and orientation of antimicrobial peptides in membranes by multidimensional solid-state N M R spectroscopy. Biochim Biophys Acta 1 4 6 2 ( 1 - 2 ) , 157-83. Brass, V . , Bieck, E . , Montserret, R., Wolk, B . , Hellings, J. A . , B lum, H . E . , Penin, F. , and Moradpour, D . (2002). A n amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis C virus nonstructural protein 5 A . J Biol Chem 2 7 7 ( 1 0 ) , 8130-9. Brignati, M . J., Loomis, J. S., Wi l l s , J. W. , and Courtney, R. J. (2003). Membrane association of VP22 , a herpes simplex virus type 1 tegument protein. J Virol 7 7 ( 8 ) , 4888-98. Carette, J. E . , Guhl , K . , Wellink, J., and Van Kammen, A . (2002a). Coalescence of the sites of cowpea mosaic virus R N A replication into a cytopathic structure. J Virol 7 6 ( 1 2 ) , 6235-43. Carette, J. E . , Stuiver, M . , Van Lent, J., Wellink, J., and Van Kammen, A . (2000). Cowpea mosaic virus infection induces a massive proliferation of endoplasmic reticulum but not Golgi membranes and is dependent on de novo membrane synthesis. J Virol 7 4 ( 1 4 ) , 6556-63. Carette, J. E . , van Lent, J., MacFarlane, S. A . , Wellink, J., and van Kammen, A . (2002b). Cowpea mosaic virus 32- and 60-kilodalton replication proteins target and change the morphology of endoplasmic reticulum membranes. J Virol 7 6 ( 1 2 ) , 6293-301. Carrere-Kremer, S., Montpellier-Pala, C , Cocquerel, L . , Wychowski, C , Penin, F. , and Dubuisson, J. (2002). Subcellular localization and topology of the p7 polypeptide of hepatitis C virus. J Virol 7 6 ( 8 ) , 3720-30. 99 Carrier, K . , Hans, F., and Sanfacon, H . (1999). Mutagenesis of amino acids at two tomato ringspot nepovirus cleavage sites: effect on proteolytic processing in cis and in trans by the 3C-like protease. Virology 258(1), 161-75. Chen, J., and Ahlquist, P. (2000). Brome mosaic virus polymerase-like protein 2a is directed to the endoplasmic reticulum by helicase-like viral protein l a . J Virol 74(9), 4310-8. Cho, M . W. , Teterina, N . , Egger, D . , Bienz, K . , and Ehrenfeld, E . (1994). Membrane rearrangement and vesicle induction by recombinant poliovirus 2C and 2 B C in human cells. Virology 202(1), 129-45. Ciervo, A . , Beneduce, F. , and Morace, G . (1998). Polypeptide 3 A B of hepatitis A virus is a transmembrane protein. Biochem Biophys Res Commun 249(1), 266-74. Cuconati, A . , Mol l a , A . , and Wimmer, E . (1998). Brefeldin A inhibits cell-free, de novo synthesis of poliovirus. J Virol 72(8), 6456-64. Curran, A . R., and Engelman, D . M . (2003). Sequence motifs, polar interactions and conformational changes in helical membrane proteins. Curr Opin Struct Biol 13, 412-417. Datta, U . , and Dasgupta, A . (1994). Expression and subcellular localization of poliovirus VPg-precursor protein 3 A B in eukaryotic cells: evidence for glycosylation in vitro. J Virol 68(7), 4468-77. Davis, S. J., and Vierstra, R. D . (1998). Soluble, highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants. Plant Mol Biol 36(4), 521-8. de Jong, A . S., Wessels, E . , Dijkman, H . B . , Galama, J. M . , Melchers, W . J., Willems, P. H . , and van Kuppeveld, F. J. (2003). Determinants for membrane association and permeabilization of the coxsackievirus 2B protein and the identification of the Golgi complex as the target organelle. J Biol Chem 278(2), 1012-21. DeGrado, W . F., Gratkowski, H . , and Lear, J. D . (2003). H o w do helix-helix interactions help determine the folds of membrane proteins? Perspectives from the study of homo-oligomeric helical bundles. Protein Sci 12(4), 647-65. Deleage, G . , Combet, C , Blanchet, C , and Geourjon, C. (2001). A N T H E P R O T : an integrated protein sequence analysis software with client/server capabilities. Comput Biol Med 31(4), 259-67. den Boon, J. A . , Chen, J., and Ahlquist, P. (2001). Identification of sequences in Brome mosaic virus replicase protein l a that mediate association with endoplasmic reticulum membranes. J Virol 75(24), 12370-81. 100 dos Reis Figueira, A . , Golem, S., Goregaoker, S. P., and Culver, J. N . (2002). A nuclear localization signal and a membrane association domain contribute to the cellular localization of the Tobacco mosaic virus 126-kDa replicase protein. Virology 3 0 1 ( 1 ) , 81-9. Echeverri, A . C , and Dasgupta, A . (1995). Amino terminal regions of poliovirus 2C protein mediate membrane binding. Virology 2 0 8 ( 2 ) , 540-53. Engelman, D . M . , Chen, Y . T., Chin, C. N . , Curran, A . R., Dixon, A . M . , Dupuy, A . D . , Lee, A . S., Lehnert, U . , Matthews, E . E . , Reshetnyak, Y . K . , Senes, A . , and Popot, J.-L. (2003). Membrane protein folding: beyond the two stage model. FEBS Lett 5 5 5 , 122-125. Gaire, F. , Schmitt, C , Stussi-Garaud, C , Pinck, L . , and Ritzenthaler, C. (1999). Protein 2 A of grapevine fanleaf nepovirus is implicated in R N A 2 replication and colocalizes to the replication site. Virology 2 6 4 ( 1 ) , 25-36. Gazina, E . V . , Mackenzie, J. M . , Gorrell, R. J., and Anderson, D . A . (2002). Differential requirements for COPI coats in formation of replication complexes among three genera of Picornaviridae. J Virol 7 6 ( 2 1 ) , 11113-22. Gonzalez, M . E . , and Carrasco, L . (2003). Viroporins. FEBS Lett 5 5 2 ( 1 ) , 28-34. Han, S., and Sanfacon, H . (2003). Tomato ringspot virus proteins containing the nucleoside triphosphate binding domain are transmembrane proteins that associate with the endoplasmic reticulum and cofractionate with replication complexes. J Virol 7 7 ( 1 ) , 523-34. Hirokawa, T., Boon-Chieng, S., and Mitaku, S. (1998). SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics 1 4 , 378-379. Hofmann, K . , and Stoffel, W . (1993). TMbase-a database of membrane spanning protein segments. Bio. Chem. Hoppe-Seyler341, 166. Jones, D . T., Taylor, W . R., and Thornton, J. M . (1994). A model recognition approach to the prediction of all-helical membrane protein structure and topology. Biochemistry 3 3 , 3038-3049. Laemmli, U . K . (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 2 2 7 ( 2 5 9 ) , 680-5. Lundin, M . , Monne, M . , Widel l , A . , V o n Heijne, G . , and Persson, M . A . (2003). Topology of the membrane-associated hepatitis C virus protein N S 4 B . J Virol 7 7 ( 9 ) , 5428-38. 101 Moradpour, D . , Gosert, R., Egger, D . , Penin, F. , B lum, H . E . , and Bienz, K . (2003). Membrane association of hepatitis C virus nonstructural proteins and identification of the membrane alteration that harbors the viral replication complex. Antiviral Res 60(2), 103-9. Osman, T. A . , and Buck, K . W . (1997). The tobacco mosaic virus R N A polymerase complex contains a plant protein related to the RNA-binding subunit of yeast elF-3. J Virol 6075-82. Ponnambalam, S., Rabouille, C. , Luzio, J. P., Nilsson, T., and Warren, G . (1994). The TGN38 glycoprotein contains two non-overlapping signals that mediate localization to the trans-Golgi network. J Cell Biol 125(2), 253-68. Ritzenthaler, C. , Laporte, C. , Gaire, F., Dunoyer, P., Schmitt, C. , Duval , S., Piequet, A . , Loudes, A . M . , Rohfritsch, O., Stussi-Garaud, C. , and Pfeiffer, P. (2002). Grapevine fanleaf virus replication occurs on endoplasmic reticulum-derived membranes. J Virol 76(17), 8808-19. Rost, B . , Casadio, R., and Fariselli, P. (1996). Refining neural network predictions for helical transmembrane proteins by dynamic programming. ISMB 4, 192-200. Rott, M . E . , Gilchrist, A . , Lee, L . , and Rochon, D . (1995). Nucleotide sequence of tomato ringspot virus R N A 1 . J Gen Virol 76 ( Pt 2), 465-73. Rust, R. C. , Landmann, L . , Gosert, R., Tang, B . L . , Hong, W. , Hauri, H . P., Egger, D . , and Bienz, K . (2001). Cellular COPII proteins are involved in production of the vesicles that form the poliovirus replication complex. J Virol 75(20), 9808-18. Salonen, A . , Ahola , T., and Kaariainen, L . (2005). Vi ra l R N A replication in association with cellular membranes. Curr Top Microbiol Immunol 285, 139-173. Sanfacon, H . (1995). Nepoviruses. In "Pathogenesis and host specificity in plant diseases, V o l . III. Viruses and Viroids" (R. P. Singh, U . S. Singh, and K . Kohmoto, Eds.), pp. 129-141. Pergamon Press, Oxford. Schaad, M . C , Jensen, P. E . , and Carrington, J. C. (1997). Formation of plant R N A virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein. EmboJ16(\3), 4049-59. Schlegel, A . , Giddings, T. H . , Jr., Ladinsky, M . S., and Kirkegaard, K . (1996). Cellular origin and ultrastructure of membranes induced during poliovirus infection. J Virol 70(10), 6576-88. Schwartz, M . , Chen, J., Lee, W . M . , Janda, M . , and Ahlquist, P. (2004). Alternate, virus-induced membrane rearrangements support positive-strand R N A virus genome replication. Proc Natl Acad Sci USA 101(31), 11263-8. 102 Shai, Y . (1999). Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim Biophys Acta 1462(1-2), 55-70. Sonnhammer, E . L . , von Heijne, G . , and Krogh, A . (1998). A hidden markov model for predicting transmembrane helices in protein sequences. ISMB 6, 175-182. Suhy, D . A . , Giddings, T. H . , Jr., and Kirkegaard, K . (2000). Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles. J Virol 74(19), 8953-65. Sun, F. , Xiang, Y . , and Sanfacon, H . (2001). Homology-dependent resistance to tomato ringspot nepovirus in plants transformed with the VPg-protease coding region. Can J Plant Pathol 23, 292-299. Tarentino, A . L . , and Maley, F. (1974). Purification and properties of an endo-beta-N-acetylglucosaminidase from Streptomyces griseus. J Biol Chem 249(3), 811-7. Teterina, N . L . , Gorbalenya, A . E . , Egger, D . , Bienz, K . , and Ehrenfeld, E . (1997). Poliovirus 2C protein determinants of membrane binding and rearrangements in mammalian cells. J Virol 71(12), 8962-72. Towner, J., Ho, T., and Semler, B . (1996). Determinants of membrane association for poliovirus protein 3 A B . JBiol Chem 271(43), 26810-8. Towner, J. S., Mazanet, M . M . , and Semler, B . L . (1998). Rescue of defective poliovirus R N A replication by 3AB-containing precursor polyproteins. J Virol 72(9), 7191-200. Tusnady, G . E . , and Simon, I. (1998). Principles governing amino acid composition of integral membrane proteins: Applications to topology prediction. J Mol Biol 283, 489-506. Voinnet, O., Rivas, S., Mestre, P., and Baulcombe, D . (2003). A n enhanced transient expression system in plants based on suppression of gene silencing by the p i 9 protein of tomato bushy stunt virus. Plant J33(5), 949-56. von Heijne, G . (1992). Membrane protein structure prediction. Hydrophobicity analysis and the positive-inside rule. J Mol Biol 225, 487-494. Wang, A . , Carrier, K . , Chisholm, J., Wieczorek, A . , Huguenot, C , and Sanfacon, H . (1999). Proteolytic processing of tomato ringspot nepovirus 3C-like protease precursors: definition of the domains for the V P g , protease and putative R N A -dependent R N A polymerase. J Gen Virol 80 ( Pt 3), 799-809. 103 Wang, A . , Han, S., and Sanfacon, H . (2004). Topogenesis in membranes of the N T B - V P g protein of Tomato ringspot nepovirus: definition of the C-terminal transmembrane domain. J Gen Virol 85(Pt 2), 535-45. Wang, A . , and Sanfacon, H . (2000). Proteolytic processing at a novel cleavage site in the N-terminal region of the tomato ringspot nepovirus RNA-1-encoded polyprotein in vitro. J Gen Virol 81 (Pt 11), 2771-81. Yamaga, A . K . , and Ou, J. H . (2002). Membrane topology of the hepatitis C virus NS2 protein. JBiol Chem 277(36), 33228-34. Zhang, S. C , Ghosh, R., and Jeske, H . (2002). Subcellular targeting domains of Abutilon mosaic geminivirus movement protein B C 1 . Arch Virol 147(12), 2349-63. Zhang, S. C , Wege, C , and Jeske, H . (2001). Movement proteins (BC1 and B V 1 ) of Abutilon mosaic geminivirus are cotransported in and between cells of sink but not of source leaves as detected by green fluorescent protein tagging. Virology 290(2), 249-60. 