Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Molecular biological studies of rubella virus structural proteins Qiu, Zhiyong 1994

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata


831-ubc_1994-953837.pdf [ 3.68MB ]
JSON: 831-1.0088226.json
JSON-LD: 831-1.0088226-ld.json
RDF/XML (Pretty): 831-1.0088226-rdf.xml
RDF/JSON: 831-1.0088226-rdf.json
Turtle: 831-1.0088226-turtle.txt
N-Triples: 831-1.0088226-rdf-ntriples.txt
Original Record: 831-1.0088226-source.json
Full Text

Full Text

MOLECULAR BIOLOGICAL STUDIES OF RUBELLA VIRUS STRUCTURAL PROTEINS  by  Zhiyong Qiu B.Sc., Fudan University, Shanghai, China, 1984  A thesis submitted in partial fuilfifiment of the requirements for the degree of Doctor of Philosophy in the Faculty of Graduate Studies Genetics Program  We accept this thesis as conforming to the required standard  The June, 1994 ©Zhiyong Qiu, 1994  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his  or  her  representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of The University of British Columbia Vancouver, Canada Date  DE.6 (2/88)  ‘  Abstract Rubella virus (RV) is a small enveloped RNA virus in the Togaviridae family. The virion contains three structural proteins, a capsid protein (C) associated with the genomic RNA to form the nucleocapsid and two membrane glycoproteins, El and E2. The RV structural proteins are translated as a polyprotein H) from a RV-speciflc 24S subgenomic RNA and derived by H C-E2-E1-COO 2 precursor p110 -(N posttranslational processing of p110. The role of N-linked glycosylation of El and E2 on their respective biological functions has been studied by expressing glycosylation mutants of El and E2 generated by oligonucleotide-directed mutagenesis on coding cDNA. Expression of the E2 mutant proteins in COS cells indicated that removal of any of the glycosylation sites resulted in slower glycan processing, lower protein stability and aberrant disulfide bonding of the mutant proteins, with the severity of defect depending on the number and location of deleted carbohydrate sites. Expressed El glycosylation mutant proteins from vaccinia recombinants were on recognized by a panel of El-specific monoclonal antibodies, indicating that carbohydrate side chains El El are not involved in the constitution of epitopes recognized by these monoclonal antibodies. All the glycosylation mutants were capable of eliciting anti-RV El antibodies in immunized mice; however, only the single glycosylation mutants were found to be capable of inducing viral neutralizing antibodies, suggesting that carbohydrates on El are important for maintaining proper protein folding and epitope exposure. Assembly of RV was found to be independent of the genomic RNA but strictly dependent upon the co-expression of C, E2 and El, in stable cell lines expressing RV structural proteins. Assembly and release of RV vinon was dramatically reduced in RV-infectecl cells treated with two Golgi transport inhibitors, brefeldin A and monensin, although there was no significant alteration for the expression and processing of the structural proteins. My finding indicates that stable association of RV El and E2 with the intact Golgi complex is essential for efficient RV assembly.  11  Table of Contents ABSTRACT LIST of TABLES LIST of FIGURES LIST of ABBREVIATIONS ACKNOWLEDGEMENTS 1. INTRODUCTION 1.1. N-linked glycosylation of viral glycoproteins Structure and biosynthesis 1.1.1. Approaches for functional analysis of N-linked glycosylation 1.1.2. Biological functions of N-linked glycosylation 1.1.3. 1.2. Targeting of viral membrane glycoproteins and virus assembly General concepts 1.2.1. Virus assembled at the plasma membrane 1.2.2. Virus assembled in the Golgi complex 1.2.3. Virus assembled in the ER, the Golgi complex 1.2.4. or a pre-Golgi compartment Closing remarks 1.2.5. 1.3. Rubella 1.3.1. 1.3.2. 1.3.3. 1.3.4. 1.3.5. 1.3.6. 1.3.7. 1.3.8. 1.3.9. 1.3.10. 1.3.11. 1.3.12.  virus biology Classification Clinical aspects Morphology and morphogenesis Nucleic acids and genome organization Non-structural proteins Structural protein expression and processing Posttranslational modification of RV structural proteins Intracellular localization of RV structural proteins Biological function of RV structural proteins Immune responses to RV infection Immunological determinants on RV structural proteins Project rationale and thesis objectives  2. MATERIALS AND METHODS 2.1. MATERIALS and SUPPLIES 2.2. METHODS Propagation of bacterial strains 2.2.1. Preparation of competent cells and transformation 2.2.2. DNA preparation and handling 2.2.3. Expression vectors 2.2.4. DNA-mediated transfection 2.2.5. Construction of vaccinia recombinants 2.2.6. 111  ii vi vii ix xi 1 1 1 5 7 15 15 19 23 25 27 29 29 29 30 31 32 33 35 40 40 41 43 45 47 47 48 48 48 49 52 53 55  2.2.7. 2.2.8. 2.2.9. 2.2.10. 2.2.11. 2.2.12. 2.2.13. 2.2.14. 2.2.15. 2.2.16. 2.2.17. 2.2.18. 2.2.19.  Metabolic labelling Immunoprecipitation Endoglycosidase digestion Immunoblotting Indirect immunofluorescence Electrophoresis RV propagation, purification and titration Electronmicroscopy Mice immunization Enzyme linked immunoadsorbant assay (ELISA) Hemagglutination and hemagglutination inhibition assays Viral neutralization assay Lymphoproliferative assay  3. RESULTS and DISCUSSIONS 3.1. Section I. Role of N-linked glycosylation on E2 processing and transport E2 cDNAs 3.1.1. Determination of functional N-linked glycosylation sites in E2 3.1.2. Expression of E2 glycosylation mutants in COS cells 3.1.3. Formation of aberrant disuffide bonding in E2 mutant proteins 3.1.4. Glycan processing and intracellular stability of E2 proteins 3.1.5. Intracellular localization of mutant E2 proteins 3.1.6. Secretion of an anchor-free form of wild-type and 3.1.7. mutant E2 proteins Summary and Discussion for section I 3.1.8. 3.2. Section II. Effect of Brefeldin A (BFA) and monensin on protein processing and virus assembly Processing of N-linked oligosaccharides on E2 3.2.1. Processing of 0-linked glycans on E2 3.2.2. Processing and secretion of an anchor-free form of E2 3.2.3. Proteolytic processing of RV structural protein precursor 3.2.4. Subcellular distribution 3.2.5. Effect of BFA and monensin on RV assembly and release 3.2.6. Assembly of virus particles 3.2.7. Summary and Discussion for section II 3.2.8. 3.3. Section III. Influence of N-linked glycosylation on the antigenicity and immunogenicity of El glycoprotein Construction of recombinant vaccinia viruses expressing 3.3.1. RV El glycosylation mutants Expression and antigenicity of El glycosylation mutants. 3.3.2. Immunogenic properties of expressed El glycosylation mutants 3.3.3. iv  57 58 58 59 59 60 61 63 63 64 64 65 66 67 67 67 67 69 71 74 76 79 81  86 86 88 91 91 93 95 97 101  106 106 106 112  3.3.4. 3.3.5. 3.3.6.  Antigenic properties of deglycosylated RV El from RV virions Effect of glycosylation on El cell surface expression Summary and Discussion for section III  3.4. Section IV. Expression and characterization of virus-like particles containing rubella virus structural proteins Isolation of BilK cell lines expressing RV structural proteins 3.4.1. Expression of RV structural proteins 3.4.2. Assembly and release of virus-like particles in 3.4.3. stable BHK-24S cells roscopic analysis of the VLPs Electron-mic 3.4.4. Antigenicity of the VLPs 3.4.5. city of the VLPs Immunogeni 3.4.6. Summary and Discussion for section IV 3.4.7.  113 115 118  122 122 125 125 128 131 131 137 140 145  SUMMARY AND PERSPECTIVES REFERENCES  v  LIST of TABLES Table 1. Table la. Table 2.  Table 3. A. B. Table 4. Table 5.  Specificity of glycosidases used in this study Summary of properties of monoclonal antibodies directed to El Comparison of the HAT and VN antibodies from mice immunized with vaccinia recombniants containing different RV El glycosylation mutant cDNA inserts HA assay of deglycosylated RV virion Effect of deglycosylation of RV El on antibody recognition by El-specific monoclonal antibodies Immunoreactivity of the VLPs with RV-specific monoclonal antibodies Comparison of antibody titres of mouse sera from mice immunized with different RV antigens  vi  Page 6 109a  114  117 132 135  LIST of FIGURES Page Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9.  Figure 10. Figure 11. Figure 12. Figure 13. Figure 14. Figure 15. Figure 16. Figure 17. Figure 18. Figure Figure Figure Figure  19. 20. 21. 22a.  Figure 22b. Figure 23. Figure 24.  Processing of N-linked oligo saccharides to a representative biantennary complex structure Topography of the genome RNA of RV General strategy for the expression and processing of RV structural proteins Schematic representation of mammalian cell expression vectors used in this study Schematic representation of wild-type and glycosylation mutants of RV E2 Determination of the number of N-linked glycans on RV E2 Expression of wild-type and glycosylation mutants of E2 in COS cells Formation of aberrant disulfide bonding in E2 glycosylation mutants Western blot analysis of steady-state wild-type and mutant E2 proteins in transfected cells under reducing and non-reducing conditions Time course for glycan processing of wild-type and mutant E2 proteins Intracellular stability of wild-type and mutant E2 proteins Indirect immunofluorescence of wild-type and mutant E2 proteins in COS cells Intracellular processing and secretion of a soluble form of wild-type and mutant E2 proteins Effect of BFA and monensin on processing of E2 Glycosidase digestion of E2 from BFA- and monensin-treated cells Effect of BFA and monensin on processing and secretion of an anchor-free form of E2 Effect of BFA and monensin on the proteolytic cleavage of the polyprotein precursor for RV structural proteins Indirect immunofluorescence of RV structural proteins in cells transfected with pCMV5-24S and treated with BFA or monensin Release of virus particles in infected cells Titration of cell-associated and released virus Electron microscopic analysis of virus assembly Schematic representation of wild-type and glycosylation mutants of RV El Expression of El glycosylation mutants by vaccinia recombinants Immunoblot analysis of El glycosylation mutants expressed by vaccinia recombinants Immunoblot analysis of sera from mice immunized with El vaccinia recombinants vii  3 34 36 54 68 70 72 73  75 77 78 80 82 87 89 92 94 96 98 99 100 107 108 110 112  Effect of deglycosylation on the antigenicity of RV virion Indirect immunofluorescence of El glycosylation mutants in infected CV- 1 cells Figure 27. Diagrammatic representation of RV cDNAs used in the construction of recombinant plasmids Figure 28. A. Imrnunoblot analysis of proteins expressed in transformed BHK cells B. Immunoblot analysis of proteins sedimented by ultracentrifugation Time course of VLPs secretion from BHK-24S cells Figure 29. Figure 30. Purification of VLPs and RV on sucrose density gradient centrifugation Electron microscopic analysis of the VLPs in BHK-24S cells Figure 31. Figure 32. Radioimmunoprecipitation of RV structural proteins expressed in COS cells Lymphoproliferation responses of mice immunized with VLPs Figure 33. Figure 25. Figure 26.  viii  116 119 124  126 127 129 130 134 136  LIST of ABBREVIATIONS ampicillin adenosine triphosphate 5-bromo-5-chloro-3-indolyl phosphate 13-mercaptoethanol baby hamster kidney cell line base pair bovine serum albumin concanavalin A dihydrofolate reductase Dulbecco’s modified Eagle’s medium deoxyribonuclease Dithiothreitol ethylene diaminetetraacetic acid enzyme-linked immunosorbent assay endoplasmic reticulum fetal calf serum Golgi complex hemagglutination hemagglutination inhibition kilobase kilodalton L broth molar concentration milliamp monoclonal antibody minimum essential medium multiplicity of infection nitro blue tetrezolium absorbance at 405 nm wavelength polyacylamide gel electrophoresis phosphate-buffered saline plaque forming unit phenylmethylsulfonyl fluoride rabbit kidney cell line ribonuclease rotation per minute room temperature rubella virus Svedberg unit sodium dodecyl sulfate Semliki Forest virus Sindbis virus  AP ATP B CIP (3-Me BHK bp BSA Con A DHFR DMEM DNase DTT EDTA ELISA ER FCS GC HA HAT kb kDa LB M mA mAb MEM MOl NET 405 0D PAGE PBS pfu PMSF RK RNase rpm RT RV S SDS SFV SIV ix  Similian virus 40 tunicamycin tetramethyfrhodamine isothiocyanate trishydroxymethylaminomethane virus-like particle viral neutralizing vesicular stomatitis virus wheat germ agglutinin  SV4O TM TRICT Tris VLP VN VSV WGA  x  ACKNOWLEDGEMENTS First, I would like to thank my supervisor, Dr. Shirley Gillam, for her advice, support encouragement and enthusiasm during my graduate studies, and for her help both in my research and my life. I also want to thank the other members of my supervisory committee, Drs. Caroline Astell, Peter Candido and Frank Tufaro for their helpful advice, suggestion and discussions. Special thanks to Dr. Aubrey Tingle for taking time to provide criticism of the manuscripts. Thank you to members of Shirley’s lab, Tom, David, D.C., Helen, Helena, Nina, Zhewei for their help, and to many people around in the Research Centre for creating such a wonderful working environment. Lastly to my wife for her understanding throughout.  This thesis is dedicated to my parents.  xi  1. INTRODUCTION This introduction contains three subsections: an overview of biosynthesis and biological functions of asparagine-linked (N-linked) glycosylation on viral glycoproteins, recent progress in viral glycoprotein targeting and virus assembly, and a review of rubella virus biology. The rationale and objectives of this thesis will conclude the chapter.  1.1. N-linked glycosylation of viral glycoproteins N-linked glycosylation is one of the most common post-translational modifications of proteins in the exocytic pathway of eukaryotic cells. Animal viruses utilize host-cell glycosylation machinery to synthesize and process oligosaccharides attached to viral glycoproteins. The expression of viral antigens in cells has proven to be a useful system for studying the stepwise events in glycan processing and intracellular transport along the exocytic pathway. From a large number of viral glycoproteins studied so far, none of the carbohydrate structures identified is unique to viral glycoproteins; they are also present in a variety of other membrane and secretory glycoproteins. However, the impact of N-linked glycosylation on conformation and subsequently on biological functions of viral glycoproteins varies with the protein in question.  1.1.1. Structure and biosynthesis of N-linked carbohydrate on viral glycoprotein Transfer of oligosaccharide from a glycan-lipid precursor All of the N-linked carbohydrates on viral glycoproteins is synthesized and processed by host cell enzymes following the general pathway for this class of glycans (for review see Komfeld and Kornfeld, 1985). As the first step in the biosynthesis of glycoproteins, a core structure  -1-  M 3 (Glc G 9 ) 2 lcNAc an is assembled on the lipid carrier dolichol phosphate in the lumen of the endoplasmic reticulum (ER). Upon the translocation of newly synthesized polypeptide into the ER, the oligosaccharide is transferred, by oligosaccharide transferase, to the asparagine residues of polypeptides at the sequence Asn-X-Ser/Thr where X can be any amino acid except proline (Fig.l) (Kornfeld and Kornfeld, 1985). The oligosaccharide is covalently attached to the asparagine residues with a N-glycosidic linkage. A survey of protein sequences has revealed that not all the potential N-linked glycosylation sites are glycosylated (Kornfeld and Kornfeld, 1985). A properly oriented and accessible Asn-X-Ser/Thr motif in the protein is believed to play a major part in determining the efficiency of glycosylation. Sequential processing of N-linked oligosaccharide Once the oligosaccharide becomes polypeptide bound, it undergoes a series of trimming reactions by glycan modifying enzymes in the secretory pathway (Fig. 1) (for review see Kornfeld and Kornfeld, 1985). Three glucose residues are first removed in the ER by glucosidases, followed by the removal of mannose residues by ER mannosidase or by x-mannosidase I present in the Golgi cisternae. At this stage, the oligosaccharide intermediate containing 5 to 8 mannoses is recognized by the Golgi enzyme, acetylgiucosidase. This enzyme catalyzes the addition of an acetyiglucoside residue to the free mannose linked x1-3 to the mannose residues. Afterward, two more mannose residues are removed by Golgi mannosidase II, leaving a free mannose for the further addition of acetyiglucoside. After the acetylglucoside is added, the oligosaccharide becomes biantennary and is subjected to further addition of residues of galactose, fucose or sialic acid, catalyzed by glycosyl-transferases in the trans Golgi or trans-Golgi network, completing  -2-  ‘cD Turncamycin blocks lhe syn thesis of lhis  I Do  y ER t.1annosidase  EnoopIsmsc Reticulum  H  Goc Complex  Fig.1 Processing of N-linked oligosaccharides to a representative biantennary complex structure. The scheme depicts the processing from the transfer of 2 Gl M 3 G 9 lcNAc an c from its dolichol pyrophosphoryl derivative to the nascent polypeptide chain still bound to the ribosome, followed by processing reactions in the ER and GC. Oligosaccharide processing enzymes are listed above the line; the reaction they catalyze is depicted below the line (except for the alternate processing reaction, 3a). The subcellular localization of processing events are dipicted by the brackets. Structures susceptible or resistant to digestion by endo H, an enzyme frequently used as a diagnostic test for processing to complex structures, are indicated. Symbols: v, glucose; 0, mannose; , N-acetylglucosamine; •, galactose; fucose; O, sialic acid. (Moreman and Touster, 1988) ,  -3-  the assembly of the complex type oligosaccharide. Depending on the accessibility to glycan modifying enzymes and cell types, the extent of processing can be varied for different glycoproteins (for review see Kienk, 1990) or for oligosaccharides at different sites on the same protein species (Pollack and Atkinson, 1983), and this may explain the vast diversity of glycan differentiation in viral glycoproteins. Characterization of the structure of N-linked glycans The extent of N-linked oligosaccharide processing can be monitored using glycosidase digestion or lectin binding assays (Montreuil et al., 1986). Glycosidases are excellent tools to elucidate the primary structures of glycans by sequential degradation of oligosaccharides bound to the polypeptide backbone. Basically two types of enzymes are used: exoglycosidases hydrolyse glycosidic bonds of monosaccharides in terminal non-reducing positions and may achieve a stepwise degradation of the glycans; endoglycosidases hydrolyse internal glycosidic bonds. Each endoglycosidase usually recognizes a certain type of glycosidic linkage (Montreuil et al., 1986) (Table 1). By analysing sugar moieties that are liberated from the protein backbone with paper chromatography or FPLC, the structure of N-glycans on a protein can be defined. Lectins are sugar-binding proteins or glycoproteins of non-immune origin which agglutinate cells and/or precipitate glycoconjugates (Lis and Sharon, 1984). Lectins are powerful tools for characterizing structure of oligosaccharides on glycoproteins because they bind with high specificity to certain types of glycoconjugates (Montreuil et al., 1984). Practically, immobilized lectins are widely used for isolating sugar components whereas fluorescence reagent conjugated lectins are used for visualizing subcellular compartmentation.  -4-  1.1.2. Approaches for functional analysis of N-linked glycosylation Several approaches have been used to define the functional roles of N-linked carbohydrate addition in cells. These include the use of agents that interfere with glycosylation (for review ee Elbein, 1987), elimination of each N-linked glycan addition site on DNA or cDNA by oligonucleotide-directed mutagenesis and the use of glycan-processing-deficient cell lines. Tunicamycin, a nucleoside analog, is the most widely used glycosylation inhibitor (Elbein, 1987). This antibiotic inhibits the transfer of G1cNAc-1-P from UDPG1cAc to dolichol phosphate (Fig. 1). Since this step is the first in the lipid-linked pathway, tunicamycin treatment results in the synthesis of proteins that lack N-linked glycosylation (Elbein, 1987). Drugs that inhibit specific steps in the processing pathway have become available and have been extensively used in the study of the role of oligosaccharide processing intermediates in biological functions and transport of glycoproteins (Elbein, 1987). Furthermore, compounds such as brefeldin A (Fujiwara et al., 1988) and monensin (reviewed by Mollenhauer et al., 1990) which disrupt vesicular structures of cells and thus interfere with the normal distribution of resident glycan processing enzymes, have also been widely used in analyzing the effect of aberrantly processed glycans on the transport and function of glycoproteins. While treatment with the above mentioned drugs may lead to altered cell metabolism (Elbein, 1987; Mollenhauer et al., 1990), the elimination of glycosylation sites on coding DNA or cDNA of a protein by site-directed mutagenesis has proven to be a valuable method to analyze the influence of carbohydrate site chain addition on glycoproteins in cells under normal growth conditions, especially when proteins with more than one N-linked glycosylation site are being examined. Expression and characterization of mutant viral glycoproteins with partial or  -5-  Table 1. Specificity of glycosidases used in this study (Montreuil et al, 1986)  Endoglycosidase H  (Man)—Man \ z-Man—G1cNAc- 1—G1cNAc-Asn / x—Man  I y Active on N-linked oligosaccharides of glycopeptides. Enzyme cleaves only high mannose structures [n=2-150, x=(Man) , y and 12 z=H] or hybrid structures (n=2, x and/or y =NANA-Gal-G1cNAc or similar, z=H or G1cNAc). Glycopeptidase F  x \ w—Man \ u—Man--G1cNAc--G1cNAc- 1—Asn / y—Man / z Active on N-linked oligosaccharides of glycopeptides. Enzyme cleaves high mannose structures (w, x and y= one or more Man residues, u and z=H) or hybrid structures (w and x=Man, y and/or z=NANA-Gal-G1cNAc or similar, u=H or G1cNAc) or complex structures (y and w=NANA-Gal-G1cNAc or similar, x and z=H, NANA-Gal-G1cNAc or similar, u=H or GlcNAc).  Neuraminidase  NANAt2-1—X or NGNAa2-1—X Enzyme cleaves terminal sialic acid residues which are cL2,3-, a2,6or x2,8-linked to: Gal, G1cNAc, Ga1NAc, NANA, NGNA, oligosaccharide, glycolipid or glycoprotein.  -6-  complete depletion of N-linked glycosylation sites have provided greater understanding of the function of N-glycans. A large number of cell lines have been isolated that are deficient in glycan processing enzymes. Infection of these cells with viruses and the analysis of protein expression, processing and virus production have yielded a great deal of useful information about the function of oligosaccharide side chains on proteins (Kennedy, 1974; Schlesinger et al., 1976; Hsieh and Robbins, 1984).  