104 C H A P T E R 3 Characterization of membrane-association domains within the Tomato ringspot nepovirus X2 protein, an endoplasmic reticulum-targeted polytopic membrane protein2 3.1 INTRODUCTION Many positive strand R N A viruses replicate in association with membranes derived from the E R (Salonen et al., 2005a; Sanfacon, 2005). Various ER-derived structures including spherules (Brome mosaic bromovirus, B M V ) and membranous vesicles or rosettes (Rott et al.) are found in infected cells (Bienz et al., 1987; Bienz et al., 1992; Carette et al., 2002a; Restrepo-Hartwig and Ahlquist, 1996; Ritzenthaler et a l , 2002; Schwartz et al., 2002). Vi ra l nonstructural proteins and R N A synthesis localize to these modified E R structures suggesting that they are the sites of viral replication. Compartmentalization of viral R N A synthesis in the viral replication complexes (VRCs) provides an environment for increased local concentration of replication components and offers protection from R N A degradation by the host. The diverse nature of membranous replication complexes suggests highly specific interactions between viral proteins and intracellular membranes. Exogenous expression of viral non-structural proteins individually or in combination has been used to investigate the role of these proteins in membrane association during V R C biogenesis. For example, the l a protein of B M V was shown to induce the formation of membranous spherules on the E R and to recruit the polymerase and viral R N A template to these structures, suggesting that it is a key organizer of the V R C s (Chen and Ahlquist, 2 A version of this chapter has been published. Zhang, G. and Sanfacon, H. (2006) Characterization of membrane association domains within the Tomato ringspot nepovirus X2 protein, an endoplasmic reticulum-targeted polytopic membrane protein. J Virol. 2006 Nov;80(21): 10847-57. 105 2000; Chen et a l , 2001). Similarly the 3 A B , 2 B C and 2C proteins of poliovirus and other picornaviruses, the 6 kDa protein of potyviruses and the 60 kDa and 32 kDa proteins of Cowpea mosaic comovirus ( C P M V ) target to the E R membranes in the absence of other viral proteins and induce modifications of intracellular membranes similar to these found in viral infection (Carette et al., 2002b; Cho et al., 1994; Datta and Dasgupta, 1994; Schaad et al., 1997). Membrane-binding domains have been identified in some viral membrane proteins and these include transmembrane hydrophobic helices, amphipathic helices and other less-well defined sequences (den Boon et al., 2001; Echeverri and Dasgupta, 1995; Teterina et al., 1997; Towner et al., 1996; Zhang et al., 2005). However, the mechanisms of membrane binding and ER-targeting are still poorly understood. Tomato ringspot nepovirus (ToRSV, a member of the family Comoviridae) has a bipartite genome (Rott et al., 1995; Rott et a l , 1991b). Each R N A is first translated into a large polyprotein which is subsequently cleaved into mature and intermediate proteins by a viral-encoded cysteine proteinase (Pro) (Chisholm et al., 2001; Hans and Sanfacon, 1995). R N A 1 encodes proteins necessary for R N A replication which include the R N A -dependent R N A polymerase, the proteinase, the genome-linked protein (Chisholm et al.) and a putative nucleoside triphosphate-binding protein (NTB) (Wang et al., 1999). In vitro processing studies have also revealed the presence of two additional protein domains(Chisholm et al.) in the N-terminal region of the RNA1-encoded polyprotein (Fig. 3.1 A ) (Wang and Sanfacon, 2000). Similar to other plant picorna-like viruses, T o R S V infection induces severe morphological alterations of E R membranes, and T o R S V V R C s are associated with ER-derived membranes (Han and Sanfacon, 2003; Stace-Smith and R, 106 1984). Several viral proteins containing the N T B domain have been detected in infected plants including the mature N T B protein, the predominant N T B - V P g polyprotein and a 90 kDa polyprotein which may correspond to the X 2 - N T B - V P g intermediate polyprotein (Han and Sanfacon, 2003). These proteins are tightly associated with E R membranes and co-fractionate with ER-associated V R C s (Han and Sanfacon, 2003). When expressed independently of other viral proteins, the N T B - V P g protein localizes to E R membranes (Zhang et al., 2005). ER-binding of the protein is mediated by two regions present in the N T B domain: a C-terminal transmembrane helix and an N-terminal amphipathic helix (Wang et al., 2004; Zhang et al., 2005). These results led us to suggest that N T B and/or a polyprotein containing the N T B domain act as membrane anchors for the replication complexes. This suggestion is in agreement with the observation that the 60 kDa protein (equivalent to N T B - V P g ) from C P M V , another member of the family Comoviridae, is an ER-targeted protein (Carette et al., 2002b). The T o R S V X 2 protein domain shares conserved amino acid motifs with the C P M V 32 kDa protein (Rott et al., 1995). Both proteins are highly hydrophobic and are situated immediately upstream of the N T B domain in the R N A 1-encoded polyprotein. The C P M V 32 kDa protein is an E R -associated protein and has been suggested to play a key role in V R C assembly (Carette et al., 2002b; Pouwels et al., 2004). In this chapter, we have investigated the membrane-association of X 2 in planta and in vitro. We show that X 2 is targeted to the E R . ER-binding is mediated by multiple domains including two C-terminal transmembrane helices and a less well defined domain 107 further upstream. In vitro glycosylation experiments confirm that X 2 is a polytopic membrane protein that traverses the membrane at least three times. 3.2 M A T E R I A L S A N D M E T H O D S 3.2.1 Plasmids construction Plasmid psmRS-GFP (S-K), psmRS-GFP (B-K) and pER-dsRed2 have been described (Zhang et a l , 2005). Plasmids psmRS-GFP(S-K) and psmRS-GFPn ( B - K ) have unique restriction sites to facilitate in frame fusions to the C- and N-termini of G F P , respectively. Plasmid p G F P - X 2 was constructed by amplifying the entire coding region of X 2 (GenBank accession number: DQ469829) using Pfu polymerase (Stratagene) and primers 3 and 4 which include an Sstl and a Kpnl site, respectively (see Table 3.1 for the sequence of all primers). The amplified c D N A fragment was digested with these enzymes and inserted into the corresponding sites of psmRS-GFP(S-K) . Plasmid p X 2 -G F P was constructed in a similar manner using primers 18 and 21 which include a BamHI and a Kpnl site, respectively. The amplified fragments were digested with these enzymes and inserted into the corresponding sites of psmRS-GFP (B-K) . Mutated derivatives of p X 2 - G F P were constructed as described for p X 2 - G F P using the following pairs of primers: 38/39 for T M 1 , 42/43 for m X 2 , 40/46 for T M 2 , 40/41 for T M 2 - 3 , 47/41 for T M 3 and 44/21 for c X 2 . A PCR-based site-directed mutagenesis method (Fisher and Pei, 1997) was used to generate deletion mutants of p X 2 - G F P using the following pairs of primers: 36/37 for A T M 1 , 28/29 for A T M 2 , and 30/31 for A T M 3 . To construct the ATM1-2-3 mutant, two rounds of site-directed mutagenesis were conducted: in the first 108 round the TM2-3 region was deleted using primer pair 28/31 and in the second round the T M 1 region was deleted using primer pair 36/37. Agroinfiltration vectors p B I N - G F P - n N and pBIN-nN-GFP which contain the SstVKpnl or BamHl/Kpnl sites have been T a b l e 3.1. P r i m e r s used i n th is s tudy fo r p l a s m i d cons t ruc t i ons NO Sequence (5' to 3') a Comments 3 4 13 18 21 28 29 30 31 36 37 38 39 40 41 42 43 44 46 47 52 53 54 55 56 57 79 80 81 82 83 84 85 86 T T A T A T G A G C T C G G T G G C G G A T C A G G T T T C G G C A A T T T T T T G A G T C G GCGCGCGGTACCTTACTGAGTTGGGGCTCGTCCACC CTAGCCATGGGTT TCGGCAATTT TTTG CGGGATCCATGGGTTTCGGCAATTTTTTG CGGGTACCCTGAGTTGGGGCTCGTC T G A A C C T C G C A G C A C G CAGTTGCTTCGCTCTTTTG C A G G C C A A A A G A G C G A A G GGTAGTATGGCTGGAATTTTTG A A G G C A T C A G A A G T C A T T G T T G A T A A G A G C A C T C T T G C C T C G A C T C A A A A A A T T G CGGGATCCATGGCTATTAATTTAGCTAGTGGTC C G G G T A C C A T T C A C A A C A T G A T T G G C A C C A G A A A C CGCG^ATCCATGGCCCTCGTTGGGGTCGGTTTAC A G C G G T A C C G G C A T A A G C C A A T A G A C A G C C T C C CGGGATCCATG A A G G C A T C A G A A G T C A T T G T T G CGGGTACC T G A A C C T C G C A G C A C G C CGCGGATCCATG GGTAGTATGGCTGGAATTTTTG A G C G G T A C C C T C C G C A A A A T A A A G A A T G C C AGCGGATCCATGCTAATTGTAGCAGGTTCTTTTA TATTACTCGAG CTGAGTTGGGGCTCGTC CACC TATTATGGCCAGTGGTTTCGGCAATTTTTTGAGTCG T A T T A C T C G A G ATGATCTGGGTATCTTG T C A A A A T T T T C T G T C A G T G C C C A T T A A C A T C A C C T G C T C T A G A T T A C G C G T A A T C T G G G A C G T C A T A T G G G T A C T G A G T T G G G G C T C G T C T C G A C T C A A A A A A T T G C TCTGGTGCCAATCATGTTG CCAGATCCGTTTTTTC C T G C T C A T C A A A A A T T C CTGAGATCCTTGTG CTTGGGAATCCATTTAC GCCCTCGTTGGGG 5' X2 +GGGS (+) 3' X2 +Kpn\ +TAA (-) 5' X2 + Ncol (+) 5' X2 +BamH\ + ATG (+) 3' X2 +Kpn\ +TAA (-) ATM2 (-) ATM2 (+) ATM3 (-) ATM 3 (+) ATM1 (+) ATM1 (-) aa 13X2+/towHI + ATG (+) aa37X2 + KpnI (-) TM2 X2 + BamHI + ATG (+) TM3 +Kpnl (-) mX2 + BamHI + ATG (+) mX2 + Kpn\ (-) cX2 + BamHI + ATG (+) TM2 + Kpn\ (-) TM3 + BamHI + ATG (+) 5' X2 + Msc\ + N-glyc.site +ATG (+) 3' X2 +Xho\ (-) 5' X2 +Msc\ +ATG (+) 3' X2-GFP Xhol (-) Mutate GFP aa 23-25 NGH to NGS (+) Mutate GFP aa 23-25 NGH to NGS (-) 3' X2+Xba\+TAA +HA (-) A aa 7-29 (-) A aa 7-29 (+) ATM4 (+) ATM4 (-) AN (-) AM (-) AM (+) a Introduced restriction sites are underlined. Insertion of an amino acid spacer (GGGS), an N -glycosylation site and a H A tag are shown in bold. b Features of the primers are briefly indicated including the presence of introduced restriction sites, start codon (ATG) or amino acid spacer (GGGS). The strand to which the primers corresponds (+ for coding sequence and - for non-coding sequence) is also indicated. Primers used for deletion of specific regions of X2 are indicated (e.g., ATM1). Primers used for deletion of specific amino acids within X2 are also indicated (e.g. Aaa 7-29). described (Zhang et al., 2005). These restriction sites were used to insert c D N A fragments containing the G F P fusions mentioned above. Plasmid p B i N 1 9 - p l 9 containing 109 the Tomato bushy stunt virus suppressor of gene silencing has also been described (Zhang et a l , 2005). To construct p B I N - X 2 - H A , the entire coding region of X 2 was amplified as above using primers 13 (containing an Ncol site) and 79 (containing an Xba\ site and the coding sequence for the H A tag). The R T - P C R fragment was digested with Ncol and Xbal and ligated into the corresponding sites of plasmid pBBI525. A Kpnl-EcoRl fragment from the resulting plasmid was then transferred into the binary vector pBIN19. To construct plasmids pT7-X2, fragments containing the X 2 coding region were amplified as above using primers 54 (containing an Mscl site) and 53 (containing an Xhol site). The amplified fragments were digested with Mscl and Xhol and introduced into the corresponding sites of plasmid pCITE-4a (Campanella et al.) (Novagen). Plasmid pT7-G l n - X 2 was constructed in a similar manner using primers 52 (containing an Mscl site and the coding region for an introduced N-glycosylation site) and 53. To construct pT7-X 2 - G l n , the PCR-based site-directed mutagenesis method was used to mutate N G H to N G S in the N-terminus of G F P in the p X 2 - G F P plasmid using primers 56/57. This resulted in the introduction of an N-glycosylation site. The resulting plasmid was then used as a template to amplify a fragment with primer pair 13/55. The amplified fragment contained the entire X 2 coding region and a small portion of the G F P coding region which includes the introduced glycosylation site. The fragments were digested with Ncol and inserted into the corresponding site of pCITE-4a (Campanella et a l ) . Other plasmids were produced by PCR-based mutagenesis. Plasmids p T 7 - X 2 A T M 2 - G l n , p T 7 - X 2 A T M 3 -Gln , p T 7 - X 2 A T M 2 - 3 - G l n , p T 7 - X 2 A T M l - G l n and p T 7 - X 2 A N - G l n were obtained using pT7-X2-Gln as a template and primer pairs 28/29, 30/31, 28/31, 80/81 and 84/86, respectively. Similarly, p T 7 - X 2 A T M 2 - 3 - G l n was used as a template to produce pT7-110 X 2 A T M 2 - 3 - 4 using primers 82/83. Plasmids p T 7 - G l n - X 2 A T M l , p T 7 - G l n - X 2 A M , pT7-G l n - X 2 A N , pT7Gln-X2 A T M 3 and pT7-Gln -X2ATM2-3 were constructed using template pT7-Gln-X2 and primer pairs 80/81, 85/86, 84/86, 30/31 and 28/31, respectively. Finally, plasmid p T 7 - X 2 A T M 3 , pT7-X2ATM2-3 and p T 7 - X 2 A T M l were constructed using plasmid pT7-X2 as a template and primer pairs 30/31, 28/31 and 80/81, respectively. 3.2.2 Agroinfiltration ofN. benthamiana plants and confocal microscopy Binary vectors containing the plant expression cassettes with the X 2 fusion proteins were transformed into Agrobacterium tumefaciens L B A 4 0 4 4 (Invitrogen) by electroporation. The transformed bacteria were then used for agroinfiltration as previously described (Zhang et al., 2005). Three days after agroinfiltration, G F P and dsRed2 fluorescence was analyzed with a confocal microscope (Leica) as described (Zhang et al., 2005). The acquired images were processed with Leica confocal software and Photoshop 7.0 (Adobe). 3.2.3 Subcellular fractionation and membrane flotation assays Three to 4 days post-agroinfiltration, plant tissues were extracted and fractionated into post-nuclear soluble (S30), and membrane-enriched (P30) fractions as previously described (Han and Sanfacon, 2003; Schaad et al., 1997). The P30 fraction was resuspended in a volume of homogenization buffer equivalent to that of the S30 fraction or treated with an equal volume of 1 M N a C l or 0.1 M Na2CC>3 (pH 11). Membrane flotation assays were conducted essentially as described (Zhang et al., 2005). Briefly, 800 111 ul of S3 or P30 fraction was adjusted to a final volume of 1.9 ml of 71.5 % sucrose (wt/vol) in N T E buffer and overlaid with 7 ml of 65 % sucrose in N T E and 3.1 ml of 10 % sucrose in N T E . After centrifugation at 100,000 x g for 18 h, 12 1-ml fractions were collected from the bottom of the tube. Separation of proteins by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis ( P A G E ) and immunodetection were conducted as previously described (Han and Sanfacon, 2003) using a mouse monoclonal anti-GFP antibody (BD Biosciences), a rat anti-HA antibody (Roche) or a rabbit polyclonal anti-Bip antibody (donated by M . Chrispeels). The secondary antibodies were goat anti-mouse, goat anti-rat or goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase (Bio/Can). 3.2.4 In vitro translation assays and deglycosylation assays Coupled in vitro transcription-translation reactions in the presence or absence of canine microsomal membranes and deglycosylation assays of translation products were conducted as previously described (Wang et al., 2004). 3.2.5 Computer-assisted multiple sequence alignements and prediction of putative transmembrane helices and amphipathic helices Transmembrane helices in nepovirus and comovirus proteins were predicted using the following programs: PHDhtm (Rost et al., 1996), Sosui (Hirokawa et al., 1998), Tmpred (Hofmann and Stoffel, 1993), T M A P (Lundin et al., 2003), T M H M M (Sonnhammer et al., 1998), and H M M T O P (Tusnady and Simon, 1998). Prediction and projection of 112 amphipathic helices were conducted using the Antheprot program (Deleage et al., 2001). Multiple protein sequences were aligned using the ClustalW program (Chenna et al., 2003). 3.3 RESULTS 3.3.1 Computer-assisted prediction of hydrophobic regions within X2 Computer analysis of the amino acid sequence of the T o R S V X 2 protein revealed the presence of four hydrophobic regions ( T M 1 , T M 2 , T M 3 and T M 4 ) as shown in the hydrophilicity profile (Fig. 3.1 A ) . Two of these regions had very high hydrophobicity values (TM2 and T M 3 ) and were predicted to be transmembrane helices by all programs, although the exact borders of the predicted transmembrane helices differed among programs (Fig. 3.IB). Two other possible transmembrane segments (TM1 and T M 4 ) were less hydrophobic and were each predicted by only one of the six programs considered. The equivalent protein domain of four distinct nepoviruses and the C P M V 3 2 kDa protein were also examined for the presence of putative transmembrane helices (Fig. 3.2). A previously identified conserved sequence motif (F -X28 -W -X11 -L-X23 -E) (Rott et al., 1995) was present in all sequences. The sequences aligned to the T o R S V T M 2 and T M 3 regions were also predicted to be trans-membrane helices for all the proteins analyzed. In the case of the comovirus 32 kDa protein, a third possible trans-membrane helix was also identified by the majority of the programs at a position corresponding to that of the weakly predicted T o R S V T M 4 domain. 113 X1 X2 NTB VPg Pro . Pol .1 I i r I I TM1 TM2 TM3 TM4 * 0 20 40 60 80 100 120 140 160 180 3 Amino acid position B GFGNFLSRGKSAAIHLASGLSSFVGEKWSGANHWNKASEV" D l f n h f m HMMTOP SOSUI TMpred r g k s a a i n l a s g l s s f v g e -IVDKLLREHFDDTIGKWIPKLLGATQKIEELWRWSLEWAQNM1" PHDhtm HMMTOP SOSUI TMpred TMAP TMHMM SKKLDVSLRVLRGSALVGVGLLLVSGILYFAEQLLRSFGLLI' 2' PHDhtm ALVGVGLLLVSGILYFAEQ LLI HMMTOP GSALVGVGLLLVSGILY FA LLI SOSUI LRGS ALVGVGLLLVSGILYFAEQ LLI TMpred LRGSALVGVGLLLVSGILYFA FGLLI TMAP VLRGSALVGVGLLLVSGILYF GLLI TMHMM LRVLRGSALVGVGLLLVSGILYF LLI VAGS FISMFVGGCLLAYAGSMAGIFDEQMMRVRGILCEIPML1GB PHDhtm VAGS FI SMFVGGCLLAYA mmrvrgilceipml HMMTOP VAGS FI SMFVGGCLLAYAGSMA SOSUI VAGSFISMFVGGCLLAYAGS TMpred VAGSFISMFVGGCLLAYAG TMAP VAGS FISMFVGGCLLAY TMHMM VAGSFISMFVGGCLLAYAGS LYLKAQPDPFFPKKSGGRAPTQ 1 5 0 PHDhtm l y l HMMTOP SOSUI TMpred TMAP TMHMM Fig. 3.1. Computer-assisted prediction of transmembrane helices (TM) in the ToRSV X2 protein. (A) Schematic representation of putative transmembrane helices within the X2 domain. The RNA1-encoded polyprotein is shown at the top of the figure with the indicated individual protein domains. Vertical lines represent the cleavage sites recognized by the ToRSV proteinase. The X2 protein domain is shown below with strongly and weakly predicted transmembrane helices represented by black and grey squares, respectively. The hydrophobicity plot of X2 is shown at the bottom. Hydrophobicity was calculated using the algorithm of Kyte and Doolittle with a window size of 17 a.a. (Kyte and Doolittle, 1982). (B) Prediction of transmembrane helices within X2. The entire deduced amino acid sequence of X2 (GenBank accession number: DQ469829) is shown at the top of the figure. Amino acids are numbered from the first amino acid of the X2 protein domain according to the previously proposed XI-X2 cleavage site (Wang and Sanfacon, 2000). Predicted transmembrane domains are shown for each program (as indicated on the left of the figure). Upper case letters indicate a very high prediction score while lower case letters indicated a lower prediction score. The amino acid sequence deleted in the TM1, TM2, TM3 and TM4 deletion mutants is underlined in the X2 sequence. A naturally occurring putative N-glycosylation site (NMS) is shown by the arrow. 114 A X1 X2 NTB VPg Pro Pol ToRSV I I • ! II I 1 BRSV I I • ! II I I GFLV | | • i II 1 I CPMV I * l II I I 32 kDa 66 kDa B ToRSV LREJ- E DDTIGKWIPKLLGATQKIEELWRWSLE K ?\QNMSKKL88 BRV LKSF E FEFLKPYIQHAIYASAEIEKYWAFIHG RATKMWNNV BRSV MINE E RECIKMIHKELGCAMELIEVMIKKVKE V, YNSMLEKL ArMV CQK\E NATMAPYLS HLAEASN11S KIWKKLAEK MESLKGKA GFLV C K R 1 E DVTMAPYLQHLASAHSILKKIWEKLSE Vi MESLKSKA91 CPMV FRKE EEKMVQEYVPMAHRVCSWLSQLWDKIVC RISQASETM 2 3 0 ToRSV BRV BRSV ArMV GFLV CPMV DVS|r4RVLRGSALVGVGLLLVSGILYF?4flQLLRSFGLLIVAG GVETOALGDAAWWAIGITMVCGIVTLVHKLLVYLGALNAGG HCG L ^ TLGTYAMYALAILLGCGLTTLIE RCIGGAGILTKLF GL?L EVLAQHAIFALGAIVVGGVVVLVE KVLVACKVIPNCG SLAP4EVMRQHAI FALGAMVIGGWVLv E KVLIAAKII PNCG 1 3 2 GWFttJDGCRDLMTWGIATLATCSALSLM E 1 K L L V A M G F L V E P F 2 ToRSV SFISMFVGGCLLAYAGSMAGIFDEQMMRVRGILCEIPMLLY170 BRV ILCSLMLTGLLGAAGLLATGKFAEASSTLVGAMRSLIFTLF BRSV VT GV FAAIGLHAAGGFDGLQREMVQMCTALAAGIFDVHHKG ArMV IVLGAFLTLFFASLGLTALECTAEEIFRMHQCCKGAIYSMY GFLV IILGAFLTLFFASLGLTALECTAEEIFRMHACCKSAIYSMY 1 7 3 CPMV GLSGIFLRTGWAAACYNYGTNSKGFAEMMALLSLAANCVS312 Fig. 3. 2. Multiple protein sequence comparison of the X2 protein domains of nepoviruses and the 32 kDa protein of comoviruses (A) Schematic representation of the RNA 1-encoded polyprotein of nepoviruses of subgroup C (ToRSV), B (BRSV, Beet ringspot nepovirus) and A (GFLV, Grapevine fanleaf nepovirus) and of a comovirus (CPMV). Vertical lines represent cleavage sites identified by in vitro processing experiments (Hemmer et al., 1995; Margis et al., 1994). In the case of BRSV and GFLV, two hypothetical cleavage sites are shown by the dashed lines. The star represents the highly conserved region shown in B. (B) Multiple protein sequence alignment of the conserved region present within the X2 protein domain of five nepoviruses belonging to subgroup C (ToRSV and Blackcurrant reversion nepovirus, BRV), subgroup B (BRSV) and subgroup A (GFLV and Arabis mosaic nepovirus, ArMV) and within the CPMV 32 kDa protein. The following accession numbers were used to retrieve the sequences from the database: NC003509 for BRV, NC003693 for BRSVNC003615 for GFLV, NC006057 for ArMV and P03600 for CPMV). The conserved amino acids present in the "protease co-factor conserved motif (F-X28-W-X11-L-X23-E) are boxed (Rott et al., 1995). Dots above the sequence represent amino acids which are similar in all the sequences. Underlined sequences represent the core regions of trans-membrane helices predicted as shown in Fig. 3.IB. Only trans-membrane helices predicted by the majority of the programs are shown. Numbering of amino acids is shown for ToRSV and GFLV according to the proposed X1-X2 cleavage site (Margis et al., 1994; Wang and Sanfacon, 2000) and for CPMV according to the start codon. For other sequences, the XI-X2 cleavage site has not been identified and amino acids within the sequence were left unnumbered. 1 1 5 3.3.2 Subcellular localization of GFP-tagged X 2 proteins To determine whether X 2 is able to associate with intracellular membranes in planta, G F P was fused in frame to either the N - or the C-termini of X 2 (Fig.3.4A). The fusion proteins were expressed in N. benthamiana plants using agroinfiltration. G F P fluorescence in transfected epidermal cells was analyzed using a confocal laser scanning microscope three days after agroinfiltration. A previously described E R marker (ER-dsRed2 in which the red fluorescent protein is targeted to the lumen of the E R ) (Zhang et a l , 2005), was co-expressed with the G F P fusion proteins. Confocal images show that ER-dsRed2 labelled both the perinuclear and the cortical E R network, a result consistent with our previous observations (Fig 3.3, dsRed2). The fluorescence associated with free G F P was present in the cytosol (cytoplasm is usually pressed against the plasma membrane due to the presence of large vacuoles in mature epidermal cells that occupy most of the intracellular space) and inside the nucleus (Fig. 3.3, panel 5). The green fluorescence was sometimes found in proximity to the E R cortical network but did not coincide with the E R marker (Fig. 3.3, panel 6). In contrast, the fluorescence associated with G F P - X 2 and X 2 - G F P overlapped with the E R marker in both the perinuclear area and the cortical E R network (Fig. 3.3, panels 1-4). Similar fluorescence patterns wereobserved when X 2 - G F P and G F P - X 2 were transfected in cultured tobacco B Y - 2 cells by biolistic bombardment (data not shown). These results suggest that X 2 is targeted to E R membranes, at least in the context of the G F P fusions. Subcellular fractionations were performed to further study the membrane association of the fusion proteins. A s above, X 2 - G F P and G F P - X 2 were expressed in N. benthamiana using agroinfiltration. Three days after agroinfiltration, the leaves were extracted and soluble 116 1_ j""** ' - n 1 ... > ""Vr *n ^Jri 3 • . . . . ' U /• \ 4 * / K HI M A pa (S30) and membrane-enriched (P30) fractions were produced as described in Materials and Methods. Proteins were separated by S D S - P A G E and analyzed by immunoblotting using anti-GFP antibodies. In agreement with the FIG. 3.3. Subcellular localization of GFP-X2 and X2-GFP. GFP fusions and ER-dsRed2 (an ER marker) were expressed in leaves of N. benthamiana by using agroinfiltration as described in Materials and Methods. Epidermal cells were examined 3 days after agroinfiltration by confocal microscopy. In the merge panel, the colocalization of the GFP fluorescence (green) and of the ER marker fluorescence (red) results in a yellow color. Panels 2, 4, and 6 are close-up views of regions included in the white squares in panels 1, 3, and 5. Bars on the merged images represent 10 urn. confocal images, unfused G F P was detected mainly in the S30 fraction while the full-length G F P - X 2 and X 2 - G F P (49 kDa) were detected only in the membrane-enriched P30 fractions (Fig. 3.4B, lanes 3-8). Larger forms of the proteins (about 100 kDa) were also detected in P30 fractions of X 2 - G F P and G F P - X 2 , which may correspond to dimers as many membrane proteins can maintain their oligomeric forms in the presence of SDS 117 A GFP (27 kDa) V//////////////M GFP-X2 (49 kDa) I X2-GFP (49 kDa) i v///////mmm X2-HA (23 kDa) I I B ck GFP X2-GFP GFP-X2 ck X2-HA kDa 1 2 3 4 5 6 7 8 9 10 11 12 10 11 12 X2-GFP Extr. Buffer 1M NaCl Fig. 3.4. Subcellular fractionation of X2 fusion proteins. (A) Schematic representation of X2 fusion proteins. The open box represents the X2 domain, while the hatched and black boxes represent the GFP and H A domains, respectively. The predicted molecular mass of each fusion protein is indicated in parentheses. (B). Subcellular fractionation of X2 fusion proteins. Plant tissues expressing the various fusion proteins were fractionated into a soluble (S) and membrane-enriched (P) fraction as described in Material and Methods. Proteins were separated by SDS-PAGE (12% for lanes 1-8 and 15% for lanes 9-12) and detected by immunoblotting using anti-GFP (lanes 1-8) or anti-HA (lanes 9-12) monoclonal antibodies. Migration of molecular mass standards is indicated on the left (lanes 1-8) or right (lanes 9-12) sides, ck: negative control transfected with pBin-pl9 only. (C) Membrane flotation assays. Equal volume of post-nuclear (S3) fractions derived from plants expressing GFP-X2, X2-GFP, X2-HA or unfused GFP were used for membrane flotation assays as described in Material and Methods. Fractions were collected from the step sucrose gradient and proteins present in each collected fraction (as indicated at the top of the figure) were separated by SDS-PAGE and immunodetected using anti-GFP, anti-HA or anti-Bip antibody. Only the relevant part of the gel is shown. In the case of X2-GFP, P30 fractions of X2-GFP were incubated for 30 min at 4°C in extraction buffer (Extr. Buffer), 1 M NaCl or 0.1 M N a 2 C 0 3 (pH 11) before the flotation assay. 