1.1.3. Biological functions of N-linked glycosylation N-linked glycosylation influences a number of properties of proteins, including stability, intracellular transport, biological activity and antigenicity (for review see Olden et al., 1982; Klenk, 1990). There are no general rules existing regarding the consequences of changing the normal glycosylation pattern on a protein. Results from experiments using tunicamycin have indicated that the requirement for glycosylation is intrinsic to a given protein (Olden et al., 1982). Furthermore, in some cases, the biological effect of N-linked glycosylation depends on the particular site within the polypeptide chain. Role of carbohydrate in initiating protein folding and maintaining protein stability One of the most important functions of N-linked glycosylation is to initiate and maintain protein folding into its proper configuration. N-linked sugars are added co-tianslationally to the polypeptide chain as the consensus sequences emerge on the luminal side of the ER membrane, prior to or during folding. Inhibition of N-linked glycosylation, using tunicamycin or elimination  -7-  of N-linked glycosylation sites by mutagenesis, leads to misfolded proteins, such as aggregated or disulfide cross-linked complexes (Machamer et aL, 1985; Rose and Doms, 1988; Ng et aL, 1990; Vidal et aL, 1992). It is believed that the hydrophilic oligosaccharides render folding intermediates more soluble and less likely to form irreversible aggregates (Rose and Doms, 1988). The importance of individual N-linked oligosaccharide side chains depends on the protein in question as well as the location of the glycosylation site within the protein. Elimination of some sites may result in little or no defect whereas others may be essential for correct folding and stability (Ng et aL, 1990; Sodora et al., 1991; Roberts et aL, 1991; Pique et al., 1992). Elimination of individual glycosylation sites can also lead to the generation of temperaturesensitive (ts) mutants (Gallagher et al., 1988; Machamer and Rose, 1988; Ng et al., 1990) and the effect of such elimination can be additive and strain-specific (Machamer et al., 1985; Gallagher et al., 1992). Oligosaccharides on glycoproteins are believed to provide protection against protease attack (reviewed by Olden et al., 1982). After treatment with glycosidases to remove glycans on glycoproteins, the polypeptide backbones become more susceptible to protease digestion (Olden et al., 1982). On the other hand, a higher turnover rate has been observed for many glycosylation mutant proteins and for glycoproteins from cells treated with glycosylation inhibitors (Ng et al., 1990; Sodora et al., 1991; Roberts et al., 1991). The deleterious effect of elimination of glycosylation on protein stability parallels that of protein folding (Ng et al., 1990; Sodora et al., 1991). Proteins misfolded due to inhibition of glycosylation by tunicamycin or deletion of glycosylation sites have a low solubiity and tend to form aggregates in the ER and be degraded  -8-  (Ng et al., 1990; Sodora et al., 1991; Roberts et al., 1991). Role of carbohydrate in facilitating intracellular transport and processing It is now clear that carbohydrate itself does not determine the subcellular destination of viral glycoproteins. However, glycosylation greatly enhances the movement of many glycoproteins out of the ER, although this often results through effects on protein folding. It is possible though, that oligosaccharides could have the effect of increasing transport rates by generating affinities towards enzymes sequestered in the exocytic pathway. The requirement for carbohydrate in the transport of membrane and secretory proteins is not universal and is highly protein-specific (reviewed by Olden et al., 1982). Some proteins are transported and function normally when glycosylation is inhibited with tunicamycin, whereas others exhibit folding defects, frequently resulting in protein aggregation in the ER or rapid degradation (Machamer et al., 1985; Rose and Doms, 1988; Hurtley and Helenius, 1989; Ng et al., 1990). It has also been shown that the arrest of transport of misfolded viral protein in the ER can be rescued at a lower temperature (Gallagher et al., 1988; Machamer and Rose, 1988; Ng et al., 1990), which supports the concept that retention in the ER is due to protein misfolding. Addition of supernumerary carbohydrate side chains, (e.g. for VSV G and influenza virus HA), can also have the similar effect, disrupting folding and transport (Doms et al., 1988; Gallagher, et al., 1988; Machamer and Rose, l988a). However, some degree of flexibility has been observed in the position on the polypeptide backbone at which carbohydrate chains are required (Doms et al., 1988, Machamer and Rose, 1988b). A single amino acid substitution of VSV G protein has been found to eliminate the stringent carbohydrate requirement for cell surface expression of the  -9-  mutant VSV G protein (Pitta et al., 1989). Carbohydrates may affect in two ways, the proteolytic processing of viral precursor glycoprotein: as a general requirement for proper protein folding and subsequently transport to the site of cleavage, or as a factor modulating protein configuration within or adjacent to the cleavage site for the access of protease. Inhibition of N-linked glycosylation inhibits the processing of Sindbis virus envelope protein E2 (Leavitt et al., 1977) and the Newcastle Disease virus F glycoprotein (Morrison et al., 1985) due to failure in transport of nonglycosylated proteins to the Golgi complex for cleavage. Using oligonucleotide-directed mutagenesis, others have found the site-specific influence of glycosylation on cleavage of the CKIPenn strain of avian influenza virus HA (Deshpande et al., 1987), Friend murine leukemia virus envelope protein (Kayman et al., 1991) and measles virus fusion protein (Alkhatib et al., 1994). Role of carbohydrate in modulating biological activities of proteins The envelope glycoproteins of animal viruses may be involved in the attachment of virus to the host cell receptor, and fusion between the virus envelope and the cell membrane; they also serve as the target for host immune surveillance (discussed below). The importance of N-glycosylation of viral glycoproteins in receptor binding has been illustrated in some detail for human immunodeficiency virus type I (HIV- 1) envelope glycoprotein gp 120 (Fenouillete et al., 1990). Deglycosylation of HW-1 gpl2O by glucosidase digestion results in a less than 10-fold reduction of the ability to bind to CD4, the cellular receptor for HP/-i (Fenouillet et al., 1990). Deglycosylation also significantly reduces but does not abolish, HIV- 1 binding to and infectivity of CD4 cells (Fenouillete et al., 1990). Anticarbohydrate monoclonal antibodies (mAbs) can  -  10  -  block infection by cell-free virus as well as inhibiting syncytium formation, probably through steric hindrance (Hansen et al., 1990). However, nonglycosylated HIV-1 gpl2O synthesized in the presence of tunicamycin fails to bind to CD4 (Li et al., 1993). These data suggest that glycosylation of gp 120 is essential to create a conformational epitope to which CD4 binds, but is not directly involved in CD4-binding. In contrast, treatment of respiratory syncytial virus with N-glycanase and 0-glycosidase under mild conditions to remove readily accessible carbohydrate from respiratory syncytial virus glycoprotein results in a significant loss of virus infectivity (Lambert, 1988), suggesting that carbohydrate exerts a considerable influence on the attachment and/or penetration function of the viral  glycoproteins.  Similarly,  enzymatic removal  of N-linked oligosaccharide from  hemagglutinin-neuraminidase (HN) glycoprotein of human parainfluenza virus type 1 leads to a change in the interaction of HN with the host receptor, sialic acid (Gorman et al., 1991). Site-specific contribution of N-linked glycosylation on a viral protein towards their biological activities has been studied in several viruses employing mutagenesis approach. Mutations at particular sites in envelope glycoproteins have been found to be responsible for loss of infectivity of Friend murine leukemia virus (Kayman et al., 1991), and a decreased induction of polykaryon formation in measles virus (Alkhatib et al., 1994) and HW-1 (Dedera et al., 1992). Finally, lack of glycosylation at a conserved site in influenza virus neuraminidase has been found to confer strain-specific neurovirulence in mice (Li et al., 1993). Role of carbohydrate in influencing antigenic properties Carbohydrates on viral glycoproteins can modulate the antigenicity and immunogenicity of viral  -  11  -  glycoproteins, directly or indirectly. The indirect influence refers to the fact that oligosaccharide can promote and maintain the correct folding of viral glycoproteins and thus stabilize the epitopes or facilitate epitope exposure. It is often found that synthetic peptides bearing neutralizing epitopes of viral glycoproteins or viral glycoproteins expressed from E.coli fail to induce neutralizing antibodies, suggesting that lack of glycosylation may reduce the immunogenicity of proteins. Since the contribution of each glycosylation site within a protein possessing multiple glycosylation sites may be different (see, the site-specific effect of glycosylation on the immunoreactivity has been characterized for a number of viral glycoproteins. Using site-directed mutagenesis to alter N-linked glycosylation sites, it has been found that one glycosylation site on bovine herpesvirus type 1 glycoprotein gIV is important for its immunoreactivity (Tikoo et a., 1993). Mutant proteins lacking glycans at residue 102 show altered reactivity with conformation-dependent gIV-specific mAbs and also induce significantly lower neutralizing antibody responses than wild-type (Tikoo et al., 1993). On the other hand, evidence has been obtained that carbohydrates can shield antigenic sites from immune recognition by steric hindrance. Addition of carbohydrate side chains at novel sites on influenza virus hemagglutinin results in the shielding or disruption of functional epitopes on the surface of hemagglutinin (Gallagher, et al., 1988). One of the Sindbis virus neutralization escape mutants selected with mAbs shows a codon change which results in the gain of a new glycosylation site at amino acid residue 203 of the E2 protein (Davis et al., 1987). A more common approach utilized in studying carbohydrate shielding of epitopes on viral glycoproteins is the use of glycosidases to remove glycans from expressed glycoproteins or from assembled virions, and analysis of the antibody binding activity as well as the immunogenicity of these  -  12  -  deglycosylated proteins or virions. Deglycosylation does not affect the binding of Rauscher leukemia virus envelope glycoproteins to neutralizing antibodies from sera; and in fact, deglycosylated virions induce a faster neutralizing antibody response than that of untreated control virus (Elder et al., 1986). The immunoreactive conformation of envelope glycoproteins of HIV- 1 remains unaltered after deglycosylation (Ferouillet et al., 1990). However, rabbits immunized with these deglycosylated glycoproteins produce lower viral neutralizing (VN) antibodies that inhibit HIV- 1 infectivity or syncytium formation in infected cells (Benjouad et aL, 1992). Besides total removal of glycans from glycoproteins, partial removal of sugar moieties from carbohydrate side chains or inhibition of oligosaccharide processing have also been found to interfere with the fine conformation of domains in the polypeptides within or adjacent to glycosylation sites and to result in an altered antigenic properties. The glycoprotein gIV of bovine herpesvirus 1 expressed from recombinant baculovirus infected insect cells, which are devoid of sialyl transferases for the addition of terminal sialic acid to the N-glycans, reacts less efficiently with niAbs that recognize conformation-dependent epitopes, and induces lower overall or neutralizing antibody titres, than the protein from virion grown in mammalian cells (van Drunen Littel-van den Hurk, et al., 1991). In cells treated with N-methyl-1-deoxynojirimycin, an inhibitor of a-glucosidase, the normal carbohydrate trimming is inhibited. Under these conditions, SFV E2 protein expressed from SFV-infected chicken cells contains three additional glucose residues in the oligosaccharide side chain and has a significantly changed antigenicity (Kaluza et al., 1980). Carbohydrates can serve directly as targets for recognition by the immune system.  -  13  -  Because of their host specificity, carbohydrate epitopes are responsible, by a mechanism of molecular mimicry, for cross-reactivities with host components or with other viral glycoproteins carrying similar oligosaccharides. This has been well documented in the case of influenza virus (Klenk, 1990). Carbohydrate epitopes are rare; more frequently, the sugar moieties are part of the constitution of conformation-dependent epitopes on viral glycoproteins. Depletion of N-linked glycan either by changing the consensus sequence for N-glycosylation or by total inhibition of N-glycosylation using tunicamycin results in the reduced reactivity of bovine herpesvirus 1 gIV to mAbs that recognize conformation-dependent epitopes but not those which react to linear epitopes (Tikoo, et at, 1993). Immunization of animals with mutant protein in which oligosaccharide side chains involving conformation-dependent epitopes are deleted, results in no significant difference in total antibody responses; however, the neutralizing titre of the antibodies is much lower (Tikoo, et al., 1993).  -  14  -  1.2. Targeting of viral membrane glycoproteins and virus assembly  1.2.1. General concepts Assembly of enveloped viruses Enveloped viruses package their genomes within a protein shell, and this nucleocapsid (or core structure) is then enveloped by a lipid bilayer at the final step of virus maturation, the budding process, during which the nucleocapsid core extrudes itself through a certain region of the cellular membrane. The envelope of viruses is made up of a regular lipid bilayer derived from, and similar in structure and composition to, one of the host cell membranes. However in this process, the host membrane proteins are effectively excluded and replaced with virus-specific membrane proteins (Suomalainen et al., 1992). The mechanism of virus budding is still largely unknown. It is believed that the interaction between the nucleocapsid and viral membrane glycoproteins, in most cases, is the driving force for virus assembly. Accumulation of viral proteins within a specific subcellular compartment may provide ground for such an interaction and may, at least partially, determine the site of virus maturation. In general, the unambiguous proof of virus maturation at any particular subcellular site is provided by the demonstration of electron microscopic profiles of virus particle accumulation and budding. Virus assembly at almost all cellular membrane structures, plasma membrane (PM), Golgi complex (GC), endoplasmic reticulum (ER) and inner nuclear membrane has been reported. By comparing the electron microscopic profiles of virus budding with data from light- and electron-microscopic immunolocalization of the distribution of viral glycoproteins as well as the nucleocapsid, a strong correlation between the site of virus budding and glycoprotein targeting  -  15  -  has been found for a number of viruses. Targeting of viral envelope glycoproteins Viral proteins destined for the plasma membrane follow the general secretory pathway also utilized by host cell plasma membrane proteins. In fact, viral spike proteins have been instrumental in dissecting the various steps involved in this exocytic pathway. The organelles within this system include the rough and smooth ER, the cis- medial- and trans-Golgi, the trans Golgi network (TGN), secretory vesicles and granules, and the PM (reviewed by Dunphy and Rothman, 1985). The ER represents the point of entry for the proteins that will traverse this complex organellar pathway (reviewed by Pfeffer and Rothman, 1987). Proteins synthesized on the polysomes associated with the ER membrane, are cotranslationally inserted into the ER due to the presence of a signal sequence (Singer et al., 1987). During and after the process of translocation itself, components of the ER play a variety of roles in catalyzing posttranslational modifications such as proteolysis and N-linked glycosylation of the extruded proteins. In addition it has become increasingly clear that molecules residing within the lumen of the ER assist in the correct folding of translocated polypeptides and their assembly into oligomeric complexes (Ng et al., 1990; Earl et al., 1991). The attainment of a correct structure appears to be critical for transport out of the ER to the Golgi, and may in fact be the rate-limiting step in the process (see of the introduction) (reviewed by Rose and Doms, 1988). Proteins normally targeted to the PM exit the ER and move to the Golgi cisternae via vesicle-mediated membrane fusion. The GC is a set of subcompartments that comprises at least three biochemically distinct units (reviewed by Dunphy and Rothman, 1985), based on the enzymes that transform N-linked  -  16  -  oligosaccharide chains into their mature form. As glycoproteins are transported from cis through medial to trans Golgi, carbohydrate modifications are carried out by resident enzymes. A large variety of other processes such as 0-linked glycosylation, acylation, suiphation, and proteolytic cleavage/activation also occur in the GC. The majority of membrane-spanning proteins are transported to the PM in a constitutive, unregulated fashion, whereas secretion of soluble proteins involves the TGN, which is a branchpoint directing the final destination of proteins (Pfeffer and Rothman, 1987). Not all the proteins that enter the secretory pathway end up at the cell surface; a number of cellular and viral glycoproteins are retained in one of the subcellular compartments of the exocytic pathway (the ER, the GC or the TGN). The intracellular retention of glycoproteins is believed to be mediated by “retention signals” located in the primary structure of proteins (Lewis and Pelham, 1989; Swift and Machamer, 1991). Many ER-resident proteins bearing an amino acid motif of Lys-Asp-Glu-Leu (KDEL) or similar sequences interact with the KDEL receptor which normally resides in the cis Golgi cisterae (Lewis and Petham, 1989). Ligand binding induces a change of conformation in the KDEL receptor and results in the retrograde movement of the receptor-ligand complex back to the ER (Lewis and Pelham, 1991). The Golgi retention signal has been found to be located in the transmembrane domains of proteins (Swift and Machamer, 1991) while the cytoplasmic tail has been shown to be important in retention in the TGN. In most studies of protein targeting, the criteria of cell surface expression of  glycoproteins is defined using inimunofluorescence techniques while subcellular localization of proteins can be determined using light- and electron-microscopic immunolocalization.  -  17  -  Biochemical analysis such as analysis of the extent of N- or 0-linked glycosylation, organelle specific proteolytic processing and the oligomeric state of the proteins in question have also been extensively used to study protein transport. Experimental approaches Conventional electron-microscopic profiles of virus particle accumulation and budding of infected cells at different stages during virus replication provide adequate information on the site of virus budding. Recent developments in irnmuno-electron microscopic techniques allow the fine localization of virus-specified proteins in virus-infected cells. This methodology has been applied to transfected cells expressing viral protein from DNA or cDNA constructs derived from partial or intact viral genomes. Comparison of protein localization with or without virus assembly has shed light on which viral proteins determine the site of budding. While morphological studies create a steady-state image of virus budding and virus particle accumulation, data from biochemical analysis have been proven to be very informative on the stepwise processes from the synthesis of viral proteins to their incorporation into virions. In such studies, two protein transport inhibitors, brefeldin A (BFA) and monensin have been extensively utilized.  Monensin,  a monovalent ion-selective ionophore, facilitates the  transmembrane exchange of principally sodium ions for protons, which results in the neutralization of acidic intracellular compartments such as the trans Golgi apparatus and associated elements, combined with a disruption of the normal functions of these compartments. Late Golgi processing events such as terminal glycosylation and proteolytic cleavages are most susceptible to inhibition by monensin (reviewed by Mollenhauer et al., 1990).  -  18  -  BFA, a fungal metabolite, is another compound which has been extensively utilized in studying ER-Golgi trafficking in recent years. At low concentrations (ijig/mi) this lipophilic molecule retards protein secretion, apparently acting specifically on transport from the ER to the Golgi (Misumi et al., 1986). At an intermediate concentration (2.5 ig/m1) the Golgi is disassembled, while at 10 pg/mI, transport is completely blocked and morphological changes including dilation of the ER as well as loss of the Golgi structure (Fujiwara et al., 1988, Misumi et al., 1986). At high concentration, BFA causes the movement of resident Golgi proteins back into the ER (Doms et al., 1989). This inhibitory effect of BFA is reversible. It should be noted that although these observations are applicable to most type of cells, the effect of BFA treatment in each cell type is cell-type specific.  1.2.2. Virus assembly at the PM Most  enveloped  animal  viruses  (e.g.  aiphaviruses,  arenaviruses,  orthomyxoviruses,  paramyxoviruses, rhabdoviruses, and retroviruses) acquire their envelope at the PM. After budding has been completed, virus particles are released directly into the extracellular space. Morphogenesis and assembly For virus maturation at the PM, in most cases the pre-assembled nucleocapsid is transported to the cell surface and interacts with viral glycoprotein(s) embedded in the PM, leading to formation and release of the virus particles. Matrix or membrane (M) proteins are involved, for some viruses, in this nucleocapsid-glycoprotein interaction. In the unusual case of lentiviruses, assembly of the core and virion occurs simultaneously, and does not require the envelope glycoprotein.  -  19  - Assembly mediated by spike-nucleocapsid interactions Alphaviruses are enveloped RNA viruses belonging to the Togaviridae family. The virions appear as essentially spherical, 60-65 nm diameter particles  .  The genome, a 49S RNA molecule with  positive polarity, is encapsidated by a single species of capsid protein arranged in an icosahedral configuration. This nucleocapsid is enveloped by a lipid bilayer derived from the host cell plasma membrane. Projecting from the bilayer and embedded in it are the viral encoded glycoproteins designated El and E2. For alphaviruses replicating in vertebrate cells, the assembly of nucleocapsid takes place in the cytoplasm while the budding occurs primarily at the plasma membrane (Smith and Brown, 1977). Several ts mutants of alphaviruses have been isolated which have maturation defects associated with the spike proteins at the restrictive temperature and thus are defective in the release of infectious virions. In some ts mutants, the mutated spike proteins fail to be transported to the plasma membrane at the restrictive temperature, and electron microscopic (EM) analysis of these cells shows no budding structure under these conditions (Brown and Smith, 1975; Saraste et al., 1980; Smith and Brown, 1977). In one Sindbis virus ts mutant (ts2O)-infected cells, the altered spikes are able to reach the cell surface, and in these cells, the plasma membrane is seen to be lined with nucleocapsids engaged in the budding process (Smith and Brown, 1977; Saraste et al., 1980). These results indicate that the nucleocapsid cannot bud from the cell without correct nucleocapsid-spike protein interaction at the cell surface. Amino acid substitutions in the cytoplasmic tail of E2 have been shown to lead to defects in virion assembly (Gaedigk-Nitschko and Schlesinger, 1991). Recently, using recombinant SFV genomes lacking the nucleocapsid protein gene or, alternatively, the spike genes, Suomalainen et al. (1992) demonstrated that virus release is strictly dependent on the  -  20  -  coexpression of the nucleocapsid and spike proteins and concluded that the budding of aiphaviruses is mediated by nucleocapsid-spike interaction. Matrix or membrane (M) protein mediated nucleocapsid-glycoprotein interaction Rhabdoviruses, orthomyxoviruses and paramyxoviruses are negative-stranded RNA viruses. Although there are differences in their genomic organization (segmented or nonsegmented), virion structure (spherical or bullet-shaped) and gene expression strategy, they are similar in that virion maturation occurs at the cell surface and is mediated by respective M proteins. The ribonucleoprotein cores are assembled in the nucleus (orthomyxoviruses) or cytoplasm (rhabdo and paramyxoviruses) and transported to a region of the plasma membrane which contains newly inserted but randomly distributed viral glycoproteins. The M proteins bridge the gap between the ribonucleoprotein core and the cytoplasmic extension of the glycoproteins, leading to the envelopment of nucleocapsid with glycoprotein-covered plasma membrane. For vesicular stomatitis virus (VSV), a rhabdovirus, the M (matrix) protein binds to progeny nucleocapsid and the nucleocapsid-M-protein complex migrates to the cell surface to initiate budding (Newcomb et al., 1982). In paramyxoviruses, the M (membrane) proteins aggregate in the inner aspect of the cell surface and noncovalently associate with glycoprotein. The complex ‘captures’ the newly arrived nucleocapsid to activate the budding process (reviewed by Dubois-Dalq et al., 1984). Spike glycoprotein transport Much of what is known about stepwise transport of glycoproteins from the site of biosynthesis to the cell surface has been learned from the study of some viral glycoproteins: VSV G protein,  -  21  -  influenza HA and alphavirus p62 and El. Although the movement of these proteins from the ER to the cell surface is fast, with a t 112 of less than 30 minutes, they are subjected to a number of structural modulations. The lumen of the ER provides an environment optimized for protein folding and multi-subunit assembly. The ectodomain of the polypeptide must fold correctly in order to be transport competent (Earl et al., 1989). Protein misfolding induced by inhibition of glycosylation (by TM or mutagenesis) results in the aggregation and cross-linking of proteins by disulfide bonds (Hurtley et at, 1989; Machamer and Rose, 1988b). These defective proteins are retained in the ER and eventually degraded (Lippincott-Schwartz et al., 1988). Retention prevents delivery of nonfunctional viral membrane proteins to the site of virus budding. Movement from the ER to GC is the rate-limiting step in the exocytic pathway for viral membrane proteins that are targeted to the cell surface, whereas intra-Golgi transport is fast (Pfeffer and Rothman, 1987). Transport between Golgi compartments is a vesicular process during which proteins are exposed to the sequential action of N-linked glycosylation modification enzymes residing in each subdivision of the GC (Dunphy and Rothman, 1985). Compartmentalization is also important in that it allows protein cleavage/activation to occur at a specific stage of maturation within the Golgi, as in the case for the conversion of alphavirus p62 to E2 and E3 (Hakimi and Atkinson, 1982).  2.2.3. Studies with inhibitors, BFA and monensin BFA and monensin effectively block the incorporation of the VSV G protein and El and E2 proteins of SW into their respective virions as a result of the inhibition of transport of these  -  22  -  proteins to the plasma membrane. In the presence of BFA, the acquisition of endo H resistance by VSV G protein and the proteolytic conversion of SIV pE2 to E2 (a Golgi-specific event) are inhibited, suggesting that the transport of these envelope proteins is arrested in the ER (Oda, et al., 1990). In monensin-treated cells, fatty acid attachment to VSV G and SIV pE2, and the posttranslational removal of mannose residues from oligosaccharides on VSV G occurs normally, whereas proteolytic cleavage of SW pE2 to E2 is inhibited, suggesting that monensin acts during late Golgi processing (Johnson and Schlesinger, 1980).  