118 (DeGrado et al., 2003). This possibility was not investigated further. The presence of the G F P fusion proteins in P30 fractions may result from true membrane association or simply protein aggregation. To distinguish between these two possibilities, we used a membrane flotation assay. In this assay, total plants extracts are overlaid with a sucrose gradient and subjected to centrifugation. Low-density membranes and proteins associated with these membranes float to the upper part of the gradient while soluble proteins or aggregated proteins remain at the bottom. We used Bip (an endogenous E R luminal protein) (Staehelin, 1997) and unfused G F P as controls. A s shown in Fig. 3.4C, B i p rose towards the top of the gradient (fractions 8 and 9) while G F P remained at the bottom of the gradient (fractions 1 and 2). G F P - X 2 and X 2 - G F P were found in fractions 8 and 9 confirming that they are membrane associated. To investigate the nature of the association of X 2 - G F P with membranes, we treated the P30 fractions containing X 2 - G F P with N a 2 C 0 3 (0.1 M , p H 11) and N a C l (1 M ) and then conducted membrane flotation assays. These chemicals are known to release peripheral membrane proteins from the membranes but not integral membrane proteins (Howell and Palade, 1982; Sankaram and Marsh, 1993). X 2 - G F P was found in the membrane fraction after both treatments suggesting that it interacts directly with the lipid bilayer of the membrane (Fig. 3.4C). To provide further evidence that X 2 is a membrane-associated protein, we also fused the entire protein to a smaller epitope tag ( X 2 - H A , in which the H A epitope tag is fused to C-119 terminus of X 2 , F ig 3.4A). X 2 - H A was mainly detected in P30 fraction (Fig. 3.4B, lanes 11 and 12) and floated to the top of the gradient in membrane flotation assay (Fig. 3.4C). 3.3.3 X2 contains multiple ER-target ing domains The tight association of X 2 G F P fusion proteins to E R membranes prompted us to investigate sequence elements within X 2 mediating the membrane association. We first generated several mutants of X 2 - G F P in which the hydrophobic segments T M 1 , T M 2 and T M 3 were deleted either individually or in combination (Fig. 3.5A, constructs A T M 1 , A T M 2 , A T M 3 and A T M 1-2-3). We found that all four X 2 derivatives retained the ability to associate with the E R , i.e., they had similar patterns of fluorescence compared to the wild-type X 2 - G F P in confocal images (compare Fig . 3.3 and Fig . 3.6). They also partitioned to membrane-enriched fractions in subcellular fractionation experiments (Fig. 3.5B). We then fused different portions of X 2 (Fig. 3.5A, constructs T M 1 , T M 2 - 3 , T M 2 , T M 3 , c X 2 and mX2) to the N-terminus of G F P and tested whether any given fragment could target G F P to the E R . The fluorescence associated with the T M 1 and c X 2 fusion proteins did not overlap with that of ER-dsRed2 (Fig. 3.6). The proteins were detected both in the S30 and P30 fractions (Fig. 3.5C). However, the presence of these proteins in the P30 fraction was probably due to protein aggregation rather than to membrane association as the proteins remained at the bottom of the gradient in the membrane flotation assays (Fig. 3.5D). The highly hydrophobic T M 2 and T M 3 domains targeted the G F P to the E R membrane when fused to G F P individually (TM2 and TM3) or in combination (TM2-3, F ig . 3.6). Targeting to the E R was partial when only one of the hydrophobic regions was included (as evidenced by the presence of some fluorescence 120 A M r X 2 a a X 2 - G F P ammm^^^zzzwttL^^Ki^^^ 49 k D a 1-190 A T M 1 • J T ^ M — i 4 6 k D a 1-190 ( A 1 3 - 3 7 ) A T M 2 c l | mmm | 4 6 5 k D a 1-190 ( A 9 9 - 1 1 7 ) A T M 3 c j | 4 6 6 k D a 1-190 ( A 1 2 5 - 1 4 4 ) A T M 1 - 2 - 3 • 1 - l 1 own l 4 0 k D a 1-190 ( A 1 3 - 3 7 + A 9 9 - 1 4 4 ) T M 1 mmm 3 0 k D a 1 3 - 3 7 m X 2 I I 3 5 . 8 k D a 3 8 - 9 8 T M 2 mm 2 9 . 5 k D a 9 9 - 1 1 7 T M 3 mmt- 2 9 6 k D a 1 2 5 - 1 4 4 T M 2 - 3 mmnmmt- 3 3 k D a 9 9 - 1 4 4 c X 2 i mmm i 3 2 . 8 k D a 1 4 5 - 1 9 0 Fig. 3.5. Subcellular fractionation of X 2 - G F P mutants derivatives. (A) Schematic representation of X2-GFP derivatives. Only the X2 domains are shown in the figure. The GFP domain (not shown) is fused to the C-terminus of each X2 derivative. The predicted transmembrane domains are shown with grey and black boxes as in Fig. 3.1. The predicted molecular mass of each GFP fusion protein and the amino acids of X2 included in each fusion proteins are shown on the right. (B) and (C). Subcellular fractionation of X2-GFP derivatives. Soluble (S) and membrane-enriched (P) fractions were prepared from plants expressing mutated X2-GFP proteins as described in Material and Methods. Proteins were separated by SDS-PAGE (12%) and immunodetected with anti-GFP antibody. Migration of molecular mass standards is shown on the right of each gel. (D) Membrane flotation assays. For TM1, mX2 and cX2, post-nuclear (S3) fractions were used for the flotation assays. In the case of TM2, TM3 and TM2-3, P30 fractions were used. Fractions were collected from the step sucrose gradient and proteins present in each collected fractions were separated by SDS-PAGE (12 %) and immunodetected with the anti-GFP antibody. (E) Biochemical treatments of membrane-enriched fractions derived from plants expressing mX2. Membrane-enriched (P30) fractions from Fig. 4C were treated with 0.1 M N a 2 C 0 3 (pH 11) or 1 M NaCl for 30 min at 4°C. After separation of membrane-bound (P) and soluble (S) proteins, the presence of mX2 in these fractions was revealed by immunoblotting with the anti-GFP antibody. 121 within the nucleus with T M 2 and T M 3 , Fig. 3.6 and data not shown). The T M 2 and T M 3 proteins partitioned in both the S30 and P30 fractions (Fig. 3.5C). The full-length 33 kDa TM2-3 fusion protein was found predominantly in the P30 fraction. A 30 kDa truncated protein which may correspond to degradation products of the full-length protein was also detected in the S30 fraction. The T M 2 , T M 3 and the full-length TM2-3 fusion proteins present in the P30 fraction floated to the top of the gradient in membrane flotation assays confirming that they are membrane-associated (Fig. 3.5D). Surprisingly, although no hydrophobic sequence was predicted in this Fig. 3.6. Subcellular localization of X2-GFP derivatives in epidermal cells of A7, benthamiana. Plants expressing X2-GFP derivatives and an ER marker (ER-dsRed2) were examined using confocal microscopy 3 days after agroinfiltration. Pictures represent portions of a single cell including the nucleus (shown by the arrow) and the cortical ER network GFP DiKni: Mcrjt region, m X 2 was found to associate with the E R in confocal pictures, fractionated with 122 the membrane-enriched P30 fraction and partitioned with the membranes in the flotation assays (Fig. 3.6, F ig . 3.5C and 3.5D). We treated the P30 fractions of m X 2 with N a 2 C 0 3 (0.1 M , p H 11) and N a C l (1 M ) which were separated subsequently into S30 and P30 fractions. We found that m X 2 was present in the P30 fraction after the treatment, suggesting a direct interaction between m X 2 and the l ipid bilayer of the membranes (Fig. 3.5E). Taken together, these results suggested that X 2 contains three ER-targeting domains including two highly hydrophobic C-terminal regions and an additional domain further upstream. 3.3.4 T o p o l o g y o f X 2 in ER membranes inferred from the pattern of glycosylation in vitro Our result that X 2 contains three ER-targeting domains suggests that it is a polytopic membrane protein. To investigate the topology of X 2 in the E R membrane, we chose to examine the glycosylation patterns of X 2 derivatives, into which N-glycosylation sites were introduced at different locations. Because the active site of the glycosyltransferase is situated on the luminal side of E R membranes, glycosylation of introduced N -glycosylation sites could only occur i f they were translocated into the E R lumen. To test the glycosylation status of a protein, in vitro translations can be conducted in the presence or absence of microsomal membranes, which consist predominantly of E R membranes (Fig. 3.7). If glycosylation occurs, an additional slower migrating band (about 3 kDa larger) should be detected in S D S - P A G E when translated in the presence of membranes. Glycosylation can be further confirmed by treatment of the reaction mixture with PNGase F, resulting in the disappearance of this additional protein. The wild-type X 2 protein contains a possible N-glycosylation site ( N M S , Fig . 3.1). However, this sequence was not 123 recognized in vitro (Fig. 3.7, construct X2) . We then introduced an N-glycosylation site either at the N-terminus (Gln-X2) or at the C-terminus of the protein (X2-Gln) (Fig. 3.7). G l n - X 2 was glycosylated indicating that the N-terminus of the protein is translocated into the E R lumen. In contrast, X 2 - G l n was not glycosylated. The observed translocation of the N-terminus of the protein in the E R lumen confirms that X 2 is a transmembrane protein. M M PNGase F W T X 2 Gln-X2 X2-Gln X2ATM3-Gln X2ATM3 Gln-X2ATM3 X2ATM2-Gln X2ATM2-3-Gln X2ATM2-3 t X2ATM2-3-4-Gln t Gln-X2ATM2-3 % Gln-X2AN X2AN-Gln X2ATM1 X2ATM1-Gln Gln-X2ATM1 Gln-X2AM % =2-X2 a.a. 1-190 1-190 1-190 1-190 (A125-144) 1-190 (A125-144) 1-190 (A125-144) 1-190 (A99-117) 1-190 (A99-144) 1-190 (A99-144) 1-190 (A99-144 + A154-171) 1-190 (A99-144) 99-190 1-190 (A9-99) 1-190 (A13-37) 1-190 (A13-37) 1-190 (A13-37) 1-190 (A63-98) N T . N T . Fig. 3.7. In vitro glycosylation assays of wild-type or mutated X2. On the left, is a schematic representation of the various X2 constructs. The predicted transmembrane domains are shown with grey and black boxes as in Fig. 3.1. Amino acids inserted at N - or C-termini of the protein are shown with dark lines. Introduced N-glycosylation signals are represented by the solid letter Y. A naturally occurring putative N-glycosylation site is shown by the open letter Y, although this site was not recognized in any of the mutants tested. The dashed lines represent deleted regions within X2. The name of each construct is indicated on the left and the amino acids of the X2 domain contained in each construct are indicated in the middle. In vitro glycosylation assays are shown on the right side of the figure. Each protein was translated in the presence (+) or absence (-) of canine microsomal membranes (MM). The translation products were further treated with endoglycosydase F (PNGase F), separated by SDS-PAGE and detected by autoradiography. Only the relevant portion of the gel is shown. N. T. not tested. A s mentioned above, computer predictions strongly suggest that T M 2 and T M 3 traverse the membrane and form a hairpin structure. Lack of glycosylation of X 2 - G l n suggests 124 that i f T M 2 and T M 3 form a hairpin structure in the membrane, the loop is in the E R lumen. To confirm this orientation, we deleted T M 2 and T M 3 individually or in combination. We hypothesized that deleting the second transmembrane domain of the hairpin ( X 2 A T M 3 - G l n mutant) would result in the translocation of the C-terminus of the protein in the lumen of the membranes. A s expected, glycosylation of this mutant readily occurred in the presence of the membranes. This glycosylation was not due to the recognition of the internal N M S sequence, as the control X 2 A T M 3 mutant remained unglycosylated. Introduction of this mutation in the G l n - X 2 protein did not alter its state of glycosylation, suggesting that deletion of T M 3 did not affect the orientation of the N -terminal region of the protein (compare mutants G l n - X 2 A T M 3 to Gln-X2) . Similarly, deletion of T M 2 from the X 2 - G l n protein resulted in the reorientation of the C-termini of the protein in the lumen ( X 2 A T M 2 - G l n mutant). These results provide support for the suggestion that T M 2 and T M 3 form a hairpin in the membrane. To confirm this, we deleted both domains from the X 2 - G l n protein (X2ATM2-3-Gln) . Unexpectedly, glycosylation was still observed although it was much reduced compared to the X 2 A T M 3 - G l n mutant. A s above, this glycosylation was not due to the recognition of the internal N M S sequence as the X 2 A T M 2 - 3 mutant was not glycosylated. To determine whether the putative T M 4 domain played a role in the translocation of the C-terminus of the protein in the membrane lumen, we constructed a triple mutant in which T M 2 , T M 3 and T M 4 were deleted. L o w levels of glycosylation were still observed in this new mutant, suggesting that T M 4 was not a primary determinant of the membrane topology. We conclude that an additional domain upstream of T M 2 is likely responsible for the low level of glycosylation observed in the X 2 A T M 2 - 3 - G l n and X 2 A T M 2 - 3 - 4 - G l n proteins. 125 To investigate which region of X 2 is responsible for the translocation of the N-terminus of the protein in the lumen, we introduced a series of mutation in the G l n - X 2 protein. First, we deleted both T M 2 and T M 3 (Gln-X2ATM2-3) . Glycosylation of this mutant was still observed suggesting that a region of X 2 present between the N-terminus of the protein and the T M 2 domain acts as a transmembrane domain (Fig. 3.7). We then deleted the entire N-terminal region of X 2 (Gln-X2AN) . Translocation of the N-terminus of the protein was eliminated confirming the presence of a trans-membrane segment in this region. This result is consistent with the in planta observation that an ER-targeting domain is present in the m X 2 - G F P fusion protein. T M 1 is the only hydrophobic region predicted by computer. However, deletion of T M 1 in the context of G l n - X 2 (Gln-X 2 A T M 1 ) did not prevent the glycosylation. The observed glycosylation was due to the recognition of the introduced N-terminal glycosylation site as the control X 2 A T M 1 mutant remained unglycosylated. A stretch of 35 amino acids located immediately upstream of the T M 2 domain was also deleted (mutant G l n - X 2 A M ) . Glycosylation of this mutant was still observed. Based on these results, we tentatively suggest that a region confined within amino acids 38 to 62 may be involved in the membrane association and in the translocation of the N-terminus of X 2 in the membrane lumen. Finally, the A N and A T M 1 were also introduced in the X 2 - G l n protein. The X 2 A N - G l n and X 2 A T M 1 - G l n mutants remained unglycosylated, suggesting that deletion of the N-terminal region of the protein did not affect the orientation of its C-terminus. 3.4 D I S C U S S I O N 126 In this study, we use G F P fusion proteins to show that the T o R S V X 2 protein contain several ER-targeting sequences. We acknowledge that our experimental system differs from a natural viral infection in several important aspects. First, the protein was translated from an m R N A rather than produced through polyprotein processing. A s a result, a methionine was inserted at the N-terminus of the protein. Second, fusion of X 2 to G F P may affect the biological function of X 2 and/or its intracellular localization in planta. However, it should be noted that both N - and C-terminal fusions of the protein to G F P resulted in similar fluorescence patterns. Also , several independent ER-targeting elements were identified within X 2 using in vivo G F P fusion assays and the presence of these membrane-association domains was confirmed by in vitro glycosylation assays. Finally, the membrane-association of X 2 in vivo was confirmed using a smaller epitope tag (HA) . We have previously shown that ER-derived membranes play a key role in T o R S V replication (Han and Sanfacon, 2003). X 2 shares many sequence similarities with the C-terminal region of the 32 kDa protein of C P M V , which is also targeted to the E R when fused to G F P (Carette et a l , 2002b). In infected plants, the C P M V 32 kDa protein is found at or near ER-derived membrane vesicles which contain V R C s (Pouwels et al., 2004). The C P M V 32 kDa protein has been suggested to act as a second membrane-anchor for the replication complex in addition to the 60 kDa protein (Carette et al., 2002b). B y analogy it is tempting to suggest that the T o R S V X 2 protein is also associated with ER-bound V R C s in infected plants although we are unable to confirm this suggestion at this time, due to difficulties encountered in producing antibodies against this very hydrophobic protein. 127 Although the T o R S V X 2 protein and the C P M V 32 kDa protein both target G F P to E R membranes, the fluorescence pattern of these proteins is somewhat different. Fluorescence associated with the T o R S V X 2 - G F P fusion protein is evenly distributed in the cortical E R network and in the perinuclear E R . N o obvious membrane proliferation or alteration of membrane morphology was observed. In contrast, the C P M V 32 kDa protein-GFP fusion is specifically targeted to the cortical E R (Carette et al., 2002b). It also induces aggregation of cortical E R and formation of small bodies near the nucleus. One possibility is that the different behaviors of the two fusion proteins are due to intrinsic properties of the two proteins, possibly modulated by divergent sequences outside of the conserved motif. Alternatively, the differences observed could be due to the experimental system used. In this study, the fusion proteins were expressed by agroinfiltration, while in the C P M V study, the fusion proteins were expressed from a viral vector. In this study we have identified three distinct membrane-association domains within X 2 : two C-terminal trans-membrane helices (TM2 and T M 3 ) and a third less-well defined domain within the m X 2 region. Each of the three elements could direct G F P to the E R membranes independently (Fig. 3.6). This observation is further supported by our in vitro glycosylation study which suggests that all three domains have the ability to traverse the membranes (Fig. 3.7). Based on these results, we propose a model for the topology of X 2 in the membrane (Fig. 3.8A). In this model, the N-terminus of X 2 is oriented in the lumen while the C-terminus is cytosolic. The protein traverses the membrane three times. The highly hydrophobic T M 2 and T M 3 regions were strongly predicted to form a hairpin 128 in the membrane not only in the T o R S V X 2 protein domain but also in the equivalent protein domains of other nepoviruses and in the C P M V 32 kDa protein (Fig. 3.1 and 3.2). Our in vitro results suggest that the hairpin loop resides in the lumen of the membrane. This proposed topology is supported by the following evidence. First, the C-terminus of A ( K ) C h a r g e d © H y d r o p h o b i c Fig. 3.8. Topological model of X2 in ER membranes. (A) Proposed topological model of X2. On the top of the figure is the linear representation of membrane-association domains of X2. The light grey regions represent hydrophobic domains (TM1 and TM4 as in Fig 3.1) that do not traverse the membranes. Transmembrane a-helices TM2 and TM3 are shown by the black boxes as in Fig. 1, The star represents a putative amphipathic helix. Below is the topological model of X2 in ER membranes. The double-lipid layer of the membranes is represented by the two horizontal shaded lines. The predicted orientation of the various transmembrane domains within the membrane is shown. (B) Helical wheel projection of a putative amphipathic helix located between amino acids 46 and 63. the wild-type protein is exposed to the cytosolic face of the membrane. Second, deletion of T M 3 or T M 2 reversed the orientation of C-terminus of X 2 from cytosol to lumen. 129 Third, deletion of T M 2 and T M 3 resulted in a reduction of the translocation of the C -terminus of the protein in the lumen although it did not completely eliminate it (see below for a possible interpretation of this result). Finally, the N-terminus of the Gln -X 2 A N protein which includes the T M 2 and T M 3 domains but not the upstream membrane-association domain is oriented towards the cytosolic face of the membrane The observation that the central region of X 2 (mX2) contains ER-targeting sequences and probably traverses the membrane at least in vitro is surprising as this region is largely hydrophilic and is not predicted to contain transmembrane domains (Fig. 3.1). A putative amphipathic helix is present between amino acid 46 and 63 and may be responsible for the translocation of the N-terminus of the protein in the lumen (Fig. 3.8B). Similar putative amphipathic helices were also found at equivalent positions in the X 2 protein domain of other nepoviruses and in the C P M V 32 kDa protein (data not shown). Amphipathic helices initially orient parallel to the membrane with their hydrophobic face towards the membrane and their hydrophilic face towards the cytosol. Translocation of amphipathic helices across membranes usually involves the oligomerization of the amphipathic helix at the membrane surface followed by insertion of the oligomers into the membrane in a post-translational manner through the barrel stave mechanism (Bechinger, 1999; Shai, 1999). The X 2 putative amphipathic helix may insert in the membrane in either orientation, providing a possible explanation for our observation that both G l n - X 2 A T M 2 - 3 and X 2 A T M 2 - 3 - G l n are glycosylated. In fact, dual orientation of trans-membrane segments has been documented (Mottola et al., 2002). In the context of -the wild-type X 2 protein, the presence of the T M 2 and T M 3 domains may force the 130 putative amphipathic helix to adopt a type I topology (in-out, F ig . 3.8A). A similar situation was reported for the human band 3 protein, in which a downstream transmembrane domain dictated the orientation of upstream transmembrane segments (Ota et al., 1998). Further experimentation wi l l be required to confirm the role of the proposed amphipathic helix in membrane association. In this study, the topology of the mature X 2 protein was analyzed. However, the protein is initially produced as a polyprotein in which X 2 is located immediately upstream of the N T B domain. Also , intermediate polyproteins containing both the X 2 and N T B domains are likely to be present in infected cells. In fact, in addition to the N T B and N T B - V P g proteins, a 90 kDa membrane-associated protein containing the N T B domain was previously detected in infected plants which may correspond to the X 2 - N T B - V P g polyprotein (Han and Sanfacon, 2003). Previous analysis of the topology of N T B - V P g in E R membranes using in vitro glycosylation assays revealed that the N-terminus of N T B is translocated into the E R lumen (Zhang et al., 2005). This would be in apparent contradiction with the results presented here indicating that the C-terminus of the mature X 2 protein is oriented towards the cytosolic face of the membrane. One possible explanation is that the C-terminus of X 2 or the N-terminus of N T B adopts a different orientation in the context of the polyproteins from that observed with the mature proteins. Dual topology has been observed for the p7 protein of Hepatitis C virus in which the protein adopts a different orientation when it is present within a larger polyprotein that also includes the E2 domain (E2-p7) (Isherwood and Patel, 2005). We have previously shown that the translocation of the N-terminus of N T B - V P g in the lumen is directed by a 131 putative amphipathic helix, which probably requires oligomerization to traverse the membrane (Zhang et al., 2005). Although it is tempting to suggest that this process is inhibited in the context of larger polyproteins that contain the X 2 domain, further experimentation w i l l be required to resolve this issue. The polytopic nature of X 2 is reminiscent of that of the 2B protein of poliovirus. Both proteins are located immediately upstream of the N T B domain (2C in the case of poliovirus). The 2B protein of poliovirus has been shown to increase membrane permeability by forming a pore in the membrane (Agirre et al., 2002). Recent evidence suggests that pore formation regulates the calcium concentration of endoplasmic reticulum membranes and may play a role in preventing defensive apoptotic host cell response (Campanella et al., 2004). It w i l l be interesting to investigate whether X 2 has the ability to modify membrane permeability or not. 132 3.5 B I B L I O G R A P H Y Agirre, A . , Barco, A . , Carrasco, L . , and Nieva, J. L . (2002). Viroporin-mediated membrane permeabilization. Pore formation by nonstructural poliovirus 2B protein. JBiol Chem 277(43), 40434-41. Bechinger, B . (1999). The structure, dynamics and orientation of antimicrobial peptides in membranes by multidimensional solid-state N M R spectroscopy. Biochim Biophys Acta 1462(1-2), 157-83. Bienz, K . , Egger, D . , and Pasamontes, L . (1987). Association of polioviral proteins of the P2 genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography. Virology 160(1), 220-6. Bienz, K . , Egger, D . , Pfister, T., and Troxler, M . (1992). Structural and functional characterization of the poliovirus replication complex. J Virol 66(5), 2740-7. Campanella, M . , de Jong, A . S., Lanke, K . W. , Melchers, W . J., Willems, P. H . , Pinton, P., Rizzuto, R., and van Kuppeveld, F. J. (2004). The coxsackievirus 2B protein suppresses apoptotic host cell responses by manipulating intracellular Ca2+ homeostasis. JBiol Chem 279(18), 18440-50. Carette, J. E . , Guhl , K . , Wellink, J., and Van Kammen, A . (2002a). Coalescence of the sites of cowpea mosaic virus R N A replication into a cytopathic structure. J Virol 76(12), 6235-43. Carette, J. E . , van Lent, J., MacFarlane, S. A . , Wellink, J., and van Kammen, A . (2002b). Cowpea mosaic virus 32- and 60-kilodalton replication proteins target and change the morphology of endoplasmic reticulum membranes. J Virol 76(12), 6293-301. Chen, J., and Ahlquist, P. (2000). Brome mosaic virus polymerase-like protein 2a is directed to the endoplasmic reticulum by helicase-like viral protein 1 a. J Virol 74(9), 4310-8. Chen, J., Noueiry, A . , and Ahlquist, P. (2001). Brome mosaic virus Protein l a recruits viral R N A 2 to R N A replication through a 5' proximal R N A 2 signal. J Virol 75(7), 3207-19. Chenna, R., Sugawara, FL, Koike , T., Lopez, R., Gibson, T. J., Higgins, D . G . , and Thompson, J. D . (2003). Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 31(13), 3497-500. Chisholm, J., Wieczorek, A . , and Sanfacon, H . (2001). Expression and partial purification of recombinant tomato ringspot nepovirus 3C-like proteinase: comparison of the 133 activity of the mature proteinase and the VPg-proteinase precursor. Virus Res 7 9 ( 1 - 2 ) , 153-64. Cho, M . W. , Teterina, N . , Egger, D . , Bienz, K . , and Ehrenfeld, E . (1994). Membrane rearrangement and vesicle induction by recombinant poliovirus 2C and 2 B C in human cells. Virology 2 0 2 ( 1 ) , 129-45. Datta, U . , and Dasgupta, A . (1994). Expression and subcellular localization of poliovirus VPg-precursor protein 3 A B in eukaryotic cells: evidence for glycosylation in vitro. J Virol 6 8 ( 7 ) , 4468-77. DeGrado, W . F., Gratkowski, PL, and Lear, J. D . (2003). H o w do helix-helix interactions help determine the folds of membrane proteins? Perspectives from the study of homo-oligomeric helical bundles. Protein Sci 1 2 ( 4 ) , 647-65. Deleage, G . , Combet, C. , Blanchet, C. , and Geourjon, C. (2001). A N T H E P R O T : an integrated protein sequence analysis software with client/server capabilities. Comput Biol Med 3 1 ( 4 ) , 259-67. den Boon, J. A . , Chen, J., and Ahlquist, P. (2001). Identification of sequences in Brome mosaic virus replicase protein 1 a that mediate association with endoplasmic reticulum membranes. J Virol 7 5 ( 2 4 ) , 12370-81. Echeverri, A . C , and Dasgupta, A . (1995). Amino terminal regions of poliovirus 2C protein mediate membrane binding. Virology 2 0 8 ( 2 ) , 540-53. Fisher, C. L . , and Pei, G . K . (1997). Modification of a PCR-based site-directed mutagenesis method. Biotechniques 2 3 ( 4 ) , 570-1, 574. Han, S., and Sanfacon, H . (2003). Tomato ringspot virus proteins containing the nucleoside triphosphate binding domain are transmembrane proteins that associate with the endoplasmic reticulum and cofractionate with replication complexes. J Virol 7 7 ( 1 ) , 523-34. Hans, F. , and Sanfacon, H . (1995). Tomato ringspot nepovirus protease: characterization and cleavage site specificity. J Gen Virol 7 6 ( 4 ) , 917-27. Hemmer, O., Greif, C , Dufourcq, P., Reinbolt, J., and Fritsch, C. (1995). Functional characterization of the proteolytic activity of the tomato black ring nepovirus RNA-l -encoded polyprotein. Virology 2 0 6 ( 1 ) , 362-71. Hirokawa, T., Boon-Chieng, S., and Mitaku, S. (1998). SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics 1 4 , 378-379. 134 Hofmann, K . , and Stoffel, W . (1993). TMbase-a database of membrane spanning protein segments. Bio. Chem. Hoppe-Seyler 347', 166. Howel l , K . E . , and Palade, G . E . (1982). Hepatic Golgi fractions resolved into membrane and content subfractions. J Cell Biol 92(3), 822-32. Isherwood, B . J., and Patel, A . H . (2005). Analysis of the processing and transmembrane topology of the E2p7 protein of hepatitis C virus. J Gen Virol 86(Pt 3), 667-76. Kyte, J., and Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J Mol Biol 157(1), 105-32. Lundin, M . , Monne, M . , Widel l , A . , V o n Heijne, G. , and Persson, M . A . (2003). Topology of the membrane-associated hepatitis C virus protein N S 4 B . J Virol 77(9), 5428-38. Margis, R., V i ry , M . , Pinck, M . , Bardonnet, N . , and Pinck, L . (1994). Differential proteolytic activities of precursor and mature forms of the 24K proteinase of grapevine fanleaf nepovirus. Virology 200(1), 79-86. Mottola, G . , Cardinali, G . , Ceccacci, A . , Trozzi, C. , Bartholomew, L . , Torrisi, M . R., Pedrazzini, E . , Bonatti, S., and Migl iaccio, G . (2002). Hepatitis C virus nonstructural proteins are localized in a modified endoplasmic reticulum of cells expressing viral subgenomic replicons. Virology 293(1), 31-43. Ota, K . , Sakaguchi, M . , von Heijne, G . , Hamasaki, N . , and Mihara, K . (1998). Forced transmembrane orientation of hydrophilic polypeptide segments in multispanning membrane proteins. Mol Cell 2(4), 495-503. Pouwels, J., van der Velden, T., Willemse, J., Borst, J. W. , van Lent, J., Bisseling, T., and Wellink, J. (2004). Studies on the origin and structure of tubules made by the movement protein of Cowpea mosaic virus. J Gen Virol 85(Pt 12), 3787-96. Restrepo-Hartwig, M . A . , and Ahlquist, P. (1996). Brome mosaic virus helicase- and. polymerase-like proteins colocalize on the endoplasmic reticulum at sites of viral R N A synthesis. J Virol 70(12), 8908-16. Ritzenthaler, C , Laporte, C , Gaire, F. , Dunoyer, P., Schmitt, C , Duval, S., Piequet, A . , Loudes, A . M . , Rohfritsch, O., Stussi-Garaud, C , and Pfeiffer, P. (2002). Grapevine fanleaf virus replication occurs on endoplasmic reticulum-derived membranes. J Virol 76(17), 8808-19. Rost, B . , Casadio, R., and Fariselli, P. (1996). Refining neural network predictions for helical transmembrane proteins by dynamic programming. ISMB 4, 192-200. 135 Rott, M . E . , Gilchrist, A . , Lee, L . , and Rochon, D . (1995). Nucleotide sequence of tomato ringspot virus R N A 1 . J Gen Virol 7 6 ( P t 2 ) , 465-73. Rott, M . E . , Tremaine, J. FL, and Rochon, D . M . (1991). Nucleotide sequence of tomato ringspot virus R N A - 2 . J Gen Virol 7 2 ( P t 7 ) , 1505-14. Salonen, A . , Ahola, T., and Kaariainen, L . (2005). Vi ra l R N A replication in association with cellular membranes. Curr Top Microbiol Immunol 2 8 5 , 139-173. Sanfacon, H . (2005). Replication of positive-strand R N A viruses in plants: Contact points between plant and virus components. Can JBot 8 3 ( 1 2 ) , 1529-1549. Sankaram, M . B . , and Marsh, D . (1993). Protein-lipid interactions with peripheral membrane proteins. In "Protein-lipid interactions" (A. Watts, Ed.), pp. 127-162. Elsevier Science Publishers B . V . , Amsterdam. Schaad, M . C , Jensen, P. E . , and Carrington, J. C. (1997). Formation of plant R N A virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein. Embo J 1 6 ( 1 3 ) , 4049-59. Schwartz, M . , Chen, J., Janda, M . , Sullivan, M . , den Boon, J., and Ahlquist, P. (2002). A positive-strand R N A virus replication complex parallels form and function of retrovirus capsids. Mol Cell 9 ( 3 ) , 505-14. Shai, Y . (1999). Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim Biophys Acta 1 4 6 2 ( 1 - 2 ) , 55-70. Sonnhammer, E . L . , von Heijne, G . , and Krogh, A . (1998). A hidden markov model for predicting transmembrane helices in protein sequences. ISMB 6 , 175-182. Stace-Smith, and R (1984). Tomato ringspot virus. CMI/AAB Description of Plant Viruses 2 9 0 . Staehelin, L . A . (1997). The plant E R : a dynamic organelle composed of a large number of discrete functional domains. Plant J 1 1 ( 6 ) , 1151-65. Teterina, N . L . , Gorbalenya, A . E . , Egger, D . , Bienz, K . , and Ehrenfeld, E . (1997). Poliovirus 2C protein determinants of membrane binding and rearrangements in mammalian cells. J Virol 7 1 ( 1 2 ) , 8962-72. Towner, J., Ho, T., and Semler, B . (1996). Determinants of membrane association for poliovirus protein 3 A B . JBiol Chem 2 7 1 ( 4 3 ) , 26810-8. 136 Tusnady, G . E . , and Simon, I. (1998). Principles governing amino acid composition of integral membrane proteins: Applications to topology prediction. J Mol Biol 283, 489-506. Wang, A . , Carrier, K . , Chisholm, J., Wieczorek, A . , Huguenot, C , and Sanfacon, H . (1999). Proteolytic processing of tomato ringspot nepovirus 3C-like protease precursors: definition of the domains for the V P g , protease and putative R N A -dependent R N A polymerase. J Gen Virol 80 ( Pt 3), 799-809. Wang, A . , Han, S., and Sanfacon, H . (2004). Topogenesis in membranes of the N T B - V P g protein of Tomato ringspot nepovirus: definition of the C-terminal transmembrane domain. J Gen Virol 85(Pt 2), 535-45. Wang, A . , and Sanfacon, H . (2000). Proteolytic processing at a novel cleavage site in the N-terminal region of the tomato ringspot nepovirus RNA-1-encoded polyprotein in vitro. J Gen Virol 81(Pt 11), 2771-81. Zhang, S. C , Zhang, G. , Yang, L . , Chisholm, J., and Sanfacon, H . (2005). Evidence that insertion of Tomato ringspot nepovirus N T B - V P g protein in endoplasmic reticulum membranes is directed by two domains: a C-terminal transmembrane helix and an N-terminal amphipathic helix. J Virol 79(18), 11752-65. 137 C H A P T E R 4 General discussion and future projects 4.1 G E N E R A L D I S C U S S I O N In this thesis, the membrane association and topology of two T o R S V proteins (i.e. N T B -V P g , Chapter 2 and X 2 , Chapter 3) were investigated using G F P fusion approaches in planta and glycosylation site mapping in vitro. The studies in planta demonstrated that both N T B - V P g and X 2 , when expressed individually in N. benthamiana epidermal cells, localize to the E R membranes, and the ER-binding of both proteins is mediated by multiple domains. In combination with in vitro glycosylation studies, the membrane topology of the two proteins was established and the proposed topological models are presented (Fig.2.10 and Fig.3.8). The results discussed in this thesis bring novel ideas about the interaction between the E R membranes and the T o R S V N T B - V P g and X 2 proteins, and raise new questions for future studies. To our knowledge, this is the first systematic identification of membrane-binding domains and membrane topology analysis of viral membrane replication proteins for a plant picorna-like virus. Several lines of evidence suggest that both N T B - V P g and X 2 are integral membrane proteins that localize to the E R . First, both proteins, when fused to G F P , colocalize with ER-dsRed2; second, the N V - G F P and G F P - N V fusion proteins are present predominately in membrane enriched P30 fraction and several X 2 fusion proteins including G F P - X 2 , X 2 - G F P and X 2 - H A co-fractionate with the E R marker (Bip) in membrane flotation assays, even after treatment with N a C l and Na2C03 which release peripheral and luminal proteins, respectively; third, the N - and C-terminal truncated proteins of N T B - V P g and 138 three segments of X 2 (mX2, T M 2 and TM3) target G F P to E R membranes suggesting that these sequences are functional in the entire protein; finally, glycosylation of both full-length and deletion glycosylation mutants of N T B - V P g and X 2 are detected in in vitro glycosylation assays and ER-specific glycosylation of the G F P - c N V construct was confirmed in vivo. ER-association of N T B - V P g and X 2 is in agreement with the observation that the genome replication of T o R S V occurs in association with E R or E R -derived membranes (Han and Sanfacon, 2003). For the N T B - V P g protein, the behaviour of this protein is similar when it is expressed independently or during viral infection, in which the protein is translated as part of polyprotein and released by proteolytic processing. In viral infection, the N T B - V P g along with other NTB-containing proteins are integral membrane proteins, localize to E R membranes and co-fractionate with the active viral replication activities and translocate the V P g domain into the E R lumen (Han and Sanfacon, 2003). For the X 2 protein, it is unknown whether this protein behaves the same during viral infection due to the difficulties in generating antibodies against this very hydrophobic protein. However, it should be noted that the C P M V 32K protein, which shares homology with the X 2 protein, associates with E R or ER-derived membranes both during viral infection and when expressed as a G F P fusion in plant cells (Carette et al., 2002b). Experiments with deletion mutants of N T B - V P g and X 2 G F P fusions in planta indicate that N T B contains two membrane binding segments located at each of the N - and C-termini of the protein (Chapter 2), while X 2 contains three membrane-targeting sequences located in the central part of the protein (Chapter 3). Each of the elements is 139 necessary and sufficient for targeting to and association with E R membranes. The sequences other than these membrane-binding elements play little, i f any, role in the membrane-targeting and association as evidenced by the solubility of the fusion proteins including G F P - m N , c X 2 - G F P and T M 1 - G F P . These results correlate very well with in vitro studies conducted before and during this thesis showing that each of the membrane binding elements can traverse the membranes. The N-terminal transmembrane domains of X 2 and N T B probably form an amphipathic helix as suggested by computer analysis. In contrast, the transmembrane segments towards the C-termini of the two proteins consist of about 20 hydrophobic amino acids, which have enough hydrophobicity and length to form transmembrane a- helices. These membrane binding elements are likely targeted and inserted into E R membranes in different manners. The transmembrane a- helix in N T B - V P g is located at the C-terminal end. Proteins with a C-terminal membrane anchor are usually targeted and inserted in the membrane post-translationally (High and Abe l l , 2004). In plant cells, tail-anchored proteins are predominately associated with E R membranes and also with plastid membranes (Borgese et a l , 2003). Interestingly, dual membrane localization with the E R and chloroplast was reported for the C P M V 60k protein (Carette et al., 2002b). In the case of T o R S V N T B - V P g , we have also observed occasional chloroplast association of some of our truncated constructs containing only the C-terminal transmembrane domain (Zhang, unpublished data). Post-translational translocation of the C-terminal transmembrane domain is also in agreement with cleavage between the junction of N T B and V P g by the downstream viral proteinase as those proteins are initially translated as a 140 polyprotein in which N T B - V P g is located upstream of the viral proteinase domain (Fig. 1.4). For the same reason, the N - termini of N T B - V P g and X 2 are probably translocated post-translationally. The identification of amphipathic helix-dependent translocation of the N-termini of N T B - V P g and X 2 provides evidence for a post-translational translocation mechanism. Post-translational protein folding and interactions are required for membrane translocation of amphipathic helices which usually lay parallel to the membranes with the hydrophobic side inserting into the membranes and the hydrophilic sides facing the cytoplasm unless they form membrane-embedded oligomers leading to the formation of an aqueous pore across the membrane (Bechinger, 1999; Shai, 1999). A different mechanism may be used for the insertion of the two C-terminal transmembrane domains in X 2 , which form a hairpin resulting in both the N - and C-termini oriented into the cytoplasmic side and accessible to the downstream viral proteinase. A hairpin structure is a common structural element found in many proteins. It is usually targeted and inserted as a unit in E R membranes in a SPR-dependent, co-translational manner (Saaf et al., 2000). Those observations raise the possibility that the two transmembrane domains in X 2 may target the T o R S V P l polyprotein to the E R membrane before processing occurs. Membrane association of T o R S V X 2 and N T B - V P g is in agreement with classification of this virus as a picorna-like virus. The results presented in this thesis reveal remarkable similarities to other picornaviruses such as C P M V and poliovirus. X 2 and N T B - V P g of T o R S V are equivalent to the C P M V 32K and 60K proteins and to proteins encoded by the poliovirus 2 B C 3 A B region (Fig. 4.1). X 2 and N T B - V P g , C P M V 32K and 60K 141 proteins (Carette et al., 2002a) and 2B, 2C, and 3 A B proteins of poliovirus target to the E R or ER-derived membranes (de Jong et al., 2003; Echeverri and Dasgupta, 1995; Towner et al., 1996). Moreover, two membrane-binding determinants with A * 2B 2C 3A 3B A * * * 32 K 60K VPg P o l i o v i r u s C P M V T o R S V X2 NTB VPg Fig. 4.1. Comparison of the membrane-binding domains of proteins from Poliovirus 2BC3AB region, the CPMV 32k and 60K proteins and the ToRSV X2 and NTB-VPg proteins. The boxes represent protein domains and the vertical lines in C P M V and ToRSV represent cleavage sites. The blue triangles indicate the putative amphipathic helices and the red asterisks represent stretches of hydrophobic amino acids. The hatched areas represent conserved NTB motifs. properties that parallel those identified in T o R S V N T B - V P g have been mapped to similar positions in 2 C 3 A B , a functional equivalent of N T B - V P g . A n N-terminal amphipathic helix is required for membrane association of the poliovirus 2C (Echeverri and Dasgupta, 1995), and a stretch of hydrophobic amino acid mediates the membrane association of 3 A B (Towner et al., 1996). The C P M V 60K protein also contains an amphipathic helix at the N-terminus and a stretch of hydrophobic amino acids at the C-terminus immediately upstream of V P g , suggesting that the two elements are involved in membrane-binding of this protein (Carette et al., 2002b). Similarity in membrane-binding determinants has also been found in the X 2 proteins, the C P M V 32K protein and the poliovirus 2B protein. The membrane determinants in both X 2 and 2B consist of an N-terminal amphipathic helix 142 and transmembrane a-helice(s) which are located very close to the amphipathic helix. The amphipathic helices in poliovirus 2B are suggested to traverse the membrane leading to formation of aqueous pores in the membranes and an increase in membrane permeability. Studies on X 2 indicate that the amphipathic helix can also traverse the membrane at least in vitro. A putative amphipathic helix is also observed at an equivalent position in the C P M V 32K protein which is followed by three stretches of hydrophobic amino acids (Carette et al., 2002b). However, there are also noticeable differences in membrane association between these proteins (Fig. 4.1). Although N T B - V P g , 60K and 2 C 3 A B have similar membrane-binding domains, the two membrane-binding domains in poliovirus are located within two distinct domains, 2C and 3 A B . Moreover, the N-terminal amphipathic helix in 2C and the hydrophobic domain in 3A are suggested to adopt a peripheral topology (Choe and Kirkegaard, 2004; Teterina et al., 1997) instead of the transmembrane topology observed for N T B - V P g . More variation is observed in the 2B, 32K and X 2 proteins. 2B binds to the E R with an N-terminal amphipathic helix and a transmembrane domain which are proposed to form a hairpin in the membrane with both ends oriented towards the cytosol (Gonzalez and Carrasco, 2003) while X 2 has two transmembrane domains in addition to the amphipathic helix resulting in an overall Niurrien/CCytosoi topology (Fig. 3.8). The membrane-binding domains and topology of 32K have not been determined, but it is very likely that they are different from those of X 2 and 2B since there are three predicted hydrophobic domains following the putative amphipathic helix (Carette et al., 2002b). Furthermore, no morphological changes are observed in plant cells expressing N T B - V P g 143 and X 2 G F P fusions. In contrast, 32K and 60K induce membrane proliferation and formation of membrane aggregates in cortical and perinuclear E R resembling those observed during viral infection (Carette et al., 2002a). Poliovirus 2 B C and 2C can induce membranous vesicles resembling those observed during poliovirus infection (McBride et al., 1996; Teterina et al., 1997). The 3A protein in isolation causes swelling of E R membranes but no small vesicles are formed (Suhy et al., 2000). Coexpression of 2 B C and 3 A results in formation of double-membrane vesicles most similar to the vesicles induced during viral infection (Suhy et al., 2000). Expression of 2B alone increases membrane permeability but does not induce obvious morphological changes in E R membranes (Sandoval and Carrasco, 1997). The lack of E R morphological changes may be an intrinsic property of X 2 as observed for 2B of poliovirus. This is also in agreement with the observation that all the deletion mutants of X 2 tested did not induce membrane morphological changes (Fig. 3.4). In contrast, obvious membrane morphological changes were observed when the N-terminal fragment of the N T B G F P fusion (nN-GFP) was expressed in plant cells (Fig. 2.7). This mutant expressed at a much higher level than the N T B G F P fusions raising the possibility that the expression level of N T B G F P fusions is too low to induce membrane alterations. Interestingly, the membrane rearrangement determinant has also been mapped to the N -terminal amphipathic helix of 2C (Teterina et al., 1997). In addition to protein expression level, other factors such as the interaction of N T B - V P g with lipids, viral or host proteins may be required to induce membrane alteration. A s discussed above, membrane association of 2C is different from that of N T B - V P g in that 2C binds to membranes with 144 the N-terminal amphipathic helix while N T B - V P g contains a second transmembrane domain at the C-terminal end. This transmembrane domain contains a putative signal peptide peptidase cleavage site and cleavage by this enzyme would release the transmembrane from the membrane (McLauchlan et al., 2002). It is possible that N T B -V P g requires this cleavage to activate its membrane reorganization ability by adopting a similar membrane conformation as 2C. Alternatively, polyproteins containing the N T B domain, e.g. X 2 - N T B - V P g which has been detected during viral infection, may be required for membrane alteration. This phenomenon has been observed in Semliki forest virus in which the presence of polyprotein precursors including P123 and P1234 is essential for the induction of cytoplasmic vacuoles (Salonen et al., 2003). Protein-protein interaction may also play a role in the membrane alteration. Co-expression of 2 B C and 3 A can induce membrane vesicles most similar to those formed during viral infection (Suhy et al., 2000). It has been suggested that interaction between 2 B C and 3 A is involved in the membrane induction. This suggestion is corroborated by recent studies showing a positive correlation between the interactions among 2B, 2C and 3 A and the induced membrane structures (Teterina et al., 2006; Towner et al., 2003). Given the fact that both X 2 and N T B - V P g localize to the E R membrane, it is possible that interaction between X 2 and N T B is also involved in the membrane alteration. The E R localization of X 2 and N T B - V P g has multiple implications for their roles in the viral replication cycle. X 2 and N T B are initially translated as domains within a polyprotein. If the hairpin structure in the C-terminus of X 2 functions as a co-translational ER-targeting signal as suggested above, the entire polyprotein would be 145 targeted to the E R membranes. Membranes are likely to influence the subsequent cleavage of the polyprotein and topology of the membrane proteins. The N T B - V P g precursor is detected in vivo (Han and Sanfacon, 2003) but is not released in vitro after translation of X l - X 2 - N T B - V P g - P r o or N T B - V P g - P r o - P o l precursors, rather the mature N T B protein is released alone with the VPg-Pro or VPg-Pro-Pol (Wang et al., 1999). It is possible that the different cleavages in vitro and in vivo are due to the different conformation of the proteinase and/or the substrates in a membrane environment. Membrane association facilitates the correct processing of the polyprotein by the viral proteinase of Africa swine virus (Heath et al., 2003). Precursors containing N T B and X 2 , e.g. X I - X 2 and X 2 - N T B - V P g , are likely adopting different topologies from that of mature proteins, and thus have different functions in viral life cycle. Co-translational E R -targeting of X 2 as suggested above would target not only the polyprotein but also the viral R N A to E R membranes. The proximity of R N A 1 to viral proteins and E R membranes would facilitate the recruitment of the R N A 1 to the membrane-bound replication complex. This is in agreement with the coupled translation, membrane induction and replication observed in picornaviruses (Egger et al., 2000). In B M V , an alternative template recruitment pathway involving the interaction of l a with the translating m R N A via the la-interactive N-terminal region of the nascent 2a polypeptide has been reported (Chen et al., 2003). Although no severe membrane induction and morphological changes were observed when X 2 and N T B were expressed individually in this study, E R association of the two proteins suggests they are the major players in induction of membrane structures for the viral replication complexes by analogy to membrane rearrangement activity shown in membrane proteins in other picornaviruses. 146 After membrane induction, the two proteins may act as membrane anchors for viral replication complex while other components of viral replication complexes including viral R N A s , viral and host proteins are tethered on the membrane surface through macromolecule interaction with the X 2 and/or N T B or as part of polyproteins containing these two proteins. A s mentioned above, V P g probably acts as a primer for viral R N A replication which is thought to occur on the cytoplasmic side of membranes. The luminal localization of the V P g domain in N T B - V P g suggests that N T B - V P g is not a donor for a replication active V P g . We have detected another V P g containing precursor, VPg-Pro-Pol , which is present in both soluble fractions and membrane fractions and the membrane association of this precursor is peripheral. It is likely that VPg-Pro-Pol serves as the donor of V P g for T o R S V R N A replication (Joan Chisholm, unpublished data). Finally, some viral membrane proteins, e.g. poliovirus 2B (Campanella et al., 2004) and H C V N S 5 A (Reyes, 2002), have been implicated in anti-host defence by suppressing apoptosis. It is possible that T o R S V membrane proteins such as X 2 have a similar function. A major shortcoming of this thesis is the lack of functional analysis of N T B - V P g and X 2 -G F P fusion proteins. Therefore it is not known whether the fusion proteins are biologically active or not. This concern is difficult to address for picorna-like viruses since translation and replication are coupled and complementation experiments of replication proteins are not feasible for these viruses (Cleveland et al., 2000). For T o R S V , the lack of infectious transcript also eliminates the possibility to replace N T B - V P g or X 2 with its G F P fusion form to test whether the G F P fusions can support viral replication. Infectious transcripts are available for other nepoviruses and many of the features 147 discussed in this thesis are conserved. Therefore, this concern could be addressed with these transcripts. 4.2 F U T U R E P R O J E C T S As discussed above, several important questions raised in this thesis need to be addressed in future projects to provide a better understanding of the formation of the T o R S V replication complex and the functions of viral replication proteins. The first project would be to investigate protein-protein interactions between N T B or X 2 with other T o R S V replication proteins such as Pro and Pol or with T o R S V R N A s to prove the membrane anchor function of N T B and X 2 as suggested in this thesis. This could be done in vitro by using a yeast two-hybrid system (protein-protein interaction) or three-hybrid system (protein-RNA interaction) (Hook et al., 2005). Alternatively, the interaction could be investigated in vivo by coexpression of N T B or X 2 with Pro, Pol or viral R N A in plants to see i f the latter components can be relocalized from the cytosol to E R membranes in the presence of the putative membrane anchors. Additionally, pull-down experiments could be conducted with tagged proteins. The second project would be to identify which viral protein(s) induces membrane proliferation and morphological changes during viral infection. B y analogy to C P M V and poliovirus, N T B and/or X 2 are the most likely candidates for membrane alteration. A better transient expression system for example, using the Potato Virus X vector (Mallory et al., 2002) for high, prolonged expression of the two proteins, plus good antibodies against the two proteins for immunolocalization by confocal and electron microscopy w i l l be the keys for fulfillment of this project. The third project would be to confirm the translocation of the N-termini of N T B and/or X 2 in 148 vivo and try to prove pore-forming activities and the anti-host defence function of the two proteins. To address these questions, attempts have been made to investigate self-interaction of the amphipathic helix at the N-terminus of N T B in plant cells by bimolecular fluorescent complementation (Zhang, unpublished data). Unfortunately, the results from these experiments were difficult to interpret since positive signals were observed even in the absence of fusion to one of the partners. This problem has also been reported by others (Zamyatnin et al., 2006). N o obvious glycosylation was detected when mutants of the N-terminus of N T B in which a glycosylation signal was introduced into the N-terminal end were expressed in plants, suggesting the N-terminal amphipathic helix is not translocated efficiently into the E R lumen in vivo under the condition tested. To determine the pore-forming and anti-apoptosis activity of X 2 , the methodologies established for the study of the poliovirus 2B protein would be a good example to follow (Gonzalez and Carrasco, 2003). In conclusion, the results discussed in this thesis not only provide new information about the interaction between the E R membranes and T o R S V N T B - V P g and X 2 but also raise new questions for new research directions. 149 4 . 3 B I B L I O G R A P H Y Bechinger, B . (1999). The structure, dynamics and orientation of antimicrobial peptides in membranes by multidimensional solid-state N M R spectroscopy. Biochim Biophys Acta 1 4 6 2 ( 1 - 2 ) , 157-83. Borgese, N . , Colombo, S., and Pedrazzini, E . (2003). The tale of tail-anchored proteins: coming from the cytosol and looking for a membrane. J Cell Biol 1 6 1 ( 6 ) , 1013-9. Campanella, M . , de Jong, A . S., Lanke, K . W. , Melchers, W . J., Willems, P. FL, Pinton, P., Rizzuto, R., and van Kuppeveld, F. J. (2004). The coxsackievirus 2B protein suppresses apoptotic host cell responses by manipulating intracellular Ca2+ homeostasis. JBiol Chem 2 7 9 ( 1 8 ) , 18440-50. Carette, J. E . , Guhl , K . , Wellink, J., and V a n Kammen, A . (2002a). Coalescence of the sites of cowpea mosaic virus R N A replication into a cytopathic structure. J Virol 7 6 ( 1 2 ) , 6235-43. Carette, J. E . , van Lent, J., MacFarlane, S. A . , Wellink, J., and van Kammen, A . (2002b). Cowpea mosaic virus 32- and 60-kilodalton replication proteins target and change the morphology of endoplasmic reticulum membranes. J Virol 7 6 ( 1 2 ) , 6293-301. Chen, J., Noueiry, A . , and Ahlquist, P. (2003). A n alternate pathway for recruiting template R N A to the brome mosaic virus R N A replication complex. J Virol 7 7 ( 4 ) , 2568-77. Choe, S. S., and Kirkegaard, K . (2004). Intracellular topology and epitope shielding of poliovirus 3 A protein. J Virol 7 8 ( 1 1 ) , 5973-82. Cleveland, S. M . , Buratti, E . , Jones, T. D . , North, P., Baralle, F. , M c L a i n , L . , Mclnerney, T., Durrani, Z . , and Dimmock, N . J. (2000). Immunogenic and antigenic dominance of a nonneutralizing epitope over a highly conserved neutralizing epitope in the gp41 envelope glycoprotein of human immunodeficiency virus type 1: its deletion leads to a strong neutralizing response. Virology 2 6 6 ( 1 ) , 66-78. de Jong, A . S., Wessels, E . , Dijkman, H . B . , Galama, J. M . , Melchers, W . J., Willems, P. PL, and van Kuppeveld, F. J. (2003). Determinants for membrane association and permeabilization of the coxsackievirus 2B protein and the identification of the Golgi complex as the target organelle. JBiol Chem 2 7 8 ( 2 ) , 1012-21. Echeverri, A . C , and Dasgupta, A . (1995). Amino terminal regions of poliovirus 2C protein mediate membrane binding. Virology 2 0 8 ( 2 ) , 540-53. Egger, D . , Teterina, N . , Ehrenfeld, E . , and Bienz, K . (2000). Formation of the poliovirus replication complex requires coupled viral translation, vesicle production, and viral R N A synthesis. J Virol 7 4 ( 1 4 ) , 6570-80. 150 Gonzalez, M . E . , and Carrasco, L . (2003). Viroporins. FEBS Lett 5 5 2 ( 1 ) , 28-34. Han, S., and Sanfacon, H . (2003). Tomato ringspot virus proteins containing the nucleoside triphosphate binding domain are transmembrane proteins that associate with the endoplasmic reticulum and cofractionate with replication complexes. J Virol 7 7 ( 1 ) , 523-34. Heath, C. M . , Windsor, M . , and Wileman, T. (2003). Membrane association facilitates the correct processing of pp220 during production of the major matrix proteins of African swine fever virus. J Virol 7 7 ( 3 ) , 1682-90. High, S., and Abe l l , B . M . (2004). Tail-anchored protein biosynthesis at the endoplasmic reticulum: the same but different. Biochem Soc Trans 3 2 ( P t 5), 659-62. Hook, B . , Bernstein, D . , Zhang, B . , and Wickens, M . (2005). RNA-protein interactions in the yeast three-hybrid system: affinity, sensitivity, and enhanced library screening. Rna 1 1 ( 2 ) , 227-33. Mallory, A . C , Parks, G . , Endres, M . W. , Baulcombe, D . , Bowman, L . H . , Pruss, G . J., and Vance, V . B . (2002). The amplicon-plus system for high-level expression of transgenes in plants. Nat Bio technol 2 0 ( 6 ) , 622-5. McBride , A . E . , Schlegel, A . , and Kirkegaard, K . (1996). Human protein Sam68 relocalization and interaction with poliovirus R N A polymerase in infected cells. Proc Natl Acad Sci USA 9 3 ( 6 ) , 2296-301. McLauchlan, J . , Lemberg, M . K . , Hope, G. , and Martoglio, B . (2002). Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets. EmboJ21(\5), 3980-8. Reyes, G . R. (2002). The nonstructural N S 5 A protein of hepatitis C virus: an expanding, multifunctional role in enhancing hepatitis C virus pathogenesis. J Biomed Sci 9 ( 3 ) , 187-97. Saaf, A . , Hermansson, M . , and von Heijne, G . (2000). Formation of cytoplasmic turns between two closely spaced transmembrane helices during membrane protein integration into the E R membrane. J Mol Biol 3 0 1 ( 1 ) , 191-7. Salonen, A . , Vasiljeva, L . , Merits, A . , Magden, J . , Jokitalo, E . , and Kaariainen, L . (2003). Properly folded nonstructural polyprotein directs the semliki forest virus replication complex to the endosomal compartment. J Virol 7 7 ( 3 ) , 1691-702. Sandoval, I . V . , and Carrasco, L . (1997). Poliovirus infection and expression of the poliovirus protein 2B provoke the disassembly of the Golgi complex, the organelle target for the antipoliovirus drug Ro-090179. J Virol 7 1 ( 6 ) , 4679-93. 151 Shai, Y . (1999). Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim Biophys Acta 1462(1-2), 55-70. Suhy, D . A . , Giddings, T. H . , Jr., and Kirkegaard, K . (2000). Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles. J Virol 74(19), 8953-65. Teterina, N . L . , Gorbalenya, A . E . , Egger, D . , Bienz, K . , and Ehrenfeld, E . (1997). Poliovirus 2C protein determinants of membrane binding and rearrangements in mammalian cells. J Virol 71(12), 8962-72. Teterina, N . L . , Levenson, E . , Rinaudo, M . S., Egger, D . , Bienz, K . , Gorbalenya, A . E . , and Ehrenfeld, E . (2006). Evidence for functional protein interactions required for poliovirus R N A replication. J Virol 80(11), 5327-37. Towner, J., Ho, T., and Semler, B . (1996). Determinants of membrane association for poliovirus protein 3 A B . JBiol Chem 271(43), 26810-8. Towner, J. S., Brown, D . M . , Nguyen, J. H . , and Semler, B . L . (2003). Functional conservation of the hydrophobic domain of polypeptide 3 A B between human rhinovirus and poliovirus. Virology 314(1), 432-42. Wang, A . , Carrier, K . , Chisholm, J., Wieczorek, A . , Huguenot, C , and Sanfacon, H . (1999). Proteolytic processing of tomato ringspot nepovirus 3C-like protease precursors: definition of the domains for the V P g , protease and putative R N A -dependent R N A polymerase. J Gen Virol 80 ( Pt 3), 799-809. -Zamyatnin, A . A . , Jr., Solovyev, A . G . , Bozhkov, P. V . , Valkonen, J. P., Morozov, S. Y . , and Savenkov, E . I. (2006). Assessment of the integral membrane protein topology in living cells. Plant J 46(1), 145-54. 152 

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