1.2.3. Viruses assembled in the GC Members of the Bunyavirus family are the only viruses in which budding occurs for certain in the GC. Although there are a large number of viruses in this family, they share a similar general structure and site of maturation. Morphogenesis and assembly Bunyavirus particles are 90-100 nm in diameter and contain two membrane glycoproteins, Gi and G2. The internal protein N associates with RNA to form the nucleocapsid. EM studies show that virus particles mature intracellularly by budding into smooth vesicles in a perinuclear region and the budding structure is not observed at the PM. During Uukuniemi virus infection, both Gi and G2, as well as N, probably in the form of nucleocapsids, accumulate in the GC. The helical nucleocapsids appear to line up beneath the membrane of distended Golgi vesicles. As G 1 and G2 accumulate in the GC, progressively more nucleocapsid seems to enter the GC region. Little if any N protein is seen associated with the ER or the PM. Thus specific interactions between  -  23  -  nucleocapsids and membranes containing viral glycoproteins exist only in the GC (Kuismanen et aL, 1982, 1984). Targeting of Gi and G2 to the GC Based on primary structure deduced from cDNA sequences, Gi and G2 from most of the viruses in Bunyavirus family are type I membrane glycoproteins (Pettersson, et al., 1988). The oligosaccharides on Gi and G2 are found to be heterogeneously processed, as judged by endo H digestion and analysis of terminal glycans. The presence of immature glycans may reflect the site of maturation in the GC. In virus-infected cells or cells expressing viral protein from vaccinia recombinant virus, most of the glycoproteins accumulate in the GC and cannot be chased out from there. This strong retention of glycoproteins in the GC suggests that a retention signal may reside in either Gi or G2, or both. Inhibitor studies, BFA and monensin BFA treatment does not affect the assembly of intracellular infectious virus particles of Punto Toro virus but causes a rapid and dramatic change in intracellular distribution of Gi and G2 glycoproteins, from a Golgi pattern to an ER pattern (Chen et al., 1991). In contrast, budding of bunyanvirus is inhibited by the ionophore monensin (Cash 1982; Kuismane et al., 1985, Chen et al., 1991) whereas the association of the nucleocapsid with Golgi vesicles seems to be unaffected (Kuismanen et al., 1985). This points to the possibility that the pH or the ionic milieu prevailing in the GC is critical for bunyavirus budding. Budding in the GC may thus be dependent not only on a certain concentration of glycoproteins, but also on a conformational  -  24  -  change of the glycoproteins induced by this milieu.  1.2.4. Virus assembly in the ER, the GC or a pre-Golgi compartment Members of the coronaviridae family are enveloped RNA viruses that acquire their lipoprotein coats by budding at intracellular membranes (the ER, the GC, or in a compartment between these two organelles). Coronaviruses have a single species of coat protein, N protein, which is associated with the genomic RNA in the cytoplasm to form helical, loosely coiled nucleocapsids and two membrane glycoproteins M and S. Morphogenesis and assembly Coronavirus budding occurs at intracellular membranes between the rough ER and the Golgi apparatus. At the budding site, nucleocapsids align on the cytoplasmic side of smooth membrane in the ER, or the GC where viral glycoproteins have accumulated (Massaiski et al., 1982). Budding at the cell surface has not been observed. In a detailed electronmicroscopic study of different types of cells infected with mouse hepatitis virus A59, slight differences in the sites of virus maturation were observed, either between the GC and the smooth perinuclear vesicles, or tubules in a pre-Golgi region (Tooze et al., 1984; Tooze and Tooze, 1985; Tooze et al., 1987). It appears that the location of coronavirus budding is at least in part determined by the host cell. M protein targeting determines the site of coronavirus maturation Amino acid sequences predicted from cDNA sequences reveal that coronavirus M glycoprotein is a type III membrane glycoprotein with multiple transmembrane domains (Mayer et al., 1988;  -  25  -  Wickner and Lodish, 1985). Based on the topological model and protease protection assays, it is proposed that most of the M protein is embedded in the membrane (Mayer et al., 1988). M protein  contains  either  N-  or  0-linked  oligosaccharides  depending  on  the  strain.  Immunolocalization of M protein in coronavirus-infected cells or cells transfected with a cloned cDNA has indicated that it is predominantly found in smooth membranes of the pre-Golgi region, the GC and, later in infection, also in the ER (Rottirer and Rose, 1987; Machamer and Rose, 1987; Mayer, et al., 1988). Recent studies employing immuno-electronmicroscopic and recombinant DNA techniques have shown that the M protein is targeted to the GC through a “Golgi retention signal localized in the first transmembrane domain of M protein (Machamer et aL, 1990; Swift and Machamer, 1991; Machamer et al., 1993). This retention signal” is sufficiently efficient to cause proteins normally targeted at the cell surface (e.g. VSV G) to be retained in the GC (Swift and Machamer, 1991). The site of virus budding is determined by the subcellular localization of M protein, based on several lines of evidence. M protein is never transported beyond the trans Golgi to the PM, and no virus budding has been observed at the PM, although a large amount of S protein may accumulate at the PM (Mayer et al., 1988). In coronavirus-infected cells treated with tunicamycin, M protein and the nucleocapsid protein are normally incorporated into virions in the ER and Golgi without S protein (Holmes et al., 1981). In this respect, coronavirus M protein resembles the matrix or membrane proteins of paramyxovirus and rhabdovirus in function during virus assembly, although it is a glycoprotein.  -  26  - Studies with monensin Studies by Niemann et al., (1982) show that monensin does not interfere with coronavirus budding in the rough ER or Golgi, but it does inhibit virus release and fusion of infected cells. During monensin treatment, oligosaccharides on the S protein are shown to be resistant to endo H digestion but lack fucose, indicating that transport of the S protein is inhibited between the trans Golgi and the cell surface. The M protein incorporated into virions is devoid of carbohydrate, implying that the transport of M protein is also inhibited by monensin.  1.2.4. Closing remarks In the case of viruses that bud at the plasma membrane, the viral glycoproteins are rapidly transported to the cell surface via the normal exocytic pathway (Stephens and Compans, 1988). By contrast, for most of the intracellular maturing enveloped viruses, at least one of the virusspecified glycoproteins is targeted to and accumulates in the budding compartment. Examples of such glycoproteins have been discussed earlier in this introductory section, e.g. coronavirus M protein (in a post-ER, pre-Golgi intermediate compartment, or GC) and bunyavirus Gi and G2 (GC). Thus, one important factor in determining the site of budding is clearly the targeting to and accumulation of viral glycoprotein in the compartment. Viruses incorporate functional gene products into virions. The first step in this quality control mechanism is that newly synthesized viral glycoproteins must fold into a proper conformation to obtain transport competence. For proteins destined for the cell surface, this is the rate-limiting step in the exocytic pathway, during which they undergo a series of posttranslational modifications and become biologically functional upon reaching plasma  -  27  -  membrane. For proteins targeted to an intracellular compartment, however, correct protein folding enables proteins to exit the ER and to be retained in one of the compartments. Such retention may be due to 1) the presence of specific amino acid motif (linear or conformational) that constitute a “retention signal” (as in the case of coronavirus M glycoprotein); 2) lateral interaction between viral membrane glycoproteins that result in the formation of large aggregates that exclude them from transport vesicles; 3) association with macromolecules residing in a subcellular compartment via interaction with regions of the protein other than the “retention signal”. To date, little is known about the mechanism underlying virus budding, particularly for virus budding at the intracellular membrane. It is understandable that an important prerequisite for virus budding may be the need for a critical concentration of viral glycoproteins within the budding compartment. A good explanation is that normally a particular type of virus buds only in one of the subcellular compartments. Conformational changes in glycoproteins along the transport route induced by milieu (pH, ionic conditions) or posttranslational modifications (e.g. glycan processing) may facilitate the interaction of viral glycoproteins with nucleocapsids. Lastly, compartment-specific molecules may assist the budding process.  -  28  -  1.3. Rubella virus biology  1.3.1. Classification Rubella virus (RV) is the sole member of the genus Rubivirus in the family Togaviridae (Porterfield et al., 1978). Based on morphological criteria, Togaviruses are defined as spherical, enveloped viruses with an icosahedral nucleocapsid. The genome is composed of a single infectious RNA molecule. Surrounding the nucleocapsid is the host cell-derived lipid bilayer containing viral membrane glycoproteins. Progress in the molecular characterization of viruses in the Togaviidae family has led to reclassification of these viruses on the basis of viral genome structure, organization, and gene expression. Under the current classification, the Togaviridae family consists of two genera: aiphaviruses (arthropod-borne) and rubiviruses (non-arthropod borne) (Francki et al., 1991). The two well-studied viruses, Sindbis virus (SIN) and Semliki Forest virus (SFV) are included in the aiphavirus genus whereas RV is the only known member of rubivirus genus (Francki et al., 1991).  1.3.2. Clinical aspects RV is the etiological agent of a relatively mild childhood disease known as German measles. RV infection in humans may be asymptomatic or can induce adenopathy, malaise, low grade fever, and exanthem. The most common complication following natural RV infection is transient joint involvement such as polyarthralgia and arthritis. The primary medical significance of RV infection is that the virus can cross the placenta and replicate in the fetus. Infants born to women infected during the first trimester of gestation  -  29  -  have a high incidence of birth defects, collectively known as congenital rubella syndrome (CRS). These include heart defects, cataracts, deafness, and mental retardation. Vaccination with live, attenuated virus has been successful in reducing the incidence of CRS. However, rubellaassociated arthritis and the consequence of viral persistence in vaccinees resulting from RV vaccination remain major medical concerns (Chantler et al., 1982). Furthermore, RV infection has been linked to some chronic diseases, including autoimmune diseases (Wolinsky, 1990). Although the highest correlation between RV infection and chronic disease is found in the CRS population (Wolinsky, 1990), the association of RV persistence with arthritis (reviewed by Phillips, 1989) and multiple sclerosis (Nath and Wolinsky, 1990) has been suggested.  1.3.3. Morphology and morphogenesis Early studies employing conventional electron microscopy of RV grown in BHK-21 cells indicated that RV virions are spherical, 60-70 nm in diameter, with a 30 nm electron dense core surrounded by an envelope (von Bonsdorff and Vaheri, 1969). These structures have been defined as the icosahedral nucleocapsid (the dense core) (Murphy et al., 1968) and lipid-bilayer (envelope associated with the hemagglutination activity) (Holmes et al., 1969). The mechanism of RV assembly and budding is largely unknown. Among reports on the RV budding site, there is an apparent discrepancy. In BHK-21 and Vero cells, intracellular maturation (in Golgi or vacuoles) and budding at the plasma membrane have been observed (Bardeletti et al., 1979; Payment et al., 1975). However, studies in which cells were infected with RV at high multiplicity of infection (MOl) and analyzed at different time intervals suggest that there exits a course of progression of RV maturation from the Golgi towards the plasma membrane (von Bonsdorff and Vaheri,  -  30  -  1969; Bardeletti et al., 1979).  1.3.4. Nucleic acids and genome organization The RV genome, a single-stranded RNA molecule with a sedimentation coefficient of 40 S, is infectious (Hovi and Vaheri, 1970). RV-irifected cells contain, in addition to the 40 S RV genome, an RV-specific RNA molecule which sediments at 24 S (Oker-blom et al., 1984). This subgenomic RNA is polyadenylated, capped, and identical to the 3’ one-third of the 40 S RNA (Oker-Blom et al., 1984). It serves as a messenger RNA for the synthesis of RV structural proteins (Oker-Blom et al., 1984). The molecular mechanism that results in the synthesis of the 24 S subgenomic RNA is not clear, but presumably involves a negative-sense RNA intermediate. Sequences of the 24 S subgenomic RNA have been determined from cDNA clones for wild type isolates (M33 and Therien strain) (Clarke et al., 1987; Frey et al., 1986) or vaccine strains (RA 27/3 and HPV 77) (Nakhasi et al., 1989; Zhang et al., 1989). A 95% homology at the nucleotide level is found between three reported RV 24 S RNA sequences whereas little homology was found with that of the alphavirus subgenomic mRNA (Frey and Man, 1988). Recently cDNA clones covering the entire RV genome for the Therien (Dominguez et al., 1990) and M33 strains (Yang et al., 1993) have been constructed and sequenced. Sequence data derived from cDNAs reveal that the RV genome is 9756 nucleotides (Therien) or 9764 (M33) in length and has a G/C content of 69.5%, the highest of any RNA virus sequence to date (Dominguez et al., 1990). Alignment of the 40 S sequence between the Therien and M33 strains shows homologies of 97.5% and 96.6% for overall nucleotide and deduced amino acid sequences, respectively (Yang et aL, 1993). The RV genome contains two long open reading frames (ORFs),  -  31  -  a 5’ proximal ORF (from nucleotide 41 to nucleotide 6385) which encodes the nonstructural proteins and a 3’ proximal ORF (from nucleotide 6506 to nucleotide 9694) which encodes the structural proteins (Fig.2) (Dominguez et al., 1990; Yang et al., 1993). Thus, the genomic organization of RV closely resembles that of aiphaviruses (Strauss et al., 1984).  1.3.5. Non-structural proteins The non-structural proteins of RV are encoded by the 5’ two-thirds of its genome, and translated as a polyprotein precursor. In RV infected cells, protein species with molecular masses of 200, 150, 87, 75 and 27 kDa, in addition to the structural proteins, have been detected using human convalescent serum (Bowden and Westaway, 1984). Recently, RV-specific proteins with electrophoretic mobilities corresponding to 200, 150, and 90 kDa have been expressed in cells transfected with a recombinant plasmid (pTM3/nsRUB) containing the RV 5’ proximal ORF under the control of the T7 polymerase promoter (Marr, et al., 1994). Antibodies raised against bacterial fusion proteins containing regions encoded by the 5’ proximal ORF react to the 200, 150 and 90 kDa proteins from the above mentioned cDNA transfected cells as well as RV infected cells. Mutational analysis indicates that the 150 and 90 kDa proteins are the processing products of the 200 kDa precursor and the order within the ORF is NH2-P150-P90-COOH (Man et al., 1994, Forng and Frey, unpublished results). The biological functions of these proteins is not known. Amino acid sequences predicted from cDNA sequences reveal a conserved helicase motif and a replicase motif found among well studied positive-stranded RNA viruses. In addition, a cysteine protease activity is found to be involved in the processing of the nonstructural protein precursor and an important catalytic role  -  32  -  has been assigned to Cys 1151 of the protease (Marr et al., 1994).  1.3.6. Expression and processing of structural proteins RV contains three structural proteins: a capsid protein, C (33 kDa), and two membrane glycoproteins El (57 kDa) and E2 (42-47 kDa). In RV infected cells, the structural proteins are translated as a polyprotein precursor, in the order, NH -C-E2-El-COOH, with the 24 S 2 subgenomic RNA serving as a template (Fig.3) (Oker-Blom et al., 1984). The polyprotein precursor is subsequently proteolytically processed to yield three individual structural proteins. Unlike SIN and SFV, the RV capsid protein does not possess an autocatalytic serine protease-lilce activity used to release itself from the polyprotein (Clarke et al., 1987; McDonald et al., 1990). In the absence of microsomes, in vitro translation of the 24 S subgenomic RNA produces a polyprotein precursor of 110 kDa (Oker-Blom et al., 1984; Clarke et aL, 1988). Amino acid sequences predicted from cDNAs reveals that both RV El and E2 are type I membrane proteins (Singer et al., 1987) with their N-termini preceeded by stretches of 20 and 23 hydrophobic amino acid residues, respectively (Clarke et al., 1987; Frey and Marr, 1988). These hydrophobic sequences resemble the consensus signal peptides that mediate targeting of nascent polypeptides to the ER membrane and initiate translocation of protein into the ER lumen (reviewed by Wiley, 1986). In vitro and in vivo expression of wild-type and mutant proteins lacking the signal sequences demonstrate that the presence of the signal peptides is required for translocation and processing of the polyprotein precursor (Hobman et al., 1988; Hobman and Gillam, 1989; Man et al., 1991). Furthermore, mutations at the cleavage sites (von Heijne, 1984) of either the E2 or El signal peptide resulted in the accumulation of uncleaved polyprotein  -  33  -  0.0  1.0  30  2.0  4.0  50  6.0  7.0  9.0  8.0  10.0  11.0  SG ANA  RUBo  :x:  •H  PH P90  P150  f  C  El  YY  -,—  ri  MAbQj  C  polyA  -.  J.w  1-;.Ik  E2f  E2  .  L1J  El  Fig.2 Topography of the genome RNA of RV. The scale at the top of diagram is in kilobases. Untranslated sequences are denoted by black lines and open reading frames (ORFs) by open boxes. The 5’ proximal ORF encodes nonstructural proteins and 3’ proximal ORF encodes structural proteins. The boundaries of the individual proteins processed from the precursor translated from each ORF are denoted. Within the nonstructural protein ORF, the location of global amino acid motifs indicative of replicase (R), helicase (H), and cysteine protease activity (P) as well as the small regions of homology between the deduced amino acid sequence of RV and SIV (X motif) is shown. Also shown are: positions of regions of nucleotide homology between RV and aiphaviruses, (open circle); subgenomic start site, (closed circle); the 3’ terminal stem-and-loop structure, (hatched circle). An expanded topography of the RV strucutral protein ORF is shown at the bottom of the diagram. Within the ORF, the positioning of the following domains of the structural proteins are shown: the hydrophilic region of C which contains a high concentration of basic amino acids and putatively interacts with the virion RNA; the hydrophobic signal sequences which proceed the N-termini of E2 and El; the transmembrane sequences of E2 and El; Y, potential N-linked glycosylation sites (the site marked with a Y is not present in the HPV-77 and M33 strain); a putative region for 0-linked glycosylation. Below the diagram are shown the location of domains which contain epitopes recognized by mouse mAbs. (N denotes domains containing epitopes recognized by neutralizing mAbs). (Frey, 1994). ,  ,  ,  •,  -  34  -  precursor (McDonald et al., 1991; Qiu et al., 1994). Therefore, it is clear that the cleavage of El and E2 signal peptides by cellular signalase gives rise to individual RV structural proteins during the processing of polyprotein precursor.  1.3.7. Posttranslational modification of RV structural proteins Capsid protein The capsid protein of RV is nonglycosylated and associates with the genomic RNA in RV infected cells to form nucleocapsid. The cDNA sequence indicates that the C protein has a maximal size of 300 amino acid residues with high percentages of arginine and proline residues (Clarke et al., 1987; Frey and Man, 1988). Capsid protein from virions migrates as a doublet in polyacrylamide gels (Suomalainen et al., 1990; Man et al., 1991; Maraucher et al., 1991), the differences in molecular weight presumably being due to the alternative sites for translation initiation. Recently it has been shown that capsid protein is phosphorylated, although the extent and function of phosphorylation is unclear (Sanchez and Frey, 1991). After the cleavage of the E2 signal peptide during translocation, the E2 signal peptide is found attached to capsid protein, which helps the capsid protein to become membrane-associated (Suomalainen et al., 1990). The E2-signal sequence-mediated membrane association of the C protein may be important in the transport of the C protein and in nucleocapsid formation. E2 glycoprotein On an SDS gel, E2 glycoprotein from virion migrates as a diffuse band with molecular weights ranging from 42 to 47 kDa. In the presence of tunicamycin, Vero cells infected with RV and  -  35  -  COS cells transfected with RV cDNAs produce E2 with a molecular weight of 29 to 31 kDa (Oker-Blom et al., 1983; Sanchez and Frey, 1991) and thus the carbohydrate contributes one-third of the molecular mass of E2. Amino acid sequence predicted from E2 cDNAs reveals a protein of 281 residues including three potential N-linked glycosylation sites in M33 (Clarke et al., 1987)  G 40 S (.—11000 b)  catj  1 A A(A)  -  -  —  • —  •  ®  24 S (—3500 b) AA(A)3  A  p110 NH ) 2 —  translation  I  1CQOH  processing capsid  -  C 33K  E2 30K  •  jj  envelope II  glycosylation  E2a (47K) E2b (42K)  El 53K ,  58K  Fig.3 General strategy for the expression and processing of RV structural proteins. (Oker-Blom et al., 1984).  -  36  -  and HPV77 (Zheng et al., 1989) strains as opposed to four in Therien (Vidgren et aT., 1987; Frey and Marr, 1988) and RA27/3 (Frey et aL, 1986) strains. In addition to N-linked glycans, E2 is known to contain 0-linked carbohydrates (Sanchez and Frey, 1991; Lundstrom et al., 1991). Digestion with glycosidases and lectin-binding assays reveal that N-linked glycans on E2 from virions contain high-mannose, hybrid-type and complex-type (Putnam and Therien strains) (Bowden and Westaway, 1985; Sanchez and Frey, 1991) or only complex-type, four branched sugars (M33 strain) (Lundström et al., 1991), with the majority of complex-type terminating in galactose and some fraction having terminal sialic acid (Sanchez and Frey, 1991; Lundstrom et al., 1991). The heterogeneous processing of both N-linked and 0-linked glycans on E2 contributes to the diffuse nature of E2 on an SDS gel. Expression of E2 in vitro and in vivo from cDNA constructs demonstrates that translocation of E2 into the lumen of the rough ER is mediated by a signal peptide residing in the C-terminus of the capsid protein, and this sequence can function externally as well as in its native internal context (Hobman and Gillam, 1989; Marr et al., 1991; Sanchez and Frey, 1991). Following translocation, N-linked glycosylation of E2 takes place. Processing of N-linked glycans on E2 involves at least two stable intermediates, a 39 kDa high mannose-containing precursor and a 42 kDa form bearing some complex-type sugars (Hobman and Gillam, 1989; Hobman, et al., 1990). Although it has been shown that E2 contains 0-glycans, the site of 0-linked glycosylation and the extent of processing have not been defined. So far the importance of N and 0-linked oligosaccharides on E2 in virion assembly and infectivity is unknown.  -  37  - El glycoprotein El is the dominant surface molecule of the RV virion (Ho-Terry and Cohen, 1984; Terry et al., 1988). El migrates as a discrete band with apparent molecular weight of 57 kDa. Nonglycosylated El synthesized in tunicamycin-treated cells has a molecular weight of 53 kDa (Oker-Blom et al, 1983; Bowden and Westaway, 1984; Sanchez and Frey, 1991). Deduced amino acid sequence shows that El is 481 amino acid residues in length with three potential N-linked glycosylation sites (Frey et al., 1986; Clarke, et al., 1987). 0-linked oligosaccharides are not detected in El (Lundstrom at al., 1991) whereas palmitic acid is incorporated in El (Hobman et al., 1990). There is a stretch of seven amino acids including five arginine residues (R-R-A-C-R R-R) before the putative signal peptide sequence of El and after the putative transmembrane anchor domain of E2 that may contain basic amino acid cleavage sites for endoproteases. Since the C-terminal amino acid sequence of E2 has not been determined, it is not known whether other proteolytic cleavages take place during the processing of the E2E 1 precursor polyprotein at the C-terminus of E2, besides the cleavage of the El signal peptide by host signal peptidase. A recent mutational study of this region shows that the cleavage of the E2E1 polyprotein precursor is impaired when the signal peptide cleavage site alone, or both arginine clusters are altered, whereas partial cleavage is observed in the mutants in which one of the two arginine clusters is modified (Qiu et al., 1994). These data indicate that the arginine clusters do not function as a basic protease cleavage site, rather, they contribute to maintaining the proper configuration of that region for access by cellular signal peptidase.  -  38  - Conformation of structural proteins Capsid protein forms a noncovalently bound dimer soon after translation in RV-infected cells as well as in cells infected with a vaccinia recombinant virus expressing C protein (Baron and Forsell, 1991). However, covalently linked C dimers are detected only in RV-infected cells but not in vaccinia recombinant-infected cells (Baron and Forsell, 1991). Similarly, disulfide-bound El-El homodimers and El-E2 heterodimers are routinely observed when rubella virions are subjected to non-reducing SDS-PAGE (Waxham and Wolinsky, 1983; Dorsett et al., 1985), whereas such glycoprotein complexes are not detected when El and E2 are expressed from cDNA (unpublished results). It is possible that the formation of intermolecular disulfide bonds of El and E2 occur after virions are released from the infected cells and exposed to a relatively oxidative environment in the medium. Besides intermolecular disulfide bonds, intramolecular disulfide bonding is found in El and E2 which is important to the maintenance of proper conformation for antibody binding (Green and Dorsett, 1986; Wolinsky et al., 1991), protein stability, hemagglutination activity and infectivity (Ho-Terry and Cohen, 1981; Katow and Sugiura, 1988). Intracellularly, El and E2 form noncovalently associated heterodimers in RV-infected cells as well as when they are expressed from cloned cDNAs (Baron and Forsell, 1991; Hobman et al., 1993). The association of El and E2 increases the intracellular transport rate of E2 (Hobman et al., 1990) and releases El from retention in a post-ER, pre-Golgi compartment (Hobman et aL, 1993).  -  39  -  1.3.8. Intracellular localization of RV structural proteins In an indirect immunofluorescence study, RV glycoproteins El and E2 were shown to be concentrated in the juxtanuclear region of both RV-infected cells and cDNA transfected cells expressing all three structural proteins of RV. This region represents a reticular structure which may span from the ER to the Golgi stacks. Low level cell surface expression of El and E2 is also observed (Hobman et aL, 1990). The capsid protein is also localized in the Golgi-like area, presumably due to its membrane association as well as the interaction with El and/or E2 (Hobman et al., 1990; Baron et al., 1992). However, when each protein is expressed separately from cloned cDNA, a different intracellular distribution pattern is observed. The capsid protein is found in a reticular structure extending throughout the cells (Baron et al., 1992). E2 is in the ER, the Golgi cisternae and the cell surface, whereas El is retained in a pre-Golgi structure (Hobman et al., 1990). Fine mapping of the El and E2 intracellular localization using immunogold labelling reveals that El, when expressed alone, is arrested in a novel post-ER, pre Golgi compartment near the exit site of the ER (Hobman et al., 1992). Although the co expression of El and E2 releases such retention, they are targeted to the Golgi complex, and are not efficiently transported to the cell surface (Hobman et al., 1993).  1.3.9. Biological function of RV structural proteins The major biological activities associated with the RV virion (structural proteins) are hemagglutination (HA) (Schmidt et al., 1968) and low-pH induced cell-cell fusion of infected cells (Vaananen et al., 1980). There is evidence linking both HA and fusion activities to RV El. Trypsin treatment of virions under conditions which digest El with minimum damage to E2  -  40  -  shows that such digestion results in the loss of the HA activity (Ho-Terry and Cohen, 1981). Murine mAbs that exhibit hemagglutination inhibition (HAT) activity, are all anti-El but not anti E2 (Waxham and Wolinsky, 1983; Green and Dorsett, 1986; Chaye et al., 1992). More direct evidence is that El but not E2 expressed via vaccinia virus recombinants can induce the production of hemagglutination inhibitory antibodies (Gillam, unpublished result). Brief exposure of RV infected cells to a pH of 6.0 or lower results in syncytium formation, presumably mediated by the RV glycoproteins expressed at the plasma membrane (Katow and Sugira, 1988). RV virions also induce fusion of erythrocytes (Vaananen et al., 1980) and the virions gain the ability to bind liposomes (Katow and Sugiura, 1988) after incubation in acidic media. The basis of the fusogenic activity in RV is not well defined but is thought to reside in El.  1.3.10. Immune responses to RV infection Natural RV infection or RV vaccination leaves a long-lasting immunity, which is attributed to circulating antibodies. The initial response following infection or vaccination is a transient 1gM response and in most cases is El specific (Partanen et al., 1985). Although other immunoglobulin classes (IgE, IgA) are stimulated subsequently, IgG production is the dominant serological response. Persisting IgG antibodies are directed to all three structural proteins of RV, although the predominant reactivity is against El (Katow and Suguira 1985; Zhang et al., 1992; Chaye et al., 1992), indicating an important role of El in inducing a protective immunity against RV infection. IgG antibodies to RV may have HAT and viral neutralizing (VN) properties (Green and  -  41  -  Dorsett, 1986; Waxham and Wolinsky, 1985). There is a good correlation between the levels of IgG to RV, and classically measured HAT titres and neutralizing antibody titres in seropositive sera (Stokes et al., 1969). It is assumed that these responses play a positive role in viral clearance and protection (Waxham and Wolinsky, 1985a). Circulating immune complexes containing RV specific antibody and antigen are frequently found after RV infections (Ziola et al., 1983) but in most cases their presence has not been associated with any of the complications following RV infection or vaccination (Singh et al., 1986). Much less is known about the importance of cellular responses to RV infection. RV specific cellular responses have been demonstrated using lymphocyte proliferation assays and lymphocyte mediated cytotoxicity assays (Buimovici-Klein and Cooper, 1985; Vesikari and Buimovici-Klein, 1974; Ilonen and Salmi, 1986). Cell-mediated cytotoxicity has been implicated in the pathogenicity of RV infection (Martin et al., 1989). In these studies, intact RV was used as the antigen for the analysis of proliferation responses. Only recently, Chaye et al. (1992) demonstrated antigen-specific lymphocyte proliferative responses in peripheral blood lymphocytes using an in vitro proliferative assay with vaccinia recombinants expressing individual RV structural proteins. In human populations, each individual exhibits different responses to El, E2 and C; however, El is the dominant antigen to which the majority of subjects develop lymphocyte proliferative responses (Chaye et al., 1992). Proliferative responses to purified, intact RV are major histocompatibility antigen (HLA) restricted (Ilonen and Salmi, 1986). Similarly, Ou et al. (1992a,b,c) isolated T-cell clones against E2 glycoprotein and C protein from RV seropositive individuals and found that HLA restrictions were associated with HLA DR7 for E2 epitopes, and HLA DR4 for C epitopes.  -  42  -  1.3.11. Immunological determinants on RV structural proteins To characterize the antigenic determinant on RV structural proteins, panels of murine mAbs have been generated and biological activities of these antibodies have been analyzed. These panels are made up primarily by El-specific antibodies with a rare number of antibodies recognizing E2 or C (Waxham and Wolinsky, 1985; Green and Dorsett, 1986; Chaye et al., 1992). In 1985, Waxham and Wolinsky (1985) mapped HA and VN activities to the El glycoprotein. Since this study, much effort has been focused on delineating functional epitopes on El (Terry et al., 1988, 1989; Lozzi et al., 1990; Wolinsky et al., 1991). Numerous methods to localize HA or VN epitopes have been utilized including recombinant DNA technology (Terry et al., 1989; deMazancourt and Perricaudet, 1989; Wolinsky et al., 1991), peptide analysis (Lozzi et al., 1990; Terry et al., 1988; Mitchell et al., 1992) and competitive binding assays with mAbs (Waxham and Wolinsky, 1985a). Six independent epitopes have been identified which are thought to be important for viral infectivity and HA (Green and Dorsett, 1986; Waxham and Wolinsky, 1985a). Three non-overlapping linear epitopes that react with mAbs having HAl and VN activities have been localized to El residues 245 to 285 (Terry et al., 1988). Wolinsky et al. (1991) and Chaye et al. (1991) separately mapped a region between residues 202 to 283 of El which consists of overlapping epitopes recognized by mAbs with VN, HAT or VN and weak HAT activities (Wolinsky et al., 1991; Chaye et al., 1992). Using a set of nested synthetic peptides, these two groups subsequently narrowed the epitope to a region between residues 213 and 239, or between residues 214 and 240, respectively (Chaye et al., 1992; Wolinsky et al., 1993). Contrary to those results with El, B cell-epitope mapping on E2 and C has been less informative, due to the lower number of mAbs available. Only one of the E2-specific mAbs has been found to possess VN  -  43  -  activity (Green and Dorsett, 1986). It was reported recently that an anti-C mAb recognizes a 52 kDa f3-cell antigen, implying that the C protein may be involved in molecular mimicry leading  to initiation of an immunopathological process (Karounos et al., 1993). Protective immunity to viral infection requires activation of helper T cells specific for viral antigens. Ou et al. (1992a, b, c, 1993) identified T-cell epitopes on RV structural proteins by screening a nested set of overlapping synthetic peptides with peripheral blood lymphocytes from immune donors and subsequently with T-cell lines/clones derived from the peripheral blood lymphocytes of immune donors. They identified regions between residues 358 and 377 of El, between residues 54 and 74 of E2, and between residues 255 and 280 of C, as the relatively immunodominant T-cell epitopes (Ou et al., 1992a,b,c, 1993). Mitchell et al. (1993), applying essentially the same methodology, identified immunoreactive regions on El and E2 recognized by T-cells of normal healthy individuals. McCarthy et al. (1993) took a different approach, using sets of comparatively short overlapping synthetic peptides containing predicted T-cell epitope motifs of RV structural proteins within the region bearing linear B-cell epitopes defined by RV specific mAbs (C 31 to E2 105 and El 1 to , 97 E2 C 29 CM to , C 202 to E1 ). With one exception, all of 3 the synthetic peptides were able to stimulate varied but individually specific lymphoproliferative responses in peripheral blood mononuclear cells from 25 to 50% of a population of normal, RV immune donors with diverse HLA backgrounds (McCarthy et al., 1993). These studies indicate that further fine-mapping of T-cell determinants among a larger human population with HLA diversity is necessary for future construction of an effective synthetic peptide vaccine for RV.  -44-  1.3.12. Project rationale and thesis objectives Viruses utilize the host cell machinery for the synthesis and processing of viral proteins. Viral proteins undergo a series of structural modulations during transport from the site of synthesis to the site at which they are incorporated into virions, and become functionally competent. Posttranslational modifications are a major focus of studies on structure/function relationship of proteins, and among them, glycosylation has been studied exhaustively. RV contains two membrane glycoproteins El and E2. In recent years, although studies on a) the structure of carbohydrates on El and E2 (Sanchez and Frey, 1991; Lundstrom et al., 1991), b) the intracelluar transport and processing of El and E2 (Hobman and Gifiam, 1989; Hobman et al., 1990; Sanchez and Frey, 1991; Baron and Forsell, 1991; Marr et al., 1991; Baron et al., 1992) and c) the analysis of immunological determinants on El and E2 (Wolinsky et al., 1991; Chaye et al., 1992) have greatly strengthened our knowledge about RV, little is known about the functional role of N-linked glycosylation on RV El and E2. In this study, the importance of N-linked oligosaccharides on RV El and E2 has been investigated with respect to its biological functions during replication and infection. The approaches taken involve a combination of recombinant DNA technology and mammalian cell expression. The thesis describes two lines of experiments. The first line of experiments is directed at the cell biology aspects of El and E2. Studies were initiated to define the role of N-linked glycosylation on processing and transport of E2, and these experiments were extended to investigate the correlation between the sorting of the El and E2 glycoproteins and virus assembly using two protein transport inhibitors, brefeldin A and monensin. The second line of experiments are focused on vaccine development. In view of the fact that the immunoreactivity of RV El  -  45  -  glycoprotein is very dependent on its native conformation, the influence of N-linked glycosylation of El on its antigenicity and immunogenicity was analyzed. The outcome of these experiments led to the expression and characterization of the virus-like particles containing RV structural proteins, and studies of their immunological properties. The potential application of these viruslike particles as an antigen sources for serodiagnostic assays and vaccine development will be discussed.  -  46  -  2. MATERIALS AND METHODS  2.1. MATERIALS and SUPPLIES DNA modifying enzymes and restriction endonucleases were purchased from Bethesda Research Laboratories (BRL), Promega, New England Biolabs, Boehringer Mannheim, Pharmacia and United States Biochemical Corporation. All enzymes were used as specified by the manufacturer unless indicated otherwise. L-[ S]-methionine (600-800 Ci/mmole) was from Du Pont Inc. Tissue 35 culture reagents were from Gibco (Gaithersburg, MD) or Sigma (St. Louis, IL). Brefeldin A was purchased from Boehringer Mannheim. Tunicamycin and monensin were products of Sigma. GENECLEAN (BlO 101) was obtained from Promega. Human polyclonal anti-rubella serum was provided by Dr. A. Tingle (B.C. Children’s Hospital, Vacouver, B.C.). Mouse monoclonal antibodies against RV El were generated in this lab. Mouse monoclonal antibodies against RV E2 and capsid protein were generously provided by Dr. J. Safford (Abbott Laboratories, Chicago, IL) or Dr. J. Wolinsky (University of Texas, Houston, TX). Fluorescein (FITC)-conjugated goat anti-mouse or anti-human IgGs were from Kirkegaard & Perry Laboratory. IThodamine (TRICT) conjugated goat anti-mouse or anti-human antibodies, and lectins were from Zymed. A TRICT conjugated rabbit anti-Golgi protein serum was prepared in the laboratory of Dr. F. Tufaro (University of British Columbia, Vancouver, B.C.). COS, CV-l, i1Cl43, BHK, Vero and RK cells, rubella virus M33 strain and vaccinia virus WR strain were obtained from the American Type Culture Collection.  -  47  -  2.2. METHODS 2.2.1. Propagation of bacterial strains E.coli strains DH5o from BRL were used for the propagation of recombinant clones. DH5o cells containing recombinant plasmids were grown in LB medium (1% tryptone; 0.5% yeast extract; 1% NaC1) containing 100 ug/ml ampicillin (AP) for selection of antibiotic resistance. For long term storage the bacterial strain was stored in 15% glycerol at -70°C.  2.2.2. Preparation of competent cells and transformation Competent cells were prepared using a method described in Promega technical bulletin 018. Briefly, E.coli cells were grown in 20 ml of LB medium until the absorbance at 600 nm reached 0.15-0.3. Cells were centrifuged at 5000 rpm in a Sorvall SS34 rotor at 4°C for 5 minutes, and the supernatant was discarded. The bacterial pellet was resuspended in 10 ml of cold solution A (10 mM 3-[N-morpholino] propanesulfonic acid (MOPS) (pH 7.0); 10 mM RbCl), and centrifuged as above. Cells were then resuspended in 10 ml of cold solution B (10 m’l MOPS (pH 6.5); 10 mM RbC1; 50 mM CaC1 ) and incubated on ice for 30 minutes. After pelleting the 2 cells as above, cell pellets were resuspended in 1 ml of solution B plus 15% glycerol, and quick frozen in 0.2 ml aliquots in dry ice-ethanol and stored at -70°C. For plasmid transformation, 0.2 ml of competent cells were incubated on ice with 10-50 ng of plasmid DNA for 30 minutes. After a two minute heat shock at 42°C, 1 ml of LB medium was added to the transformation mixture and the cells were allowed to recover at 37°C for 45 minutes before plating onto selective media.  -  48  -  2.2.3. DNA preparation and handling Mini-prep plasmid isolation Colonies containing plasmids were picked into 3-5 ml of LB containing 100 ig AP per ml and the bacteria were grown to saturation. Bacterial cells from 1.5 ml culture were pelleted for one minute in a microfuge. The pellet was resuspended in 100 ul of 50 mM glucose; 10 mlvi EDTA; 25 mM Tris-HC1 (pH 8.0) and lysed by the addition of 200 il of 0.2 N NaOH/1% SDS for 5 minutes at 0°C. Chromosomal DNA and proteins were precipitated by incubating the lysis mixture with 150 il of cold potassium acetate (3 M K; 5 M CH COO, pH 4.8) at 0°C for 5 3 minutes, followed by centrifuging in a microfuge for 5 minutes at 4°C. The supematant was extracted with an equal volume of phenol:chloroform (1:1), and the DNA precipitated with two volumes of ethanol at room temperature (RT) for 5 minutes. Plasmid DNA was recovered by centrifugation in a microfuge for 5 minutes at RT, washed in 70% ethanol, dried in a Speed Vac Concentrator, and resuspended in 50 il of TE (10 mM Tris-HC1, pH 8.0; 1 mM EDTA) containing 20 ag/m1 RNase A. Aliquots were used for restriction analysis or subcloning. This method is essentially that described by Maniatis et al. (1982). Large scale plasmid DNA preparations The protocol is a procedure obtained from Promega technical bulletin 009 (developed by Dr. P. Krieg and Dr. D. Melton of Harvard University) with modifications. Cells grown in selective media overnight in 100 ml cultures were pelleted by centrifugation at 5000 rpm in a Sorvall GSA rotor at 4°C for 5 minutes. The supernatant was discarded and each pellet was resuspended in 3 ml of 50 mlvi glucose; 10 mM EDTA; 25 mM Tris-HC1 (pH 8.0) containing 20 mg/mi  -  49  -  lysozyme followed by 20 minute incubation on ice. Cells were lysed by the addition of 6 ml of 0.2 N NaOH/1% SDS and incubation on ice for 10 minutes. Chromosomal DNA and proteins were precipitated with 4 ml of cold potassium acetate solution (see mini-prep procedure) on ice for 20 minutes, followed by centrifugation at 15,000 rpm in a Sorvall SS34 rotor at 4°C for 15 minutes. RNase A (100 ug) was added to the cleared lysate followed by incubation at 37°C for 20 minutes. The lysate was extracted twice with equal volumes of phenol:chloroform, and the nucleic acids were precipitated with one volume of isopropanol at RT for 5 minutes. Remaining nucleic acids were recovered by centrifuging at 15,000 rpm for 10 minutes at 4°C in a SS34 rotor.  The pellet was dried, and dissolved in 1.60 ml of sterile water. The solution was  transferred to siliconized Corex tubes and DNA was selectively precipitated by the addition of 0.4 ml of 4 M NaCl and 2.0 ml 13% polyethylene glycol (PEG, MW 8,000), mixing and incubation on ice for 60 minutes. The plasmid DNA was pelleted at 10,000 rpm for 10 minutes at 4°C in a SS34 rotor, washed with 70% ethanol, dried and dissolved in TE. Restriction endonuclease digestions and DNA modification All restriction digestion reactions were performed according to assay conditions specified by the suppliers. DNA fragments were ligated using T4 DNA ligase in 50 mM Tris-HC1, pH7.6; 10 mM ; 1 mM ATP; 1 mM DTT; 5% (w/v) polyethylene glycol for 2 hours at RT, except for 2 MgC1 blunt-ended fragments which were ligated overnight. Reactions were diluted five-fold with TE prior to transformation (Maniatis et al., 1982). DNA fragments with 5’ overhangs were blunt-ended with E.coli DNA polymerase I Kienow  -  50  -  enzyme in 50 mM Tris-HC1, pH 7.2; 10 mM MgSO ; 10 mM DTT; 50 mM BSA; 80 pM dNTP’s 4 for 30 minutes at RT. The enzyme was inactivated by heating at 70°C for 5 minutes (Maniatis et al., 1982). Fragments with 3’ protrusions were converted to flush ends using T4 DNA polymerase in 33 mM Tris-acetate, pH 7.9; 666 mM potassium acetate; 10 mM magnesium acetate; 0.5 mM DTT; 100 mg/mi BSA; for 5 minutes at 37°C. Reactions were adjusted to 25 mM EDTA, and the DNA purified by phenol:chloroform extraction and ethanol precipitation (Maniatis et al., 1982), or using GENECLEAN (BlO 101). Removal of terminal 5’ phosphates from DNA fragments with 5’ overhangs was done using calf intestinal alkaline phosphatase (CIP) in 50 mM Tris-HC1, pH 9.0; 1 mM 2 MgCI 0.1 mM ; ; 1 mM spermidine for two successive 30 minute incubation periods of 15 minute at 37°C 2 ZnC1 and 15 minutes at 56°C.  CIP reactions were terminated by addition of 0.3% SDS and  phenol:chloroform extraction followed by ethanol precipitation (Maniatis et al., 1982). Purification of DNA fragments from agarose gels or enzyme reaction mixtures was routinely done using GENECLEAN. Desired fragments were excised from ethidium bromide stained TAE agarose gels (see and the gel matrix was solubilized in 2-3 volumes of saturated sodium iodide at 55°C. DNA was removed from the agarose solutions by vortexing the mixture with a suspension of glassmilk, and a brief spin in a microfuge. Contaminants were washed away from the glass bound DNA by three successive washes with cold NaClJethanol/water solution. The DNA was eluted from the glass beads with TE or water by incubating at 55°C for 3 minutes.  -  51  -  2.2.4. Expression vectors pCMV5 For transient expression of RV cDNA in COS cells, pCMV5 (Andersson et al., 1990) was used (Fig.4a).  This vector directs transcription by the human cytomegalovirus major  immediate early gene promoter and provides polyadenylation signal from the human grown hormone gene at the 3’ terminus of the inserted sequence. pCMV5 contains the SV4O origin of replication allowing replication in COS cells as well as a prokaryotic origin of replication and AP resistance gene for growth and selection in E.coli. pGS2O For construction of vaccinia recombinants, vector pGS2O (Fig.4b) (Mackett et al., 1985) was used. This vector contains the vaccinia virus immediately early gene promoter p7.5, flanked by sequences from the vaccinia virus thymidine kinase gene. The AP resistance gene and prokaryotic replication origin allow propagation of the recombinant plasmid in E.coli. pNUT Vector pNUT (Fig.4c) (Palmiter et al., 1987) was used to construct stable transformed BHK cells. RV cDNAs were cloned into this vector at the Sma I site which is flanked by the mouse metallothionein gene (mIvlT-1) promoter and the 3’ polyA sequences of the human growth hormone (hGH). The presence of the dihydroxyfolate reductase (DHFR) cDNA permits the selection of transfected BHK cells in the presence of a high concentration of methotrexate. Sequence from the human hepatitis B virus 3’ end mediates insertion of adjacent DNA sequences  -  52  -  into the chromosome of host cells (Nagaya et al., 1987). The pUC18 backbone and SV4O origin facilitate replication of the plasmid in E.coli and mammalian cells, respectively.  2.2.5. DNA-mediated transfection Transfection of COS cells COS cells were transfected with plasmid DNA using a method described by Adam and Rose (1985). Subconfluent monolayers of cells grown in Dulbecco modified Eagle medium (DMEM) plus 5% fetal calf serum (FCS) were washed twice with Tris-saline (25 mM Tris-HC1 (pH 7.4), 140 mM NaC1, 3 mM KC1, 1 mM CaC1 , 0.5 mM MgC1 2 , 0.9 mM 4 2 HPO 2 Na ) . Cells were incubated with DEAE-dextran (Mr=5 X iO; 500 jig/mi) and plasmid DNA (4 jig/mi) in Tris-saline at 37°C for 30 minutes. The DNA solution was then removed and replaced with DMEM plus 40 uM chioroquine for 3 hours at 37°C. After removal of chioroquine solution, the cells were shocked with 10% dimethylsulfoxide/DMEM for 3 minutes at RT. Finally, the monolayer was washed three times with Tris-saline and incubated at 37°C for 40 hours in DMEM plus 5% calf serum. The expression of RV proteins was analyzed using metabolic labelling or immunoblotting (see below). Calcium-phosphate mediated DNA transfection Transfection of CV1 cells and BHK cells with plasmid DNAs were according to Gorman et al., (1982). CaPO JDNA mixture was prepared by combining 10-25 jig plasmid DNA in 219 jil of 4 O, 31 1 2 ddH il of 2M CaC1 2 and 250 jil of 2xHBSP (1.5 mM 4 HPO 10 mM KC1; 280 mM 2 Na ; NaCl; 12 mM glucose; 50 mM HEPES, pH 7.0). The mixture was allowed to stand for 30  -  53  -  11  on  on SV 40  C mMT-l  H8V3 -RV dJNA  hGH 3  pUC 18  Fig.4 Schematic representation of mammalian cell expression vectors used in this study. a. pCMV5. The vector backbone is pTZ18R (Pharmacia) and contains a bacteriophage fi ampicillin resistance gene (Amps). The CMV region consists of a promotor-regulatory region of the human cytomegalovirus major immediate early gene. b. pGS2O. The vector contains the promotor for an early gene coding for a 7.5 kDa polypeptide and is placed upstream from the unique restriction endonuclease Sma I site, flanked by vaccinia thymidine kinase (TK) DNA (thick line). c. Recombinant plasmid from pNUT vector. The backbone for this vector is pUCl8. The essential features of this vector are: DNA sequences taken from the 3’ termini of human hepatitis B virus genome (HBV 3’); a promotor region from mouse metallothionein I gene (mMT-1); and sequence for dihydrofolate reductase (DHFR). RV cDNAs were inserted into the Sma I site between the mIvIT-1 and hGH 3’ sequences.  -  54  -  minutes at RT prior to adding to the medium of cultured cells. Cells were incubated for different periods of time before removing the DNA mixture. The time of incubation depended on the cell type and nature of experiment.  2.2.6. Construction of vaccinia recombinants Vaccinia virus recombinants expressing RV El glycosylation mutant proteins were constructed following a standard procedure as described by Mackett et al., (1985). Infection/transfection procedure Confluent monolayers of CV1 cells in Minimal Essential Medium (MEM) were infected with purified vaccinia virus (WR strain) at a ratio of 0.05 p.f.u./cell. Inoculum was removed at 2 hours post infection (h.p.i.). Cells were washed twice with serum-free medium and 0.5 ml of DNA suspension (CaPO JDNA) (see was added to the cells and the cells were incubated for 4 30 minutes at RT prior to the addition of MEM/5% FCS. Cells were scraped into the medium at 48 h.p.i. and viruses were released by three cycles of freeze-thawing. Selection of recombinants One-fifth of the released viruses were layered onto monolayers of human tk 143 cells (gift from F. Graham, McMaster University) and incubated for 1 hour at 37°C. The inoculum was removed and cells were incubated with Eagles medium containing 5% FCS and 25 .ig/ml 5bromodeoxyuridine (BUdR). Progeny virus was harvested at 48 h.p.i., by scraping cells into medium and then three rounds of freeze-thawing.  -  55  - Plaque purification and virus titration Monolayers of CV1 cells were infected with 0.5 ml of a ten fold serial dilution of virus and incubated at 37°C for one hour, with occasional shaking. The inocula were removed, cells were washed with MEM once and overlaid with MEM containing 5% FCS and 1% noble agar. Cells were stained at 36 h.p.i. with 1% agarose containing 0.1% neutral red. Clear virus plaques were visualized after incubation for 2-3 hours. Plaques were counted and virus infectivity was calculated as plaque forming units/mi (pfu/ml). Well isolated plaques were picked into a Pasteur C. 0 pipette and virus in agarose plugs were eluted into 0.5 ml MEM and stored at -70 Large-scale virus purification 2 flask) were infected with wild type or recombinant Monolayers of CV-1 cells (in 175 cm vaccinia viruses at a multiplity of infection (MOl) of 5 and incubated for 48 hours. Cells were harvested and resuspended in 10 mM Tris-HC1, pH 9.0 and homogenized. The nuclear pellet was removed after centrifugation at 750xg for 5 minutes at 4°C. Trypsin (0.25 mg/mi) was added to the supernatant and incubated for 30 minutes at 37°C. The supernatant was then layered on top of an equal volume of 36% sucrose in 10 mM Tris-HC1 pH 9.0 and centrifuged at 13,500 rpm in a Beckman SW27 rotor for 80 minutes at 4°C. The pellet was resuspended in 2 ml of 1 mlvi Tris-HC1 pH 9.0 and layered onto continuous sucrose gradients (15-40% in 1 mM Tris-HC1, pH 9.0). The centrifugation was carried out at 4°C, 12,000 rpm for 45 minutes. Banded virus was collected with a syringe through the side of the tube and stored at -70°C.  -  56  -  2.2.7. Metabolic labelling (i). COS cells. Labelling of COS cells was performed according to Hobman and Gillam (1989). Briefly, 40 hours post-transfection, transfected cells (in 35 mm dishes) were washed once  and incubated with methionine-deficient DMEM for 30 minutes prior to the addition of 0.5 ml methionine-deficient DMEM containing 100 jiCi [ S]-methionine 3 5 (Du Pont) and 5% FCS dialyzed against phosphate-buffered saline (PBS). Incubation with [ S]-methionine-containing 3 5 medium was for 30 minutes. Some cells were further incubated with a chase medium containing 2 mM unlabelled methionine for various periods of time. Cells were washed with cold Tris-saline and lysed with 500 a1 of RIPA buffer (1% Triton X-100; 10 mlvi EDTA; 50 mM Tris-HC1, pH 7.5; 1% sodium deoxycholate; 0.15 M NaCI; 0.1% SDS). After keeping on ice for 5 minutes, lysates were scraped off the plates and cleared of nuclei and debris by centrifugation at 4°C for 5 minutes at 13,000 rpm. The supernatants were subjected to immunoprecipitation. (ii). Vero cells. Labelling of RV virions released from RV-infected Vero cells was carried out as described by Clarke et al. (1987). At 24 h.p.i., infected cells (in 60 mm dishes) were incubated with methionine-deficient medium for 30 minutes and labeled with 100 pCi S]35 [ methionine for 1 hour. Cells were washed with and incubated in MEM with 2.5% FCS for various periods of time. Medium samples were collected and an equal volume of 20% PEG (MW 8,000) in 2.5 M NaCl were added. RV particles were precipitated by centrifugation at 4°C for 10 minutes at 14,000 rpm in an Eppendorf centrifuge after incubation on ice for 1 hour. The virus  pellets  were resuspended  immunoprecipitated  with  human  in  RIPA  anti-RV  buffer serum  autoradiography.  -  57  -  and and  RV-specific  subjected  to  proteins  were  SDS-PAGE  and  2.2.8. Immunoprecipitation Immunoprecipitation of RV structural proteins from cell lysates was performed according to Hobman and Gillam (1989). Human polyclonal anti-rubella serum, mouse serum or fluid ascites were preincubated with Protein A-Sepharose (Pharmacia) for at least 4 hours at 4°C in binding buffer (100 mM Tris-HC1 (pH 7.4); 400 mM NaC1) with constant mixing. The antibody-coated beads were washed twice with binding buffer, and once in lysate buffer (25 mM Tris-HC1 (pH 7.4); 100 mM NaC1; 1 mM EDTA; 1% Nonidet P-40). Cells lysates or harvested media were added and mixed with the antibody-coated beads for over 8 hours at 4°C with constant rotation. Beads were washed once with lysate buffer, twice with wash buffer (25mM triethanolamine; 172 mM NaC1; 1% SDS; 1 mM EDTA), three times with 10 mM Tris-HC1 (pH 7.4), and once with water. Antigen-antibody complexes were dissociated from the Protein A-Sepharose by boiling in 1 X SDS dissociation buffer (see below) for 5 minutes, vortexing and pelleting the beads by centrifugation. Supernatants were collected and used for further analysis.  2.2.9. Endogycosidase digestion The conditions for endoglycosidase digestion were essentially those described by the manufacturer. Digestion with endoglycosidase H (endo H, Boehringer Mannheim) was carried out in 100 mM sodium citrate buffer (pH 5.5) containing 0.15% SDS. Digestion with 0glycosidase, endoglycosidase F/N-glycosidase F (endo F/PNGase F) and neuraminidase (all from Boehringer Mannheim) were performed in a buffer containing 20 mM sodium phosphate pH 7.0, 10 mlvi n-octylglucoside, 0.1%SDS. Immunoprecipitates to be digested with N-glycanase were adjusted to 100 mM sodium phosphate (pH 8.6), 1% Nonidet P-40, 100 mM EDTA, 0.5% 3-  -  58  -  mercaptoethanol, 0.1% SDS. All incubations were normally for 8 hours.  2.2.10. Immunoblotting RV antigens were separated by SDS-PAGE and transferred to nitrocellulose filters using a Bio-Rad Trans-Blot apparatus for 60 minutes at 240 mA in 25 mM Tris-HC1; 192 mM Glycine (pH 8.3); 20% methanol. The non-binding sites on filters were blocked by incubating for 30 minutes to overnight in TBS (25 mM Tris-HC1, pH 7.4; 150 mM NaCl) containing 4% powdered skimmed milk. Membranes were then incubated with human anti-RV serum or monoclonal antibodies (at appropriate dilutions) for 1 hour, washed with TBS/0.3% Tween-20 and treated with goat anti-human or goat anti-mouse IgG conjugated to alkaline phosphatase (BRL) for 1 hour. Blots were washed as above and developed with NBT (nitro blue tetrazolium)/BCIP (5bromo-4-chloro-3-indoyl phosphate). All incubations were done at RT.  2.2.11. Indirect immunofluorescence Transfected COS cells grown on polylysine-coated 9 mm glass coverslips were washed three times with PBS, and fixed for 20 minutes at RT in 2% formaldehyde/PBS, followed by washing with PBS. Some cells were permeabilized with 0.1%NP-40/PBS for 30 minutes prior to blocking with 1% BSA/PBS. BSA/PBS was substituted for PBS in all dilutions and washings after this step. Coverslips were overlaid with diluted human serum (1:200) or murine monoclonal antibodies (1:100), incubated for 60 minutes at RT, and washed. Incubation with secondary antibodies, fluorescein-conjugated goat anti-human or anti-mouse IgG (diluted 1:100) was for 60 minutes. For double-labelling using lectin-conjugates, permeabilized cells were incubated with  -  59  -  wheat germ agglutinin-rhodamine conjugated (WGA-TRICT) to visualize Golgi and post-Golgi structures or concanavalin A-rhodamine conjugated (Con A-TRICT) for ER staining at 10-15 jig/mi for 30 minutes at RT prior to blocking with BSA.  2.2.12. Electrophoresis Separation of DNA fragment The buffers used in agarose gel electrophoresis were 1XTAE (40 mM Tris-acetate, pH 8.0; 1 mM EDTA) and 1XTBE (89 mM Tris; 89 mM boric acid; 2 mM EDTA; pH 8.0) for separation of small fragments. The gel concentration varied from 1% to 2% agarose with 1 jig/mI ethidium bromide for visualization. DNA samples were diluted to 8% sucrose; 20 mM EDTA (pH 8.0); 0.05% bromophenol blue; 0.05% xylene cyanol and separated by electrophoresis on 10 cm submarine horizontal agarose gels at 75 volt. Separation of protein Proteins were separated using a discontinuous gel system described by Laemrnli (1970). Samples were adjusted to 62.5 mM Tris-HC1 (pH6.8); 10% glycerol; 2% SDS; 2% f3-mercaptoethanol and denatured at 95°C for 3 minutes. Stacking gels consisted of 4% polyacrylamide, and separating gels contained either 10% or 11% polyacrylamide. Solutions used to prepare these gels are described in (below). Gels were run at constant voltage of 115-125 volts until the markers have run to the desired position. The stacking gel was trimmed away, and the proteins were either fixed in 10% acetic acid for 15 minutes for fluorography or transferred to nitrocellulose membrane for immunoblot analysis. Fixed gels were immersed in the fluorographic  -  60  -  agent Amplify (Amersham) for 15 minutes, dried under vacuum and exposed to X-ray film at  -  70°C. Solutions used for electrophoresis: 5X Stacking gel buffer: 0.625 M Tris-HC1 (pH 6.8), 0.5% SDS 5X Separating gel buffer: 1.875 M Tris-HC1 (pH 8.8). 0.5%SDS 5X Gel running buffer: 0.125 M Tris-HC1; 0.96M glycine, 0.5% SDS (pH 8.3) Polyacrylamide Stock: 30% acrylamide, 0.8% N’N’-bis methylene acrylamide Gels were polymerized by adding ammonium persulfate to 0.05% and TEMED (N’N’N’N’ -Tetramethylenediamine) to 0.1%.  2.2.13. RV propagation, purification and titration Virus propagation Vero cells were normally used to grow RV M33 strain in this study. Subconfluent cells (70%) were infected with RV at a MOl of 5-10 at 37°C for 2 hours. The inoculum was removed and cells were incubated with MEM with 2.5% FCS after washing once with the same medium. The medium was collected at intervals of 24 hours starting at 48 h.p.i., and replaced with fresh medium after each harvesting, till 96 h.p.i. Cell debris were cleared by centrifugation at 3,000 rpm for 5 minutes and virus particles were harvested from the medium by centrifugation at 27,000 rpm for 2 hours. Pelleted virus was suspended in PBS and stored at -70°C.  -  61  - Purification of RV or virus-like particles using sucrose density gradients Pelleted RV or virus-like particles from 35 ml tissue culture supernatant were suspended in 0.35 ml TNG buffer (50 mM Tris, pH 7.5; 100 mM NaC1; 200 mM glycine) and applied onto the top of a 12 mI-sucrose gradient of 20-50% sucrose in TNG. Centrifugation was carried out using a Beckman SW41 rotor at 90,000xg for 16 hours at 15°C. Fractions  (— 0.5 mi/fraction) were  collected by puncturing the bottom of the tube and the density of each fraction was determined using a refractometer. 100 i1 samples from alternative fractions were diluted with an equal amount of TNG buffer and subjected to centrifugation at 90,000 rpm for 20 nun, on a Tabletop centrifuge. The pellets were resuspended with RIPA buffer. RV proteins in the pellets, and in the sample that loaded onto the gradient was analyzed by SDS-PAGE and immunoblotting (using human anti-RV serum). RV structural protein-containing fractions were considered to be purified virus or virus-like-particle stocks. Titration of RV The infectivity of harvested virus or RV stock was determined using an immunochemical focus assay (Fukuda et al, 1987). Briefly, monolayers of RK cells in a 96-well plate were infected with RV in serial dilutions (10 to l0) for 2 hours at 37°C. Infected cells were washed with PBS and fixed with 3% formaldehyde in PBS for 15 minutes at 72 h.p.i.. After washing twice with PBS, 0 in absolute methanol for 15 2 endogenous peroxidase was inactivated with 0.2 ml of 0.5% H minutes at RT. The non-selective immunoglobulin binding sites of the monolayers were blocked by incubation for 1 hour at 37°C with 0.2 ml of rabbit pre-immune serum (1:200 dilution in PBS/0.5% BSA). 0.2 ml human anti-RV serum (1:200 dilution in PBS/0.5% BSA) was added and  - 62 -  incubated at 37°C for 60 minutes followed by three rinses with wash buffer. Cells were then incubated with peroxidase-conjugated rabbit anti-human IgG (1:200 in PBS/0.5% BSA) for one hour at 37°C and then were rinsed three times with wash buffer. Plaques were visualized after applying peroxidase substrate (0.1 ml PBS containing 0.02% cold H 0 and 0.5 mg/mi 3,3’ 2 diaminobezidine tetrahydrochloride) and incubating cells at RT till brown deposits developed. Plaques were counted from three wells per virus dilution and averaged. Titres of virus were expressed as pfu/ml.  2.2.14. Electron microscopy For routine morphology studies, cells (RV-infected or stable transformed cells) were fixed with 2% glutaraldehyde, 3%paraformaldehyde in 100mM NaCacodylate buffer, pH 7.2, scraped from the culture dish, and pelleted in a microfuge. The cell pellets were then postfixed (one hour) in 2% 0s0 4 in the same buffer, stained in block (2 hours) with 2% uranyl acetate. Dehydration was carried out with a graded series of ethanol and sample blocks were embedded in Epon plastic resin using standard methods (Barteletti et al., 1979). A series of thin (250 nm) plastic sections were collected on Formvar-coated slot grids after cut and analyzed.  2.2.15. Mice immunization Immunization with live, purified vaccinia recombinants. High-titer vaccinia recombinants expressing wild-type or glycosylation mutant RV El proteins were purified from infected cells by centrifugation in sucrose density gradients (see The purified recombinant viruses were titered on CV-l cells by plaque assay ( and lxl0 5 pfu  -  63  -  of each recombinant (in PBS) were used to immunize individual mice (four in each group) by intraperitoneal (i.p.) injection. Mice were re-injected 4 additional times at 3 week intervals. Four mice were immunized with each vaccinia recombinant and were bled for serum 10 days after each injection. Sera from mice immunized with the same vaccinia recombinant were pooled and used for immunological assays. Immunization with RV and virus-like particles RV or virus-like particles were semi-purified from culture medium using centrifugation. Identical amounts of antigens (equivalent to 256 HA units) were emulsified in Freund’ s complete adjuvant and used to immunize mice (four in each group). Mice received three additional injections of antigens in Freund’s incomplete adjuvant at three-week intervals. Mice were bled and sera were collected for analysis.  2.2.16. Enzyme linked immunoadsorbant assay (ELISA) RV (diluted 1:600) or individual structural proteins (diluted 1:20) expressed from recombinant baculoviruses (Gillam, unpublished results) were coated onto Immulon-2 plates (Dynatech, Chantilly, VA) in carbonate buffer [15 mM , 3 C 2 Na O 35 mM NaHCO 3 (pH 9.5)]. Following one hour blocking in 0.5% skim milk-PBS, the plates were incubated with monoclonal antibodies (ascites fluid or tissue culture supernatant), mouse sera or human sera diluted in 0.5% skim milkPBS. The one hour incubation was followed by the addition of alkaline phosphatase-conjugated goat anti-mouse or anti-human IgG antibodies (BRL) diluted 1:3000. The plates were developed in substrate buffer [1M diethanolamine, 5 mM MgCl , 2 mg/mi p-nitro-phenylphosphate (pH 9.6)] 2  -  64  -  and read at 405 nm on a Bio-Rad microplate reader (Bio-Rad, Richmond, CA).  2.2.17. Hemagglutination (HA) assay and hemagglutination inhibition (HAl) assay HA and HAT assays were performed using a heparin/manganous chloride procedure (Liebhaber, 1970). RV antigens (25 tl) were serially diluted (two fold) with HSAG (25 mM HEPES, pH 6.5; 140 mM NaC1; 1 mM CaC1 ; 1% BSA; 0.0025% gelatin) and seeded on a polyvinyl plate. After 2 chilling the plate at 4°C for 15 minutes, 50 }Jl of 0.25% one day old chick erythrocyte suspension was added to each well. Aggregation of chick erythrocytes was developed after incubation at 4°C for one hour in some wells and the HA titre of the antigen was expressed as the end-point of serial dilution at which full agglutination was observed. For HAT assay, serum samples or ascites fluid (200 }Jl) were pre-treated with 200 il of /heparin solution (0.5 M MnC1 2 MnC1 , 2500 lU/mi Porcine heparin). Following the 15 minutes 2 of incubation, 200 p1 of a 50% chick erythrocyte solution in HSAG was added and incubated on ice for one hour. An additional 600 p1 of HSAG buffer was added and the serum/erythrocyte mixture was subjected to centrifugation for 10 minutes at l,000xg and the supernatant (a dilution of 1:8) was collected. 50 p1 of treated serum was serially diluted two-fold in polyvinyl plates and 25 p1 of RV antigen containing 4 HA units were added to each well. Following one hour incubation at 4°C, 50 p1 of 0.25% one day old chick erythrocytes in HSAG was added and the plates kept at 4°C for another hour before interpreting the results. The HAT titre was expressed as the end-point of dilution at which no aggregation of erythrocytes was observed.  -  65  -  2.2.18. Viral neutralization assay Purified ascites fluid and sera from pre-immune or immunized mice were heated at 55°C for 20 minutes to inactivate complement, diluted 1:5 in M199 medium with 2% FCS, centrifuged for 10 minutes at 10,000 rpm and sterilized by filtration through a 0.22 pm pore size filter. Serial dilutions (in triplicate) of the serum were performed in M199 medium with 2% FCS to which was added equal amounts of diluted RV (2 pfulpl in M199, 2% FCS) with or without rabbit complement (2.5%). The virus-antibody mixture was incubated at 37°C for one hour, then 50 p1 was layered onto subconfluent RK cells in 96-well microtitre plates, mixing for one hour at 37°C. The virus-antibody mixture was removed and monolayers were layered with M199 medium containing 2.5% FCS and incubated at 37°C for 72 to 96 hours. Plaques were detected using the immunoperoxidase method ( and the VN titre was the reciprocal of the dilution that demonstrated at least a 50% reduction in plaque formation compared to control cells.  2.2.19. Lymphocyte proliferation assay Lymphocytes were isolated from spleens of immunized mice. For the antigen-specific response, cells (2.5x 1 o per well) were incubated in 96-well flat bottom plates with varying concentrations of expressed antigens in triplicate. Following 7 day incubation at 37°C with antigen, the cells were pulse labeled with [ H1-thymidine (lpCi/well) for 16 hours, harvested and washed onto 3 glass-fibre filters with distilled water. After the filters were air dried overnight, 3 ml of Biodegradable Counting Scintillant (Amersham) scintillation fluid was added to determine the incorporation of [ H]-thymidine. 3  -  66  -  3. RESULTS and DISCUSSION  3.1. Section I. Role of N-linked glycosylation on E2 processing and transport 3.1.1. E2 cDNAs E2 cDNAs were constructed previously in this lab (Hobman and Gillam, 1990). Oligonucleotide directed mutagenesis was employed to introduce one or two nucleotide changes in the codons encoding asparagine or serine, resulting in a single amino acid substitution at each potential glycosylation site. The addition of N-linked oligosaccharides was prevented by changing the Asn X-Ser consensus sequence at asparagine residues 53, 71, and 115 to Gln-X-Ser, Asn-X-Gly, and Lle-X-Ser, respectively. The mutants in which consensus sequences were altered singly are referred to as G 1, G2, and G3; the double mutant is referred to as G 12; and the triple mutant is referred to as G123 (Fig.5). The positions are numbered sequentially from the N-terminus of E2. The wild-type and mutant E2 cDNAs were inserted into a mammalian expression vector pCMV5 (Fig.4a) (Andersson et al., 1989) and used to transfect COS cells.  3.1.2. Determination of functional N-linked glycosylation sites in E2 N-Glycanase digestion was performed to characterize the actual number of N-linked oligosaccharide side chains on E2. N-Glycanase hydrolyses the glucosylamine linkage of all types of N-linked oligosaccharides on glycoproteins to give free oligosaccharides and polypeptides. Digestion of radio-labeled and immunoprecipitated wild-type E2 expressed in COS cells with a serially diluted N-glycanase generated four species with apparent molecular weights of 37, 35, 33, and 31K (Fig.6). It is likely that these four species corresponded to E2 with three, two, one,  -  67  -  12  3 Y  WT  Gi  1  53 71  I  Y  I  I  115  281  1..----I  Asn ‘-Gin (53)  -- -  G2  Sec ‘-Gly (73)-  G3  I  YY  ..  Asn ‘-lie  .  (115) I  ..-  G12  G123  I  I  1  I 1  Sitesl&2 Altered  Sites 1,2 &3 Altered  Fig.5 Schematic representation of wild-type and glycosylation mutants of RV E2. The E2 protein contains three N-linked glycosylation sites at residues 53, 71 and 115 as depicted by branch ed structures (Y). The putative transmembrane region is located near the C-terminus of E2 ([‘). The first residue of mature E2 is glycine-1 and the C-terminal residue of E2 before El is glycine 281.  -  68  -  A 12345  6  29-  Fig.6 Determination of the number of N-linked glycans on RV E2. S]-methionin 35e labeled E2 [ was incubated with no (lane 1); 10 mU (lane 2); 20 mU (lane 3); 50 mU (lane 4); 100 mU (lane 5) and 300 mU (lane 6) N-glycanase (Boehringer Mannheim) for 10 minutes at 37°C. E2 was separated by SDS-PAGE and subjected to fluorography. The positions of molecular weight markers are shown on the left (kDa).  -  69  -  and no carbohydrate side chain(s), suggesting that wild-type of E2 glycoprotein normally has three N-linked oligosaccharide chains.  3.1.3. Expression of E2 glycosylation mutants in COS cells Analysis of the expression of E2 glycosylation mutants in COS cells was carried out according to procedures detailed in Materials and Methods. After a 30 minutes pulse-labelling period, wildtype E2 expressed as a prominent 37 kDa glycoprotein (Fig.7a, wt). The electrophoretic mobilities of the mutant proteins increased proportionally with the number of inactivated glycosylation sites (Fig.7a). Removal of any single glycosylation site at position 1, 2 or 3 resulted in the synthesis of a major 35 kDa glycoprotein, while the double mutant G23 and the triple mutant G 123 directed the synthesis of proteins which migrated at 33 and 31 kDa, respectively, (Fig.7a). To verify that the differences in electrophoretic mobility between wild-type and mutant E2 were due to the numbers of N-linked oligosaccharide side chains attached, some transfected cells were treated with tunicamycin. Tunicamycin at a low concentration efficiently inhibits N-linked glycosylation without interferring with protein synthesis in cells (Elbein, 1987). In the presence of 3 jig tunicamycin per ml, all the E2 polypeptides synthesized in cells transfected with wildtype and different glycosylation mutant cDNAs had the same molecular weight as the triple mutant, G123 (Fig.7a). Tunicamycin did not affect the apparent molecular weight of G123 from transfected cells (Fig.7a, G123; 7b, E2G123) nor did digestion with N-glycanase (Fig.7b, E2G123). These results further confirmed that all three potential N-linked glycosylation sites of E2 are normally used and that the difference in molecular weight between wild-type and mutant  -  70  -  ft  Gi -  +  B  G2  G3  G12  Gi23  -+  -+  -+  -+  wt — —  E2  wt -  +  Tm  E2G123  +  —  —  +  +  —  —  —  —  +  Tm glycanase  Fig.7 Expression of wild-type and glycosylation mutants of E2 in COS cells. (A). Transfected cells were labeled with S]-methionin 35 [ e for 30 minutes in the presence (+Tm) or absence of 3 jig/mi of tunicamycin. RV specific proteins were immunoprecipitated using human anti-RV serum and separated by 11% SDS-PAGE. (B). Some immunoprecipitated E2 proteins were treated with 100 mU N-glycanase at 37°C overnight (+ glycanase) and subjected to SDS-PAGE and autoradiography. The positions of molecular weight markers are shown on the left in kDa.  -  71  -  E2 is due to the number of carbohydrate chain attached.  3.1.4. Formation of aberrant disulfide bonds in E2 glycosylation mutants The possible formation of aberrant disulfide bonds in E2 glycosylation mutants was examined by pulse-chase analysis. Radio-labeled E2 proteins from transfected COS cells were immunoprecipitated with human anti-RV serum and separated by SDS-PAGE under reducing and nonreducing conditions (Cohen et al., 1982). Wild-type and mutant E2 proteins migrated slightly faster in the absence of 13-mercaptoethanol (nonreducing) than in its presence (reducing) (Fig.8), implying the existence of intramolecular disulfide bonds in E2 that have also been observed in many other glycoproteins (Machamer and Rose, 1988a, b; Vidal, et al., 1989). The 012 protein ran as a diffuse band, and the G123 protein was not detectable on the gel under nonreducing conditions, although in the presence of f3-mercaptoethanol, bands corresponding to these mutant proteins were readily detected (Fig.8). These results suggest that the formation of aberrant disulfide intramolecular bonds occurs causing the proteins to migrate as diffuse smears when disulfide bonds are not disrupted. The possible formation of aberrant intermolecular disulfide bonds in E2 mutants was further analyzed by immunoblotting (Towbin et al., 1979). Under reducing conditions, single glycosylation mutants had a prominent 35 kDa and minor 33.5 kDa and 31 kDa glycoprotein species (Fig.9). Two species at 33 and 31.5 kDa were detected in the double mutant (Fig.9). Only the 31 kDa unglycosylated E2 protein was observed in the triple mutant (Fig.9). Under nonreducing conditions, the samples migrated slightly faster because of the presence of intramolecular disulfide bonds (Fig.9). Although the majority of E2 remained as monomer,  -  72  -  +13-Me  -13-Me r’)  A  —  00000  I  Fig.8 Formation of aberrant disulfide bonding in E2 glycosylation mutants. Transfected cells were pulse-labeled with 100 jiCi S]-methionine 35 for 30 minutes and chased with excess methionine [ for 2 hours. RV-specific proteins were analyzed by immunoprecipitation using human anti-RV serum, separated on 11% SDS-PAGE with or without 3-mercaptoethanol and fluorographed. The positions of molecular weight markers are shown on the left in kDa and the arrow indicates the start of the separating gel.  -  73  -  wt  G123  G12  G3  G2  GI  A  -68 -43  —  -  -29  B  Fig.9 Western blot analysis of steady-state wild-type and mutant E2 proteins in transfected cells under reducing and non-reducing conditions. Transfected COS cells were lysed (40 hour post transfection) with RIPA buffer (50 mM Tris-HC1, pH 7.5, 1% Triton X-l00, 10 mM EDTA, 0.15 M NaC1, 0.1% SDS, 1% sodium deoxycholate) containing 10 mM iodoacetamide. Cytoplasmic extracts were electrophoresed on 11% reducing (A) and non-reducing (B) gels. The proteins were transferred to cellulose nitrate membranes. Membranes were blocked in 4% milk powder in TBS (0.15 M NaC1, 0.02 M Tris-HC1, pH 7.5) and incubated with human anti-RV serum (1:200 dilution). The proteins were visualized using alkaline phosphatase-conjugated anti-human IgG. The positions of molecular weight markers are shown on the right in kDa.  -  74  -  protein species corresponding to the position of E2 diiner, trimer and tetramer were readily observed (Fig.9). Deletion of any glycosylation site from E2 seemed either to abolish the binding of antibodies to E2 or reduce the amount of monomeric forms, especially in G12 and G123 (Fig.9). It is possible that these mutants proteins exist as alternatively folded structures that are not recognized by anti-RV serum and that the antigenic sites in G12 and G123 forms are detectable only after unfolding of these proteins by cleavage of intramolecular disulfide bonds. These finding suggest that the pattern of disulfide bonding for E2 glycosylation mutants is heterogeneous and the glycosylation may be important in preventing aberrant disulfide bond formation.  3.1.5. Glycan processing and intracellular stability of E2 proteins The kinetics of processing and the turnover rate of the E2 mutant proteins were examined by pulse-chase experiments followed by densitometric analysis of processed proteins. After thirtyminute pulse-labelling, wild-type E2 was found predominantly in the 37 kDa form, and removal of high-mannose glycans by endo H digestion reduced the molecular size to 31 kDa (Fig.10). Approximately 25, 40 and 50% of wild-type of E2 was found to possess complex-type sugar after 1-, 2- and 4-hour chase periods, respectively (Fig.10). In contrast, Gi, G2, and G3 mutant proteins containing complex-type glycans represented only 17, 14, and 10% of the total amount of each mutant protein after a two-hour chase (Fig. 10). No endo H resistance was observed for the double mutant, G12 (Fig. 10). As the acquisition of endo H resistance is believed to be indicative of transport of glycoproteins through the medial Golgi apparatus, it is evident that removal of glycosyl moieties impairs the transport of E2 mutant proteins. This effect is dependent  -  75  -  o  12  chase(hrs) endoH  —  WT  4  43-  — 29-  —  —R  —s  4  Gi  G2  43-  —s  29G3  4329-  ai  —s  G12  29-  —  Fig.10 Time course for glycan processing of wild-type and mutant E2 proteins. Cells were pulselabeled with SJ-methionine 35 for 30 minutes and chased for various times as indicated. Some [ immunoprecipitated samples were digested with endo H for at least 8 hours (+ endo H). Endo H-resistant (R) and sensitive (S) oligosaccharide-containing proteins are indicated. The positions of molecular weight markers are shown on the left in kDa.  -  76  -  on both the position and the number of glycosylation sites altered. To determine the turnover rate of wild-type and mutant E2, immunoprecipitates from transfected COS cells were fractionated on SDS-PAGE and quantitated by densitometric analysis of the autoradiographs (Fig. 11). Wild-type E2 was relatively stable in COS cells, with 70% of E2 remaining after a 4 hour chase. By contrast, the mutants exhibited a higher turnover rate. The half-lives (t ) for mutant proteins in the cells were: Gi, G2 and G3=3 hours; G12=2 hours; and 112 G123=30-60 minutes. It could be that the mutant proteins were not properly folded and transported due to an altered glycosylation pattern, and were rapidly degraded as has been reported for some other glycoproteins (Matzuk and Boime, 1988).  3.1.6. Intracellular localization of mutant E2 proteins. The  subcellular  localization  of E2  mutant proteins  was  examined  using  indirect  immunofluorescence. Cells expressing wild-type E2 exhibited staining throughout the cytoplasmic reticulum as well as in the juxtanuclear region (Fig.12a). The single, double and triple glycosylation mutant proteins displayed a predominantly reticular staining pattern as well as Golgi-like staining (Fig.12c,e,g). To visualize the distribution of E2 protein in the ER and Golgi, fluorescent-conjugated WGA and ConA were used as markers for the compartments, WGA has been shown to label trans Golgi cisternae, associated vesicles and the cell surface (Tartakoff and Vassalli, 1983) by binding to clustered terminal N-acetylneuraminic acid residues as well as N acetylgiucosamine-containing oligosaccharide chains on glycoproteins (Virtanen et al., 1980). Co staining of transfected COS cells with human anti-RV serum and fluorescent-conjugated WGA revealed that wild-type E2 was concentrated in the Golgi region (Fig.12b), while the mutant E2  -  77  -  120  PERCENTAGE  1  0  1  2  3  4  HOURS  Fig. 11 Intracellular stability of wild-type and mutant E2 proteins. Cells were pulse-labeled with 3hionin [ S]-met 5 e for 30 minutes and chased for various times as indicated. RV-specific proteins were immunoprecipitated using human anti-RV serum. Rates of degradation of wild-type and mutant E2 proteins were quantified by scanning densitometry of the X-ray films from three to six independent experiments as shown in Fig.l0. Different chase times are indicated. ---i--- wildtype, ---0--- Gi, 02, ---a--- G3, ---*--- G12, ---+--- G123. ---•---  -  78  -  Fig.12 Indirect immunofluorescence of wild-type and mutant E2 proteins in COS cells. Cells were permeabiized prior to the addition of rhodamine-conjugated WGA or ConA and anti-RV serum. After the cells were washed, a secondary antibody (fluorescein-conjugated goat anti human IgG) was added. (a) wild-type, anti-RV; (b) wild-type, TRICT-WGA; (c) G2, anti-RV; (d) G2, TRICT-WGA; (e) G12, anti-RV; (f) G12, TRICT-WGA; (g) G123, anti-RV; (h) G123, TRICT-ConA.  -  79  -  proteins were distributed throughout the reticulum network and Golgi region (Fig.12 d,f). A strong reticular staining, which co-localized with ConA, was observed in COS cells transfected with glycosylation mutants (Fig.12h). In addition, unlike wild-type E2, which has been shown to exhibit limited amount of cell surface expression (Hobman and Gillam, 1989), the glycosylation mutants had no detectable cell surface signal (data not shown). Elimination of any of the glycosylation sites in E2 seemed to impair the intracellular transport and to block the cell surface expression of E2.  3.1.7. Secretion of an anchor-free form of wild-type and mutant E2 proteins. To analyze the transport behavior of E2 mutants in the secretory pathway, a panel of truncated E2 glycosylation mutants were constructed, each of which had 68 amino acids deleted from the hydrophobic C-terminus (Hobman et al., 1994). The truncated form of wild-type E2 was secreted into the culture medium as a 36-kDa endo H-resistant glycoprotein (Hobman et al., 1994). The truncated form of E2 single glycosylation mutants (Gi, G2 and G3) but not double (G12) and triple (G 123) mutants were also secreted into the culture medium, although not as efficiently as the anchorless wild-type E2 (Fig. 1 3a). In addition, the efficiency of secretion appeared to depend on the position of the deleted glycosylation site. Deletion of the glycosylation site proximal to the C-terminus (G3) had a more profound inhibitory effect on the secretion than of the central site (G2) and of that proximal to the N-terminus (G1) (Fig. 13a). The intracellular forms of anchorless wild-type E2 and E2 glycosylation mutants were sensitive to endo H digestion (Fig.13b), whereas the secreted E2 was endo H-resistant (Fig.13a). Expression of the truncated triple mutant was not detected intracellularly (Fig. 13b). The 3 1-kDa  -  80  -  I  c  G12  G123  —+—+—+—+—+  —+  wt  Gi  G3  G2  endoH  29  -Ä  • -.  b Gi  G2  G3  G12  G123  wt  —+  —+  —+  —+  —+  —+  endoH  Fig.13 Intracellular processing and secretion of a soluble form of wild-type and mutant E2 proteins. Cells were labeled with S]-methionine 35 for 30 minutes and chased for 4 hours. (a). [ Immunoprecipitated samples from culture media of cells transfected with anchorless wild-type and mutant E2 cDNA constructs. (b). Intracellular forms of each anchorless E2 protein of wildtype and glycosylation mutant. Equal volumes of each sample were incubated at 37°C for at least 8 hours with or without endo H and separated on 11% SDS-PAGE. The positions of the molecular weight markers are shown on the left in kDa.  -  81  -  endo H-sensitive protein species found in the culture medium of the G2 transfected cells (Fig. 13a) is probably due to lysis of a portion of cells during the chase period and the intracelluar G2 mutant protein was released into the medium. Taken together, it was evident that the single glycosylation mutants Gi, G2 and G3 were transported out of the ER through the Golgi to the cell surface and then exited the cell into the culture medium, although not as efficiently as the otherwise unaltered anchorless E2 protein.  3.1.8. Summary and Discussion The role of N-linked glycosylation in processing and intracellular transport of RV E2 glycoprotein has been studied by expressing glycosylation mutants of E2 in COS cells. In RV M33 strain, all three sites were used for the addition of N-linked oligosaccharides. Removal of any of the glycosylation sites resulted in slower glycan processing, lower stability and aberrant disulfide bonding of the mutant proteins, with the severity of the defect depending on the number of deleted carbohydrate sites. The mutant proteins were translocated into the ER and transported to GC but were not detected on the cell surface. However, the secretion of the anchor-free form of E2 into the medium was not completely blocked by the removal of any one of its glycosylation sites. This effect was dependent on the position of the deleted glycosylation site. Protein movement from the ER to the medial Golgi apparatus has been identified as the rate-limiting step in the exocytic pathway (Rose and Bergmann, 1983), as measured by the acquisition of a variety of organelle-specific post-translational modifications. Regarding the intracellular transport rate, several viral glycoproteins that have been extensively investigated fall into two categories. The first group includes the VSV G protein (Rose and Doms, 1988) and  -  82  -  influenza virus HA (Gallagher et al., 1988) which move quickly along the exocytic pathway. After a 15 minutes chase period, 50% of the oligosaccharides on VSV G and 25% on influenza virus HA acquire endo H resistance (Rose and Doms, 1988; Gallagher et al., 1988). The second group includes the HIV envelope protein (Earl et al., 1991) and simian virus 5 hemagglutinin neuraminidase (SV5 HN) (Ng et al., 1989), for which acquisition of endo H resistance was observed within 80 minutes and 60 minutes post-labelling, respectively. We found that the carbohydrates on wild-type RV E2 were converted to complex-type sugar moieties by 1 hour  post-labelling. However, the conversion was not complete even after a 8 hours chase period (data not shown), reflecting a slow movement of RV E2 from the ER to the Golgi apparatus. The data from this study showed that E2 contains three potential oligosaccharide addition sites and all three potential N-linked glycosylation sites were utilized (Fig.6 and 7). Inactivation of these functional sites impaired the processing as well as the intracellular stability of E2 proteins (Fig. 10 and 11), the severity of the defect depending on both the number and the position of the glycosylation site deleted. Deletion of one N-linked glycosylation site on RV E2 considerably reduced the rate of transport, as determined by the fraction of proteins that acquired endo H-resistant carbohydrates (Fig. 10). The glycosylation site proximal to the N-terminus (Gi) seems to be less important than the site proximal to the C-terminus (G3), as judged by the fraction of molecules containing endo H-resistant carbohydrates for the membrane-bound form and by the secretion ratio of their anchorless counterparts (Fig. 10 and 13). That the oligosaccharide at each glycosylation site may play a different functional role has been noted previously with other glycoproteins (Matzuk and Boime, 1988; Ng et al., 1990). In addition, studies on other glycoproteins using the same approach have shown that glycosylation on all the  -  83  -  predicted sites is not a prerequisite for folding, assembly and transport of the protein (Guan et al., 1988). It has been suggested that the contribution of each carbohydrate chain varies depending on its location in a different conformational circumstance of a particular protein. RV E2 is rich in cysteine and undergoes intramolecular disulfide bonding (Fig.9). Inspection of the amino acid sequence of RV E2 reveals that the 62 and G3 glycosylation sites are flanked by two cysteine residues (Clarke et al., 1987). It is possible that the oligosaccharides attached to the G2 and G3 sites are important in preventing improper intramolecular disulfide bond formation, whereas glycosylation at the 61 site has less effect on proper folding and transport. The diffuse or smeared appearance of non-reduced mutant G12 and G123 proteins on an immunoblot (Fig.9) probably reflects the formation of aberrant intermolecular disulfide bonds. Thus, it appears that oligosaccharide addition is required for proper intramolecular disulfide bonding to promote correct folding, which in turn is essential for efficient transport (Vidal et al., 1989). Removal of a glycosylation site leads to formation of improper intramolecular disulfide bonds and protein misfolding. Dramatic alteration in protein conformation and possibly aggregation could be the consequence when glycosylation sites are inactivated. This may account for diminished antibody binding by G12 and 6123 proteins under non-reducing conditions (Fig.9B). Proteins that are transported slowly in cells display heterogeneity in endo H resistance. For example, this has been observed for SV5 HN (Ng et al., 1989), influenza virus neuraminidase (Kunda et al., 1991) and HIV gpl2O (Earl et al., 1991). Pulse-chase experiments have demonstrated that endo H-sensitive, partially endo H-resistant and endo H-resistant E2 forms represent the ER-, Golgi- and cell surface- isoforms of RV E2. Immunofluorescence of transfected COS cells expressing E2 wild-type and mutant genes showed that the majority of the  -  84  -  glycosylation mutant proteins were localized in the ER (Fig. 12). A small fraction were found in the Golgi region (Fig. 12). Transport of the E2 single glycosylation mutants into the Golgi compartment was evidenced by the presence of partially endo H-resistant bands after the chase period (Fig.1O), as well as by the secretion of the C-terminal truncated form of E2 single glycosylation mutants (Fig. 16a). Thus the transport of E2 to the Golgi apparatus appeared to be significantly affected but not completely blocked by the absence of any one of the N-linked oligosaccharides. The anchorless E2 single glycosylation mutants were secreted into the culture medium, although less efficiently than wild-type anchorless E2. The oligosaccharides on the secreted forms of wild-type and mutant E2 were completely endo H-resistant as is E2 from RV virion (Lundstrom et al., 1991), suggesting that carbohydrates attached to these proteins are modified by Golgi enzymes. This finding indicates that the soluble forms of E2 single glycosylation mutants are transported through the normal exocytic route. Inability to detect cell surface expression of E2 single glycosylation mutants could be due either to the low sensitivity of indirect immunofluorescence in our experiments, or to the fact that mutant E2 proteins were quickly and extensively internalized from the plasma membrane, as has been observed for other glycoproteins (Ng et al., 1989).  -  85  -  3.2. Section II. Effect of Brefeldin A (BFA) and monensin on protein processing and virus assembly  3.2.1. Processing of N-linked oligosaccharides on E2 in BFA- and monensin-treated cells The expression of RV E2 in pCMV5-E2 transfected COS cells was used to determine the appropriate concentrations of BFA and monensin for the study. Interestingly, BFA seemed to enhance the level of E2 protein expression (Fig. 14A), the mechanism of which is not understood. However, no difference in the glycan processing of RV E2 was found in cells treated with BFA in the range from 1 pg/ml to 12 jig/mi (Fig.14A) and thus 6 jig/mi of BFA was chosen for use in the subsequent analysis. In contrast, monensin at higher concentrations (25 pM) inhibited E2 synthesis (Fig.14C), and hence 5 pM monensin was used in the subsequent experiments. Endo H, an enzyme that removes N-linked high-mannose oligosaccharide, was used to monitor the processing of E2 glycoproteins in pulse-chase experiments. In control (untreated) cells after 30 minutes labelling, E2 accumulated as a 37 kDa protein (Fig. 14A). Digestion with endo H reduced the molecular size of the protein species to 31 kDa (Fig. 14A). By contrast, in BFA-treated cells, only the 42 kDa species was observed by the end of 30 minutes labelling (Fig.14A) and digestion with endo H decreased its molecular weight from 42 kDa to 36 kDa (Fig.14A). By 4 hour post-labelling, in control cells, about 50% of synthesized E2 was partially endo H-resistant (Fig.14C). Whereas BFA induced a rapid and complete conversion of glycans on E2 to endo H-resistant forms by 4 hour post-labelling (Fig.14C). In monensin-treated cells, pulse-labeled E2 protein had a similar electrophoretic mobility to that of the control cells (Fig.14B). However, after 3 hours of chase, monensin treatment gave rise to a novel E2 protein  -  86  -  0  A  3  1  6  12  BFA(LJg/mI)  0  monensin(LIM)  i  29-  B  1  5  — +  C —  +  —  2  25  +  —  —  zL  —  ctr I 4  4  3 +  +  —-  +  —  chase(h)  +  29ctrl  D  3  4  —+  —+  8_ —+  3 —  chase(h)  +  43’  Fig.14 Effect of BFA and monensin on processing of E2. Transfected cells were pulse-labeled with 100 pCi S]-methionin 35 [ e for 30 minutes and incubated with excess methionine for indicated time in chase experiments (C, D). BFA (in pg) (A) or monensin (in pM) (B) were added to the medium at the concentrations shown on top of A and B. In chase experiments, 6 pg/mi of BFA (C) or 5 pM of monensin (D) were present. Cells were lysed with RIPA buffer after labelling (A and B) or after chase for the indicated period of time (C, D). E2 proteins were immunoprecipitated with human anti-RV serum, subjected to SDS-PAGE and fluorography. Half of each immunoprecipitated sample was digested with endo H (+) for 8 hours at 37°C before separating on SDS-PAGE. Molecular weight markers (in kDa) are shown on the left. E2 protein bands containing endo H-sensitive (s) and resistant (r) sugar moieties are marked. The novel 34 kDa protein species after endo H digestion is indicated by a star (*).  -  87  -  band with partial endo H-resistant sugar moieties and an apparent molecular weight of 34 kDa (Fig.14D, star) instead of 36 kDa seen in control cells (Fig.14D, ctrl).  3.2.2. Processing of 0-linked glycans on E2 Since it has been shown that E2 contains 0-linked oligosaccharides (Lundström et aL, 1991; Sanchez and Frey, 1991), I then analyzed the extent of 0-glycosylation of E2 in BFA- and monensin- treated cells and in cells also treated with tunicamycin. In control cells (untreated with BFA nor monensin) after a 4 hour chase period, digestion with endo F/PNGase F reduced the molecular weights of two E2 species from 42 kDa and 37 kDa to 36 kDa and 31 kDa, respectively (Fig. 15A), similar to that observed after endo H digestion (Fig. 14C). After incubation with neuraminidase, the 37 kDa remained unchanged whereas the 42 kDa species was reduced to 40 kDa (Fig.15A). This 40 kDa species was diminished after further digestion with 0glycosidase (Fig.15A). Deglycosylation of E2 with the combination of endo F,PNGase F, neuraminidase and 0-glycosidase resulted in a major unglycosylated 31 kDa species and a 34 kDa minor band probably due to incomplete removal of 0-linked oligosaccharide (Fig. 15A).  Treatment with tunicamycin abolished the N-linked glycosylation, as well as the addition of 0linked oligosaccharide on E2, as only a 30 kDa unglycosylated E2 species was observed (Fig. 15A). The cause for the 1 kDa difference in molecular weight between deglycosylated E2 (by glycosidase digestion) and unglycosylated E2 (by tunicamycin treatment) is not known. In BFA-treated cells, the 42 kDa E2 species was reduced to 36 kDa by endo F/PNGase F digestion, to 39 kDa by neuraminidase digestion, or to 37 kDa by further incubation with 0glycosidase (Fig.15B). Digestion with a combination of endo F/PNGase F, neuraminidase and 0-  -  88  -  N—gly  —+--—+ ———++ — — ÷ •I- + —————  0-gly Neu Tm A  —— — —  ÷ +  +  ++  ÷  ill’ -,____-  29-  —  B 29-  C  Ii: 29-  —  —  Fig. 15. Glycosidase digestion of E2 from BFA- and monensin-treated cells. Transfected cells were labeled with S]-methionine 35 for 30 minutes and incubated with medium containing excess [ methionine for 4 hours before lysis with RIPA buffer. E2 proteins were immunoprecipitated with human anti-RV serum, digested with glycosidase for at least 8 hours at 37°C and subjected to SDS-PAGE and fluorography. BFA (6 ig/ml), monensin (5 jiM) and tunicamycin (3 jig/mi) were present in the medium where applicable. Samples digested with endo F/PNGase F (N-gly), neuraminidase (Neu) and 0-glycosidase (0-gly) are indicated. Molecular weight markers (in kDa) are included for reference. A, untreated cells. B, cells treated with BFA. C, cells treated with monensin. Tunicamycin was present in the medium of some cells as indicated.  -  89  -  glycosidase resulted in a single protein species of 31 kDa (Fig. 15B). In cells treated with both tunicamycin and BFA, two protein species with apparent molecular weights of 31 kDa and 35 kDa were observed (Fig. 15B). Since N-linked glycosylation was inhibited by tunicamycin, the  35 kDa species must contain only 0-linked glycans. Indeed, digestion with neuraminidase reduced its molecular weight to 33 kDa while further incubation with 0-glycosidase gave rise to a single protein species of 30 kDa (Fig. 15B). E2 from monensin-treated cells migrated as a broad band with molecular weights ranging from 37K to 42K (Fig.15C). The presence of 36 kDa E2 species in endo F/PNGase F treated samples indicated that 0-linked glycosylation occurred (Fig. 15C), in contrast to the situation seen in some other viral glycoproteins (Ogura et al., 1991; Collin and Mottet, 1992). A distinctive protein species with molecular weight of 34 kDa was observed in endo F/PNGase F digested samples as well as in samples digested with a combination of endo F/PNGase F, neuraminidase and 0-glycosidase (Fig. 15C). This could be an intermediate during processing of 0-linked glycans that accumulated in monensin-treated cells or alternatively is due to incomplete removal of 0-linked sugars from E2 in an altered conformation induced by monensin-treatment. In cells treated with monensin and tunicamycin, no 0-linked glycosylation occurred (Fig.15C). Taken together, these data suggested that E2 was first synthesized in the ER as a 37 kDa protein species containing only N-linked high-mannose type of glycans, and that as it reached the Golgi cisternae, 0-linked glycosylation took place which increased the molecular weight of E2 to 42 kDa. BFA treatment caused a redistribution of Golgi enzymes back into the ER and resulted in a rapid addition of 0-linked sugars on E2 in the ER, even when N-linked glycosylation was inhibited by tunicamycin. Whereas in monensin-treated cells, the 0-  -  90  -  glycosylation of E2 was not abolished, but was processed aberrantly.  3.2.3. Processing and secretion of an anchor-free form of E2. To study the transport of E2 along the secretory pathway in BFA and monensin treated cells, a cDNA construct encoding an anchor-free form of E2 (Hobman et al., 1994) was transfected into COS cells, and the expressed E2 protein was analyzed. In control cells, soluble E2 was secreted from the transfected cells into the culture medium at a ratio of 10-17% of total E2 protein (data not shown). The secreted E2 was found to be resistant to endo H digestion (Fig.16), indicating that it had been modified by glycan processing enzymes as it traversed the secretory pathway. The majority of intracellular E2 was endo H sensitive (Fig. 16). In contrast, although the internal form of soluble E2 in BFA treated cells was partially or completely resistant to endo H digestion (Fig.16), no secreted E2 was detected (Fig.16). Monensin completely inhibited the secretion of E2 from transfected cells, whereas intracellular E2 exhibited no obvious difference from that of control cells (Fig.16). Taken together, it is evident that BFA and monensin cause altered oligosaccharide processing during E2 transport and completely block the movement of E2 to the cell surface.  3.2.4. Effect of BFA and monensin on proteolytic processing of RV structural protein precursor The expression of the polyprotein precursor and the proteolytic processing of the precursor were analyzed by pulse-chase experiments using pCMV5-24S (Hobman et al., 1990) transfected COS cells. At the end of a 30 minutes labelling, the majority of RV structural proteins were present  -  91  -  internal ctrI —+  medium BFA  BFA Mon  ctrl  —+  —+ —+  —+  Mon —+ endoH  Fig.16 Effect of BFA and monensin on processing and secretion of an anchor-free form of E2. COS cells were transfected with plasmid containing cDNA encoding a secreted form of E2 and labeled with 100 pCi S]-methionin 35e for 30 minutes at 40 hour post-transfection. After [ incubation with excess methionine for 4 hours, medium samples were collected and cells were lysed. E2 proteins were immunoprecipitated with human anti-RV serum from medium as well as cell lysates, separated on SDS-PAGE and visualized by fluorography. Half of each sample was digested with endo H (+). Molecular weight markers (in kDa) are shown on the left. Ctrl, E2 from untreated cells; BFA, E2 from BFA-treated cells; mon, E2 from monensin-ireated cells.  -  92  -  Chase  Pulse Ct r I  ctrl  Mon —+  BFA  Mon endoH  a  El  4C, E2r  Fig. 17 Effect of BFA and monensin on the proteolytic cleavage of the polyprotein precursor for RV structural proteins. Cells transfected with pCMV5-24S were pulse labeled with S]35 [ methionine and chased for 3 hours. RV structural proteins were recovered from cell lysates by immunoprecipitation with human anti-RV serum and were subjected to SDS-PAGE and fluorography. The bands corresponding to RV structural proteins without endo H treatment (on the left) and with endo H treatment (on the right) are indicated. E2r, E2 proteins that contain endo H-resistant glycans; E2s, E2 that contain endo H-sensitive sugar moieties. Endo H digestion is indicated as +. Ctrl, untreated cells; BFA, BFA treated-cells; mon, monensin-treated cells.  -  93  -  as individual polypeptides, although some E1fE2 uncleaved precursor protein species with higher molecular weights were also observed (Fig. 17, pulse). These minor protein species were not seen after a 3 hour chase period (Fig. 17, chase). There was no significant difference in cleavage efficiency for RV-specific proteins between the control cells and BFA or monensin treated cells, indicating that BFA and monensin did not directly affect the proteolytic processing of RV structural protein precursor. This is consistent with our previous fmding that the cleavage of polyprotein precursor is carried out by cellular signal peptidases (McDonald et al., 1991; Qiu et al., 1994), which suggests that it is an ER-specific event and was not interrupted by the influx of resident Golgi proteins upon BFA treatment or an impaired Golgi upon monensin treatment. RV structural proteins appears to assemble into virus-like particles in the absence of genomic RNA and are released from the cells (see 3.4.), which probably is the reason that there was a slight decrease in the amount of RV structural proteins in control cells during chase.  3.2.5. Subceilular distribution of RV structural proteins in BFA and monensin treated cells. Indirect immunofluorescence was used to localize the RV structural proteins in cells transfected with pCMV5-24S. In control cells, E2 was found to be concentrated in the juxtanuclear region and co-localized with a Golgi marker (Fig. 18a,b), while El was localized in the pen- and juxtanuclear region corresponding to the ER and Golgi structure (Fig.18c,d). The capsid protein was distributed throughout the cytoplasm (Fig.18e). A limited amount of E2 and El was detected at the cell surface (Fig. 180. In BFA treated cells, E2 and El displayed a predominant perinuclear staining pattern which was co-localized with a Golgi marker (Fig. 18, g-k). Combined with the  -  94  -  Fig.l8 Indirect immunofluorescence of RV structural proteins in cells transfected with pCMV524S and treated with BFA or monensin. Cells were permeabilized prior to the addition of rhodamine-conjugated anti-Golgi protein serum, mouse monoclonal antibodies or human anti-RV serum. Some samples (F, L, and R) were not permeabiized for detection of cell surface expression. After cells were washed, a secondary antibody (fluorescein-conjugated goat anti mouse or anti-human IgG) was added. a-f, control cells: a) anti-E2; b) same cell, anti-Golgi; c) anti-El; d) same cell, anti-Golgi; e) anti-C; f) anti-RV, cell surface. g-1, transfected cells treated with BFA: g) anti-E2; h) same cell, anti-Golgi; i) anti-El; j) same cell, anti-Golgi; k) anti-C; 1) anti-RV, cell surface. m-r, transfected cells treated with monensin: m) anti-E2; n) same cell, anti Golgi; o) anti-El; p) same cell, anti-Golgi; q) anti-C; r) anti-RV, cell surface.  -  95  -  results from pulse-chase analysis, it appeared that in transfected COS cells, BFA treatment caused the Golgi proteins to cycle back into the ER (as shown by the perinuclear staining with the Golgi marker) and blocked the transport of El and E2 out of the ER. In the presence of monensin, a swollen Golgi morphology was observed (Fig.18n,p), along with a diffuse E2 and El distribution in the cytoplasm (Fig. 1 8m,o). Capsid protein was distributed in the cytoplasm in BFA and monensin treated cells (Fig. l8k,q). No cell surface fluorescence was detectable in BFA and monensin treated cells (Fig. 1 81,r).  3.2.6. Effect of BFA and monensin on RV assembly and release Release of radio-labeled RV particles was monitored in RV-infected Vero cells treated with BFA and monensin (see Material and Methods). In control (untreated) cells, the amount of viral structural proteins (C. E2, El) increased with time during the chase period (Fig. 19), indicating that virus particles were accumulating and that viruses were steadily assembled and released from the cells. In BFA and monensin treated cells, no viral structural proteins were detectable until 36 hours post-labelling (Fig. 19). Thus, BFA and monensin blocked RV release from the cells during the early stage of the chase. After a 36 hour chase period, the viral proteins detected may be due to lysis of BFA and monensin treated cells (Fig. 19). To further address this question, a one-step growth experiment was performed with RV infected Vero cells treated with BFA and monensin. BFA and monensin were added to the medium 8 h.p.i. and maintained for 60 hours. Cell-associated virus and virus in the medium were titred (Fig.20). In control cells, intracellular and extracellular viruses reached the highest titre at  -  96  -  ctrI  BFA  Mon  1220 2836  12202836  chase(h)  I E2” C ‘  Fig. 19 Release of virus particles from infected cells. Vero cells were infected with RV at a MOl of 10 and labeled with S]-methionine 35 for 1 hour at 24 h.p.i. Cells were further incubated in [ normal medium for the indicated times. Virus particles were recovered from the medium by polyethyleneglycol precipitation, resuspended in RIPA buffer and subjected to SDS-PAGE. RV structural proteins are indicated by arrowheads. Ctrl, untreated cells; BFA BFA-treated cells; Mon, monensin-treated cells.  -  97  -  PFU/m I  24  36  48  60  h.p.i  PFU/mI  BFA  10’ 10’ 10’ 102  0 24  36  48  60  h.p.i  PFU/ml  Monensin  1 0’ 102  24  36  S  48  60  h.p.  n.Qrna( E2;mcdum  Fig.20 Titration of cell-associated and released virus. Vero cells were infected with RV at a MOl of 10 and incubated for the indicated time. Medium samples were collected and cells were subjected to freezing and thawing three times to release intracellular virus. Virus infectivity was titrated on RK cells and is expressed as PFU/ml (detailed in Materials and Methods). , intracellular virus titre. , extracellular virus titre.  -  98  -  48 h.p.i, about 5x10 7 pfu/ml, with a slightly higher extracellular virus than intracellular titre (Fig.20). Titres of cell-associated viruses were much lower in BFA and monensin treated cells, about 2x10 5 pfu/ml (Fig.20), indicating that BFA and monensin dramatically reduced virus assembly in infected cells. Titres of extracellular virus from BFA- and monensin-treated cells represented only 0.1% of the total virus (Fig.20). Thus, BFA and monensin effectively inhibited virus release from infected cells. Infected cells became unhealthy with prolonged monensin treatment as an aberrant cell morphology was observed. This could explain the increased extracellular virus titre after incubation for 60 hours (Fig.20).  3.2.7. Assembly of virus particles in control or BFA and monensin treated cells RV particles in infected cells were visualized using conventional electron microscopy. Cells were fixed at 48 h.p.i., dehydrated and embedded. Thin sections were examined after staining. In RV infected Vero cells, virions were predominantly located in vacuoles in the proximity of the Golgi cisternae (Fig.21b) and in large transport vesicles (Fig.21a). Unenveloped nucleocapsids were rarely observed in the cytoplasm and no virus budding at the plasma membrane was observed after examining all the sections. In BFA-treated cells, the GC was disassembled and resulted in a dilated ER structure (Fig.21c). The number of enveloped virus particles was dramatically decreased and they were predominantly located in the cytoplasm, not associated with any membrane structure (Fig.2 ic). In monensin-treated cells, enlarged vesicles were observed (Fig.21d). No virus particles were found except that electron-dense particles, comparable in size to RV nucleocapsid, was observed in enlarged vesicles (Fig.21d).  -  99  -  •‘  C I  —-‘I  r  er  I.  —  .  Fig.21 Electron microscopic analysis of virus assembly. Monolayers of Vero cells were infected with RV at a MOl of 10 and some were treated with BFA or monensin. At 48 h.p.i., cells were fixed and prepared for electron microscopic analysis. A) RV-infected cells showing virus accumulated in large vacuole, x75,000. B) RV infected-cells showing virus maturation in the proximity to the Golgi stack, x25,000. C) RV infected-cells treated with BFA. Virus particle was found in the cytoplasm, near a dilated ER structure. x25,000. D) RV infected cells treated with monensin. No virions were observed. Electron dense particles were found in large vesicles. x25,000. er, endoplasmic reticulum; Gc, Golgi complex; flu, nucleus. -  100  -  3.2.8. Summary and Discussion The effect of BFA and monensin on the transport and processing of RV structural proteins as well as virus assembly and release was examined. BFA and monensin effectively blocked the cell surface expression of RV E2 and El membrane glycoproteins and the secretion of an anchor-free form of E2. A dramatic change in the intracellular distribution of RV structural proteins was also observed, although the proteolytic processing of RV structural protein precursor was not affected. In the presence of BFA and monensin, virus release from infected Vero cells was only 0.1% of the intracellular virus. Virus particles were observed predominantly in large vesicles or Golgi stacks in RV-infected Vero cells but were found in the cytoplasm in BFA treated cells. Enveloped viruses require their host’s secretory pathway for virus assembly and release from the cells. The rate of intracellular transport and processing of viral proteins may play an important role in controlling the efficiency of virus maturation, particularly for viruses that are assembled at the cell surface. Mutations that impair the transport of viral proteins to the appropriate cellular compartment have been found to significantly reduce the formation of infectious virus particles (Haggerty et al., 1991). In the Togavirus family, RV differs from the other subgroup (alphavirus) in that it has a relatively long latency period, slow replication and low virus yield in cultured cells (reviewed by Porterfield et al., 1978). This is inspite of the fact that these two subgroups share similar viion structure as well as strategies for viral gene expression. The transport rate of RV glycoproteins in transfected cells is fairly low compared to that of the envelope glycoproteins from SFV and SIV, the two prototypes of aiphaviruses. In cells transfected with RV E2 cDNA, about 50% of E2 contains endo H-resistant sugar moieties after a 4 hour chase period (Fig. 14), indicating that E2 moves slowly from the ER to the Golgi stack.  -  101  -  Immunofluorescence studies show that the majority of RV glycoproteins El and E2 are concentrated in the ER-Golgi region. The abundance of RV glycoproteins in the GC may be crucial to the virus’ capacity to bud from intracellular membranes (Bardeletti et  a,  1979).  In RV virion, E2 glycoprotein exists as multiple protein species with molecular weights ranging from 42K to 47K. It has been suggested that this is due to heterogeneous glycosylation. In transfected COS cells, a 37 kDa E2 protein with endo H-sensitive sugars and a 42 kDa E2 protein with partial endo H-resistant glycans were observed. The amount of the 42 kDa protein species increased with time in a pulse-chase experiment. Thus, it has been proposed that the 42 kDa protein represents the Golgi-form of E2 while the 37 kDa protein is the form present in the  ER (Hobman and Gillam, 1989) and that the partial resistance of E2 to endo H digestion reflects the slow transport rate of E2. However, employing both 0-glycosidase and endo F/PNGase F to remove 0- or N-linked oligosaccharides on E2 separately, I found that the 37 kDa E2 contained only N-linked endo H-sensitive carbohydrates whereas the 42 kDa species bore both N- and 0linked glycans (Fig.l5). The endo H -resistant sugar moieties present on the 42 kDa protein, in fact, are the 0-linked glycans. These data suggest that E2 is first synthesized as a 37 kDa protein with only N-linked high-mannose sugars in the ER, and is transported to the Golgi where 0linked glycosylation occurs. Thus the acquisition of 0-linked glycans can be used to monitOr the transport of E2 to the Golgi complex. In BFA treated cells, only the 42 kDa E2 protein with both N- and 0-linked sugars was observed (Fig. 15, 16). This probably results from the addition of 0linked glycans to the 37 kDa protein normally residing in the ER by the Golgi 0-glycosylation enzymes brought back into the ER by BFA (Lippincott-Schwartz et al., 1989). In the presence of BFA, the addition of 0-linked glycans to E2 took place quickly, whereas the maturation of  -  102  -  N-linked sugars to complex forms was slower, as judged by the fact that complete endo-H resistant E2 was observed after a 4 hour chase. Although monensin appeared to abolish 0glycosylation in many cases (Colins and Mottet, 1992), I found that E2 from monensin-treated cells possessed some 0-linked glycans. The intracellular distribution of RV structural proteins was dramatically altered in cells treated with BFA and monensin. In BFA treated cells, the Golgi complex was disassembled as the Golgi marker was localized in the perinuclear space (Fig.18h, 18j). A dilated Golgi morphology was found in monensin treated cells (Fig. 1 8n, l8p). There was a consistent co localization of RV membrane glycoproteins with the Golgi markers, suggesting that RV envelope glycoproteins interact strongly with Golgi macromolecules. Recently, it has been shown that unassembled subunits of RV El glycoprotein are arrested in a post-ER, pre-Golgi compartment (Hobman et al., 1992); however, co-expression of El and E2 could lead to the release of El from this retention and allow targeting to the GC (Hobman et aL, 1993). The implication of these observation is that folding and multimerization of RV glycoproteins is a slow, albeit important event necessary for transport of El and E2 out of the ER. On the other hand, although the mechanism underlying retention in the Golgi is not well understood, hydrophobic domains which may specify residence in the Golgi stack (Swift and Machamer, 1992), are present in both El and E2 primary structure. In RV-infected Vero cells, virions were steadily assembled and released into the medium, as judged by titres of virus in the medium and in association with cells (Fig.20). Slightly more extracellular virus than intracellular was found (Fig.20), which is consistent with earlier reports (Payment et al., 1975; Bardeletti, et al., 1979). In BFA and monensin treated cells, very few  -  103  -  infectious virus particles in the medium were detected (Fig.20), indicating that BFA and monensin effectively blocked the production of extracellular virus. The absence of radio-labeled virions in the medium of BFA and monensin treated cells (Fig.19) ruled out the possibility that the decreased virus titre was due to the loss of virus infectivity. Rather, it was due to a decrease in the number of virus particles during treatment A dramatic decrease in intracellular virus titre was also observed (Fig.20). This could be due to the inhibition of virus assembly at the internal membrane or aberrant virus assembly, which is yet to be investigated. Recently it has been reported that BFA affected viral RNA synthesis in poliovirus infected cells (Maynell et al., 1992) due to interruption of membrane-associated poliovirus replication complex. The nature and site of RV RNA synthesis is not well known. However, we found that the level of protein expression in cDNA-transfected and RV-infected cells was not affected by the concentration of BFA and monensin used (data not shown). In Vero cells, envelopment of RV nucleocapsid to form virions has been reported to take place both at the internal membrane or at the cell surface (Payment et al., 1975). In this study, morphological analysis was performed in RV-infected cells in the presence and absence of BFA and monensin in order to examine the correlation between viral glycoprotein targeting and the virus budding process. At 48 h.p.i., virus particles were found to be clustered in the vacuoles near the Golgi stack, with others in the transport vesicles (Fig.21). The abundance of virus particles in the post-Golgi structures suggested that the post- Golgi membrane network may be the primary source of membrane in RV assembly. Few virus particles, not associated with any membrane structure, were found in the juxtanuclear region of cytoplasm in BFA treated cells (Fig.2 1). No virus particles could be detected in monensin treated cells after examining all the sections  -  104  -  (Fig.21). The results from morphological analyses were consistent with a significant decrease in virus titre in BFA and monensin treated cells. The reduction of intracellular virus assembly in BFA or monensin treated cells could be due to the blockage of glycoprotein transport to the site of envelopment or the disruption of vesicular structure which may be required for efficient virus assembly. Taken together, the data presented in this study suggested a correlation between intracellular localization of RV structural proteins and the site of RV assembly. How cell membranes become incorporated into the envelope of the virion is as yet poorly understood. The subcellular location for acquiring membranes differ with viruses, reflecting the possibility that structural or component features of particular organelles are required to facilitate such envelopment. Alternatively, post-translational modification(s) on viral envelope proteins during transport may be essential for effective interaction with the nucleocapsids. Therefore, defining the site of virus assembly can shed some light on further studies on the mechanism of virus assembly.  -  105  -  3.3. Section III. Influence of N-linked glycosylation on the antigenicity and immunogenicity of El glycoprotein  3.3.1. Construction of recombinant vaccinia viruses expressing RV El glycosylation mutants The cDNA fragments encoding RV El glycosylation mutants (Hobman et al., 1991) were subcloned into the  .S.n  I site of vaccinia virus recombination vector pGS2O (Fig.4B), which  contains the conventional p7.5 early/late promoter (Mackett et aL, 1984). Transfection (see Materials and Methods 2.2.6.) and marker rescue by cell-mediated homologous recombination were performed as described by Mackett et al., (1985). The vaccinia recombinants containing wild-type and glycosylation mutant cDNAs were named according to the mutations as yR-El (wild-type); VR-E1G1, VR-E 102, VR-E 103 (single mutations); VR-E1G23 (double mutation) and VR-E1G123 (triple mutation) (Fig.22a). The construction of a vaccinia recombinant expressing RV E2 has been described (Chaye et al., 1992b).  3.3.2. Expression and antigenicity of El glycosylation mutants. Cells infected with the wild-type El vaccinia recombinant expressed a 57 kDa protein (Fig.22b, El), while the vaccinia recombinants containing El glycosylation mutant cDNAs expressed proteins with apparent molecular masses of 55 kDa (Fig.22b, Gi, G2, G3), 53 kDa (Fig.22b, G23) and 51 kDa (Fig.22b, 0123), similar to those observed in COS cells (Hobman et al., 1991). The differences in electrophoretic mobility between the wild-type and glycosylation mutants were due to differences in the numbers of oligosaccharides attached to the wild-type and mutant proteins. Corresponding polypeptides were absent from uninfected cells (Fig.22b, MI) and cells  -  106  -  C  E2  El  wtEl  I  I  E1G1  ,  I  E1G2  I  I  E1G3  I  El G23  I  E1G123  ,  YY  YY  Gi  G2Y  V  YG3  I  V  G2G3  I  Gi  G2G3  F  F  Fig.22a. Diagrammatic representation of RV El glycosylation mutant cDNAs used in the construction of vaccinia recombinant viruses. The respective portions of the E2 and El genes are indicated above the constructs. The translation initiation site is contained in the region proceeding the N-terminus of capsid near the left end of the constructs. Three N-linked glycosylation sites  76, 177, and 209 are indicated by Y and the mutagenized glycosylation sites are Gl, G2, and G3. The EcoR I (E) and Hind 111(H) sites flanking the 5’ and 3’ portion of the cDNAs, respectively, are indicated. The thick horizontal lines represent coding regions and the thin lines indicate noncoding regions. The thick vertical lines demarcate the regions of C, E2 and El coding regions (Hobman et al., 1991). at residues marked  -  107  -  Fig.22b. Expression of El glycosylation mutants by vaccinia recombinants. CV-l cells infected with wild-type vaccinia virus (WT), RV El vaccinia recombinants (El, Gi, G2, G3, G23 and G123) or mock infected (MI) were labeled with S]-methionine 35 for 30 minutes at 10 h.p.i. Cells [ were lysed with RIPA buffer and RV El-specific proteins were immunoprecipitated with RV El mAbs and subjected to SDS-PAGE. Molecular weight marks in kDa are shown on the right for reference.  -  108  -  infected with wild-type vaccinia virus (Fig.22b, WT). To characterize the immunoreactivity of the El mutant proteins, a panel of RV El-specific mAbs was used for western blot analysis under reducing and non-reducing conditions. Six RV El-specific mAbs (21B9H, 3D5D, 3D9F, 14D1F, 16B2C and H4C52) were used in this analysis. The mAbs were characterized as having VN (21B9H) or HAT (3D9F, 3D5D, 16B2C, H4C52) activities (Chaye et al., 1992a). Similar results were obtained from all six mAbs used and Fig.23 shows two typical blots using mAbs 21B9H and 3D9F. Under reducing conditions (Fig.23,  +  Me), all five El glycosylation mutants reacted with each of the six mAbs, suggesting that these mAbs recognize linear epitopes on the El primary structure. The capacity of these mAbs to bind to non-glycosylated El (Fig.23,  +  13-Me, G123) indicated that these epitopes were not  carbohydrate-dependent. The weak signal observed in immunoblots under reducing conditions is due solely to the decrease in antigenicity of El and not to the quantitative difference in the antigen used. Under non-reducing conditions, the majority of the wild-type and the single glycosylation mutants ran as monomers with a small fraction as dimers (Fig.23,  -  13-Me, wt, Gl,  G2 and G3), while the double mutant G23 gave a smeared appearence on the gel (Fig.23, 13-Me, -  G23) and the triple mutant G123 was not detectable (Fig.23,  -  f3-Me, G123). One possible  explanation for this observation is that mutant proteins G23 and 0123 form aberrant intermolecular disulfide bonds that cause them to migrate as diffuse smears on SDS-PAGE. G123 was not present on the top of the separating gel. Taken together, the data presented here suggest that denaturation of mutant proteins G23 and G123 in the absence of 13-mercaptoethanol led to decreased or abolished antibody binding activity of these proteins. Decreased antibody binding was also observed in native 023 and Gl23 proteins in immunoprecipitation (Fig.22b).  -  109  -  Table la. Summary of propeties of monoclonal antibodies directed to El  niAbs  VN or HAT  Epitope  21B9H H4C52 3D5D 14D1F 3D9F 16B2C  VN HAl HAT ND HAl HAl  214 to El El 233 ND ND ND 214 to 0 El El ND  -  109.a  + B-U.  -M. •1  —  E  o  00  -e,Iq=  00000  21B9H -  -  -  -  97 68 43 29  I  -  309F  97 68 43 29  -  — —  *--—  -  Fig.23 Immunoblot analysis of El glycosylation mutants expressed by vaccinia recombinants. Cells infected with vaccinia recombinants expressing wild-type El (WT) or El glycosylation mutants (Gi, G2, G3, G23, G123) were treated with 10 mM iodoactamine and lysed with RIPA buffer. Proteins were separated on SDS-PAGE under reducing (+ 13-Me) and non-reducing (- 13Me) conditions, and transferred to nitrocellulose membrane. RV antigens were probed with a panel of RV El-specific mAbs. Parental vaccinia virus infected cell lysate was used as the control (ctrl). Molecular weight markers (in kDa) are included for reference.  -  110-  Therefore glycosylation may be required for proper folding of El to allow efficient recognition of immunological epitopes on El.  3.3.3. Immunogenic properties of the expressed El glycosylation mutants. Although glycosylation did not significantly affect antibody binding, it might affect the elicitation of HAl and VN antibody responses. To address this question, vaccinia recombinant viruses were purified and used to immunize mice. After four injections, sera were collected, pooled and tested for their reactivities to RV El by immunoblot analysis. All the El glycosylation mutants except G123 were found to elicit antibodies recognizing authentic El from RV virions when RV proteins were separated under non-reducing conditions (Fig.24,  -  13-Me). However, only the antiserum  from wild-type El immunized mice reacted with El from RV virions under reducing conditions  (Fig.24,  +  13-Me). These results suggest that native immunogenic determinants of El  glycosylation mutants expressed via vaccinia recombinants are predominantly conformationdependent. This could be due to aberrant folding of mutant proteins and masking of linear epitopes on El when normal glycosylation is blocked. To further assess the production of anti-El antibodies from mice immunized with vaccinia recombinants, an ELISA was used to quantitate the El-specific antibodies of mouse sera (Table 2). The antibody titre from yR-El immunized mice was found to be three times higher than that in sera from those immunized with VR-E1G1, VR-E1G2, VR-E1G3 and VR-E1G23, and 15 times higher than that in serum from mice immunized with VR-E1G123. Table 2 shows the analysis of HAl and VN activities of antisera from mice immunized with vaccinia recombinant viruses. VN activity was observed in sera from mice immunized with VR-El, VR-EIG1, VR  -  111  -  —  ÷  B-Me  13-Me  • E I  H ffjj  97  684329-  Fig.24 Immunoblot analysis of sera from mice immunized with El vaccinia recombinants. Purified RV particles were subjected to SDS-PAGE under reducing (+ (3-Me) and non-reducing (- (3-Me) conditions, and transferred to nitrocellulose. El antigens were detected with antisera from mice immunized with El vaccinia recombinants. The immunizing vaccinia recombinants were indicated on the top of the gel. Wild-type vaccinia virus was used as a negative control (WT). El-specific mAbs were used as a positive control (mAb). El monomer (p), El-E2 heterodimer (o) and El-El homodimer (*) were indicated. Molecular weight markers (in kDa) are shown on left for reference.  - 112-  -E1G2 and VR-E1G3 (Table 2). Deletion of any single glycosylation site from El did not prevent neutralizing antibody production, as the ratio of VN titre to ELISA titre was similar in sera from mice immunized with yR-El, VR-E1G1, VR-E1G2 and VR-E1G3 (Table 2). Thus, carbohydrate side chains on El are not directly involved in the elicitation of VN responses. HAT activities of these sera were examined with regard to capacity to block the binding of RV virions to erythrocytes. Sera from mice immunized with yR-El, VR-E1G2 and VR-E1G3 showed HAl activity while that raised from VR-E1G1 did not (Table 2), suggesting that oligosaccharide attached at the Gi site is important in eliciting HAT antibody production. In contrast, no VN or HAT antibodies were detectable in the sera from mice immunized with either VR-E1G23 or VR E 1G 123, indicating that VN and HAl epitopes were not functionally active in these mutant proteins. This may be due to an altered protein conformation when most of or all of the carbohydrate side chains are absent.  3.3.4. Antigenic properties of deglycosylated RV El from RV virions. To further confirm the above conclusion, digestion of RV virions with endo F/PNGase F was performed under mild conditions which remove all N-linked oligosaccharides from proteins without disrupting protein conformation. The extent of deglycosylation and its possible effect on antigenicity were examined by immunoblotting (Fig.25). El from deglycosylated virions migrated as a 51 kDa protein [Fig.25,  +  13-Me, RV(dG)], similar to the non-glycosylated El expressed  from vaccinia recombinant VR-E1G123 (Fig.25,  +  13-Me, VR-E1G123), indicating that all  oligosaccharides attached to El had been removed (Fig.25,  -  113  -  +  13-Me). Under non-reducing  Table 2. Comparison of the HAl and VN antibodies from mice immunized with vaccinia recombinants containing different RV El glycosylation mutant cDNA inserts  VN titrec Virus used for immunization  ELISA titrea  HAl  vve VR-El VR-E1G1 VR-E1G2 VR-E1G3 VR-E1G23 VR-E1G123  <2 512 128 128 128 128 32  f 6 <l  titreb  128 <16 64 64 <16 <16  a  Comp.d  No comp.  <2 64 16 16 8 <2 <2  <2 32 4 4 4 <2 <2  Expressed as the highest dilution of antibodies yielding 0D 405 two times higher than background. b Expressed as the highest dilution of antibodies that completely inhibits hemagglutination. Expressed as the end point of antibody dilution that completely inhibits plaque formation. d Heat-inactivated rabbit complement (2.5%) was present in diluted antibodies. e Wild-type vaccinia virus. For the technique employed in the assay, this is the lowest dilution of serum that can be achieved. Values under 16 were considered negative.  -  114-  + B-Me  —  >  >  B-Me  >  EL Fig.25 Effect of deglycosylation on the antigenicity of RV virion. Untreated RV virion (RV), deglycosylated (dG), and cell lysate from yR-El and VR-E1G123 infected CV-l cells were subjected to SDS-PAGE under reducing (+ p-Me) and non-reducing (- f3-Me) conditions. Proteins were transferred onto nitrocellulose and RV El-specific antigens were probed with El-specific mAbs.  -  115  -  conditions, El from deglycosylated virions retained mAb binding activity, whereas non glycosylated El from vaccinia recombinant VR-E1GT23 had lost this activity (Fig.25,  -  13-Me).  This suggests that removal of sugar moieties after protein folding has less effect than blocking glycosylation during protein synthesis prior to folding. The observed significant decrease in antigen mass for El from deglycosylated virions (Fig.25) could be due to aggregation of deglycosylated virions that pelleted during sample preparation. This observation was further confirmed by using deglycosylated El from RV virions as antigen in ELISA (Table 3). Using six RV El-specific mAbs, I found that there was no difference in activity of binding to mAbs between the glycosylated and deglycosylated RV virions as determined in the ELISA (Table 3). The role of carbohydrate in HA activity of El was examined with deglycosylated RV virions. Deglycosylation of RV virions resulted in a significant decrease in the HA titre (Table 3), suggesting that carbohydrate was functionally involved in hemagglutination. However, no difference was observed between the binding of glycosylated and deglycosylated virions to chick erythrocytes when HAT mAbs were used to inhibit the binding (Table 3), suggesting that the binding of HAl antibodies to El is carbohydrate-independent.  3.3.5. Effect of glycosylation on El cell surface expression. To localize the subcellular distribution of the wild-type and mutant El proteins, CV- 1 cells infected with recombinant vaccinia viruses were analyzed by indirect immunofluorescence (Fig.26). In cells infected with vaccinia El recombinants (wild-type and glycosylation mutants), El proteins were found in the ER and Golgi-like region with the exception of E1G123, which was found to show a more profound ER staining (Fig.26, a-d). It has been shown previously that  -116-  Table 3.  A. HA assay of the deglycosylated RV virion  HA activitya HA inhibition assayb 3D5D H4C52  Glycosylated  Deglycosylated  320  40  2560 5120  2560’ 5120  aExpressed as the highest dilution of virus yielding hemagglutination. bExpress&l as the end point of antibody dilution that completely blocks the 4 HA units binding to chick erythrocytes (8-fold more deglycosylated viruses was used, compared to untreated virus).  B. Effect of deglycosylation of RV El on antibody recognition by El-specific monoclonal antibodies. ELISA titrea mAbs 21B9H H4C52 3D5D 14D1F 3D9F 16B2C a  Glycosylated  Deglycosylated  256 1024 1024 256 256 128  256 1024 1024 256 256 128  Expressed as the highest dilution of antibodies yielding twice higher 0D 405 than background.  -  117-  E2 is essential for transport of El to the cell surface (Hobman et al., 1990). The effect ofglycosylation on El cell surface expression was studied by infecting CV-l cells with El vaccinia recombinant viruses (wild-type and glycosylation mutants), or with El vaccinia recombinant viruses plus RV E2 vaccinia recombinant viruses. No cell surface expression was detected in cells infected with vaccinia recombinants of either El wild-type or glycosylation mutants (data not shown). In cells co-infected with both E2 and El vaccinia recombinants, the internal distribution of El antigens remained unchanged (Fig.26, e-h), while cell surface expression of wild-type and single glycosylation mutant El was observed (Fig.26i, j). Cell surface staining was not detected in cells co-infected with either VR-E2 and VR-E1G23 or VR-E2 and VR-E 1G123 (data not shown). The data presented here suggest that the cell surface expression of RV El requires at least two N-linked carbohydrate side chains on El proteins in addition to co-expression of RV E2.  3.3.6. Summary and Discussion The role of N-linked glycosylation on the antigenicity and immunogenicity of El  glycoprotein  was studied using vaccinia recombinants expressing El glycosylation. The expressed El glycosylation mutant proteins were recognized by a panel of specific monoclonal antibodies in radioimmunoprecipitation, immunofluorescence and immunoblotting, indicating that carbohydrate side chains on El are not involved in the constitution of epitopes recognized by those monoclonal antibodies. This observation was further supported by the fact that there is no significant change in the antigenicity after oligosaccharides on El from virions were removed by glycosidase digestion. All glycosylation mutants were capable of eliciting anti-RV El antibodies at different  -  118  -  Fig.26 Indirect immunofluorescence of El glycosylation mutants in infected CV-l cells. For cell surface expression, cells were fixed with 3% formaldehyde and incubated with an El-specific mAb mixture followed by the incubation with FITC-conjugated goat anti-mouse IgG. For detection of internal antigen, cells were permeabilized with 0.1% Nonidet-P40 prior to incubation with El mAbs. (a) VR-El; (b) VR-E1G2; (c) VR-E1G23; (d) VR-E1G123; (e) yR-El, VR-E2; (f) VR-E1G2, VR-E2; (g) VR-E1G23, VR-E2; (h) VR-E1G123, VR-E2; (i) yR-El, VR-E2, surface; (j) VR-E1G2, VR-E2, surface. For glycosylation mutants Gi and G3, a similar staining pattern as G2 was observed (data not shown).  -  119-  titres. The single glycosylation mutants (Gl, G2 and G3), but not the double mutant or the triplemutant (G123), were found to be capable of inducing VN responses. However, among the single glycosylation mutants, only G2 and G3 were able to induce HAT antibodies in mice. The influence of carbohydrate on the antigenicity of viral glycoproteins has been demonstrated in a number of viruses (see introduction section It has been suggested that oligosaccharide side chains mask adjacent polypeptide sequence and prevent the binding of respective antibodies. In our case, no difference in antigenicity was observed between the wildtype and mutant El proteins expressed from vaccinia recombinants, with respect to their mAb binding activities in immunoblotting under reducing conditions (Fig.23), suggesting that there are carbohydrate-independent epitopes in El for the six mAbs tested. Consistent with this interpretation, deglycosylation of RV virions with endo F/PNGase F resulted in no quantitative change in reactivity to El-specific mAbs when analyzed by ELISA (Table 3). However, a significant decrease was observed in HA activity of the deglycosylated RV virions (Table 3). It is concluded that carbohydrate on El is not involved in the constitution of epitopes recognized by mAbs but is functional in hemagglutination. However, non-glycosylated El expressed from VR-E1G123 failed to react to any of the mAbs under non-reducing conditions, presumably due to aberrant folding of the mutant protein when carbohydrate side chains were absent. Most viral glycoproteiris contain multiple N-linked glycosylation sites. Site-specific effects on the processing and intracellular transport of glycoproteins have been reported for SV5 HN (Ng et al., 1990) and also observed for RV E2 (see 3.1. in this section). Tn this study, all the RV El mutants were found to reside in the ER and Golgi-like region except VR-E1G123 which was localized predominantly in the ER region. All three single glycosylation mutants (E 11, E 1G2,  -  120  -  E1G3) were found to be transported to the cell surface when RV E2 was present in the cells (Fig.26j), suggesting that carbohydrate at each of the three sites affects intracellular transport of El equally. It has been reported previously that co-expression of RV E2 is required for cell surface expression of RV El, whereas E2 transport to the cell surface is an E 1-independent event (Hobman et al., 1990). In this study, I have shown that besides co-expression of E2, cell surface expression of El also requires any two of the three N-linked oligosaccharides. Taken together, this suggests that the interaction of El and E2 is a post-translational event rather than a co translational process and the formation of El -E2 heterodimer is probably important to release El from retention in the Golgi apparatus. I was interested in exploring the site-specific effect of glycosylation on the immunogenicity of RV El. Immunization of mice with vaccinia recombinants expressing El glycosylation mutants showed that removal of any one of the carbohydrates from El does not prevent single mutants from inducing VN antibodies in mice, suggesting that the protective immune response is probably directly toward the polypeplide backbone. Similarly, neutralizing activity is also detected in rabbit serum raised against RV El peptide expressed in E.coli (Terry et al., 1989). These results are consistent with the finding that the VN epitopes of El map to a region of the C-terminal half of El that does not contain glycosylation sites (Terry et al., 1988; Wolinsky et al., 1991; Chaye et al., 1992a). The failure of VR-E1G23 and VR-E1G123 to elicit neutralizing antibodies is probably due to improper conformation of the mutant proteins. A considerably lower antibody response was found in sera from mice immunized with VR-E1G1, VR-E1G2, and VR-E 103. Of three single mutants, VR-E1G1 did not induce HAl antibodies, indicating that oligosaccharide attached to the 01 site is either involved in hemagglutiniation directly or is critical for facilitating  -  121  -  hemagglutinin epitope exposure. The hemagglutinin epitopes of El have been defined as residing in the same region as neutralizing epitopes (Terry et al., 1988; Chaye et al., 1992a), a carbohydrate-free domain. However, I have shown that hemagglutination is carbohydratedependent (Table 3). Thus, the expression of HA epitopes for the induction of HAT antibody production is dependent on the conformation of native El protein. It has been shown that an El peptide expressed in E.coli is recognized by HAT mAbs but fails to produce HAl antibody in rabbits (Terry et al., 1988). The results presented here indicate that although the addition of carbohydrate is not essential for antibody binding to El, deletion of any one of the oligosaccharide side chains from El results in a less immunogenic state of El, probably due to improper folding. Thus, in developing an effective RV subunit vaccine using El, proper combinations of different epitopes in their immunoactive conformations must be considered.  -  122  -  3.4. Section IV. Expression and characterization of virus-like particles containing rubella virus structural proteins  3.4.1. Isolation of BHK cell lines expressing RV structural proteins Three RV cDNA constructs (Fig.27) were used in the isolation of stably transformed BHK cell  lines. These cDNAs encode the capsid protein (C), E2E1 polyprotein precursor (E2E1) or polyprotein precursor for all three structural proteins of RV (24S) (Clarke et al., 1987; Hobman et at, 1990). The cDNAs were subcloned into the  I site of transfer vector pNUT (Fig.4c)  (Palmiter et al., 1987), under the control of the metallothionein I promoter (Fig.4c). The resultant recombinant plasmids were transfected into BHK cells using the calcium phosphate method ( (Gorman et al., 1982). Twenty-four hours after transfection, methotrexate (2.5 mM) was added to the culture medium and cells were incubated with this selection medium for 10 days. Methotrexate-resistant colonies were picked and screened for the integration of RV cDNAs into their chromosomes using the polymerase chain reaction (Saiki et al., 1988) and for the expression of RV structural proteins using western blot analysis (Towbin et al., 1979). Isolated cell lines were stable under normal growth conditions as they retained the capacity to express RV structural proteins after four months of continuous culturing. Cell lines were named according to the RV cDNAs used for transfection, as BHK-C, BHK-E2E1 and BHK-24S, respectively.  -  123  -  RV cDNAs  ATG  C C ATG  E2E1  —  C  E2  El  ATG  24S C  E2  El  Fig.27 Diagrammatic representation of RV cDNAs used in the construction of recombinant plasmids. Translation initiation site (ATG) from RV capsid protein was used in all constructs. The putative signal peptides and membrane anchor domams of E2 and El are indicated as or respectively.  ,  -  124  -  3.4.2. Expression of RV structural proteins Monolayers of stably transformed cells were incubated with medium containing 30 iiM zinc sulfate for 12 hours to induce the expression of RV structural proteins from the promoter. The expression of RV structural proteins from stably transformed cell lines was analyzed by imunoblotting using human anti-RV serum (Towbin et al., 1979). In BHK-C cells, an intracellular protein species with molecular size of 34 kDa was observed (Fig.28A, lanes C). This protein may represent the capsid protein of RV. In BHK-E2E 1 cells, protein species corresponding to the ERand Golgi- isoforms of RV E2 (Hobman et al., 1990) and El glycoproteins were found in the cell lysate but not in the medium (Fig.28A, lanes E2E1), indicating that the E2E1 polyprotein precursor was synthesized and proteolytically processed to give rise to E2 and El proteins. In BHK-24S cells, protein species corresponding to the C, E2 and El proteins of RV were present in the cell lysate as well as in the medium (Fig.28A, lanes 24S), suggesting that the integrated cDNA of 24S RNA was active in directing the synthesis of RV structural proteins and these structural proteins were released from the cells. The secretion of RV structural proteins from BHK-24S increased with time and was linear over a period of 24 hours under ZnSO 4 induction (Fig.29).  3.4.3. Assembly and release of virus-like particles (VLPs) in stable BHK-24S cells The secretion of RV structural proteins into the medium was found to be dependent on the coexpression of C, E2 and El, suggesting that these proteins might be assembled into subviral particles prior to their release from the cells. To examine this possibility, medium from BHK-24S and RV-infected cells was subjected to ultracentrifugation (350,000xg for 20 minutes), in the  -  125  -  A lysate  medium -  OWNO  w  N  97  68-  29-  B  123  68-  leE 29-  —-Pc  Fig.28 A. Immunoblot analysis of proteins expressed in transformed BHK cells. Monolayers of BHK-C, BHK-E2E1 or BHK-24S cells were incubated with serum-free medium in the presence of 30 jiM zinc sulfate for 12 hours. Culture media were collected and cell monolayers were iysed with RIPA buffer. Samples were directly subjected to SDS-PAGE and immunoblotting. B. Immunoblot analysis of proteins sedimented by ultracentrifugation. Samples from medium of induced BHK-24S (Lanes 1 and 2) or RV-infected BHK cells (Lane 3) were centrifuged at 90,000 rpm for 20 minutes in the absence (Lanes 2 and 3) or presence of 1% NP-40 (Lane 1). The pellets were resuspended in RIPA buffer and analyzed using SDS-PAGE and immunoblotting. The positions of RV structural proteins are indicated by anowhead. The molecular weight markers are included for reference.  -  126  -  0 0.5  1  2  4  10 14 IS 24 RV (hour)  7  medium -4. —  ;qq.  4E1 )E2  lysate 4E1  = jØJ)E2  Fig.29. Time course of VLPs secretion from BHK-24S cells. Expression of RV structural proteins was induced by the addition of ZnSO 4 (30 jiM in the culture medium). Culture medium was collected and cells were lyzed at the indicated times (hour post-induction). Samples from medium were subjected to 90,000 rpm centrifugation for 20 minutes and resuspended in RIPA buffer. The resuspended pellets and the cell lysates were analyzed using SDS-PAGE and immunoblotting. The positions of RV structural proteins are indicated.  -  127  -  presence or absence of 1% non-ionic detergent, Nonidet P-40. Resuspended pellets were subjected to SDS-PAGE, transferred to nitrocellulose membranes and probed with human anti-RV serum. In the absence of NP-40, C, E2 and El were detected in the pellets from BHK-24S and RV infected cells (Fig.28B, lanes 2 and 3). In the presence of NP-40, El and E2 glycoproteins remained in the supernatant after ultracentrifugation (not shown), although trace amounts of C were found in the pellet (Fig.28B, lane 1). Thus the assembled viral proteins are secreted as particles that sediment in a gravitational field. To confirm that proteins El, E2 and C assembled into VLPs, samples from pelleted VLPs were centrifuged for 16 hours at 90,000xg through a density gradient from 20 to 50% sucrose. VLPs were recovered in fractions with density of 1.171.19 g/nil (Fig.30); similar to that of native RV virion (1.175-1.20 g/ml) (reviewed by Horzinek, 1981).  3.4.4. Electron-microscopic analysis of the VLPs The morphology of the VLPs was analyzed by employing conventional electron microscopic technique with routine Epon embedding of fixed BHK-24S cells. The VLPs found in BHK-24S cells were comparable in size to RV particles (60 nm) (Fig.3lA) and indistinguishable in appearance, with an electron dense core surrounded by an envelope (Fig.31B, C, D). These particles were predominantly located within the vacuoles in the juxtanuclear region (Fig.3 lD) or cytoplasm (Fig.3 1C), which may represent the Golgi structure. Some particles were distributed in the cytoplasm (Fig.3 1B), not associated with any membrane structure. Such particles were not observed in BHK-E2E1 or BHK-C cells (data not shown). Taken together, it is evident that VLPs were indeed assembled intracellularly prior to their release from the cells.  -  128  -  1  3  7  5  9  1113 15 17 19 2123 2  RV — -  —  -  •  4 El E2  ‘N VLP  II  Fig.30 Purification of VLPs and RV on sucrose density gradient centrifugation. Pelleted RV or VLPs from 35 ml culture medium were resuspended in 0.35 ml TNG buffer (50 mM Tris, pH7.5; 100 mM NaC1; 200 mM glycine) and applied onto the top of a 12 mi-sucrose gradient of 20-50% sucrose in TNG. Centrifugation was carried out using a Beckman SW41 rotor at 90,000xg for 16 hours at 15°C. Fractions (— 0.5 mI/fraction) were collected by puncturing the bottom of the tube and the density of each fraction was determined. 100 il sample from alternative fractions were diluted with equal amount of TNG buffer and subjected to centrifugation at 90,000 rpm for 20 minutes. The pellets were resuspended with RIPA buffer. RV proteins in the pellets, and in the samples that loaded onto the gradient (load), were analyzed by SDS-PAGE and immunoblotting (using human anti-RV serum). Fractions are numbered from the bottom (#1) to the top (#23) of the gradient. The positions of RV structural proteins are indicated.  - 129  %_‘.  4  Fig.31 Electron microscopic analysis of the VLPs in BHK-24S cells. RV-infected BHK cells (A) or induced BHK-24S cells (B, C, D) were fixed with formaldehyde/glutaraldehyde, postfixed with osmium tetroxide, ethanol dehydrated and Epon embedded. Thin sections were analyzed by electron microscopy after staining.  -  130  -  3.4.5. Antigenicity of the VLPs. HA activity of the VLPs was examined using a heparin/manganous chloride procedure (Liebhaber, 1970) and the HA titre was expressed as the endpoint of serial dilutions of sera at which erythrocyte aggregation was observed. The VLPs from BHK-24S cells displayed HA activity of 64, while RV particles retained HA activity when diluted to 1/32. This difference is due to the higher yield of VLPs from induced BHK-24S cells than that of RV from infected cells. To compare the antigenicity of VLPs with that of RV, equal amounts of RV or VLPs (with respect to HA unit) were used in each assay. Table 4 shows the antibody binding activities of VLPs and RV in immunoblot and ELISA analysis using twelve mAbs against RV El, E2 or C. Two of the E2 mAbs showed differences in western blotting between the VLPs and RV (Table 4) and VLPs displayed a higher ELISA titre with a mAb against C protein than did RV (Table 4). VLPs were also used in a solid phase immunoassay to measure the IgG response in humans. With 200 human serum samples, it was found that the correlation coefficient between the VLPs and whole RV antigens was 0.96 using a non-parametric regression analysis method (data not shown). This indicated that the antigenic determinants on the VLPs resemble those of authentic RV.  3.4.6. Immunogenicity of the VLPs. To evaluate the immunogenic properties of the VLPs, we immunized mice (BALB/c, four in each group) with the VLPs, RV or soluble El protein expressed in transfected BHK cells (Gillam, unpublished results), respectively, with comparatively same amounts of antigens (equivalent to  -  131  -  Table 4. Immunoreactivity of the VLPs with RV-specific monoclonal antibodies. Western blota  ELISA  titreb  mAb  RV  VLP  RV  VLP  H15C22(C) H32C43(El) 21B9H(E1) 3D5D(E1) 14B1F(E1) 3D9F(E1) 16A1OE(El) E2-2(E2) E2-4(E2) E2-5(E2) E2-6(E2) H46C64(E2)  +  +  -  -  128 256 256 1024 256 256 1024 160 320 320 160 320  256 256 256 1024 256 256 1024 160 320 320 160 320  +  +  -  -  +  +  +  +  +  +  -  -  +  + + +  -  -  +  +  a  Monoclonal antibodies were used at a dilution of 1:100 for ascites fluid or 1:5 for tissue culture supernatant. +, positive reactivity was detected. -, negative reactivity was detected.  b  Expressed as the highest dilution of antibodies yielding 0D 405 two times higher than background.  -  132  -  256 HA units), The presence of anti-RV antibodies in the sera of immunized mice was analyzed using radio-immunoprecipitation. As shown in Fig.32, mice immunized with the VLPs produced antibodies against all three structural proteins of RV (Fig.32, lane 3), as did mice immunized with RV (Fig.32, lane 4). Mice immunized with El protein also developed some anti-El antibody response (Fig.32, lane 2). ELISA was used to quantify the antibody titres against each of the RV structural proteins, by using individual purified RV structural proteins expressed in Spodoptera frugiperda cells infected with baculovirus recombinants (Gillam, unpublished data) as antigens. In the sera from mice immunized with VLPs, a significantly higher anti-C antibody titre was found, whereas antiEl and E2 antibody titres were slightly lower (Table 5). The biological functions of these antibodies were analyzed. Sera from VLP-immunized mice displayed VN activities (Table 5) as determined by plaque reduction assays (Fukuda et al., 1987). HAl activities were also present in the sera from mice immunized with the VLPs, as well as in the sera from mice immunized with RV (Table 5). These results suggested that although VLPs were less active in inducing overall anti-El and E2 antibodies compared to RV, they induced the production of both VN and HAT antibodies. We have also determined cell-mediated immune responses against RV in VLP-immunized mice in a lymphocyte proliferation assay (Chaye et al., l992a; Ou et  a,  1992). Lymphocyte  proliferative responses of mice were determined in vitro by direct stimulation of lymphocytes with UV-inactivated RV or individual RV structural proteins (C, E2 and El) purified from recombinant baculovirus infected insect cells. Lymphocytes from VLP-immunized mice responded strongly to UV-inactivated RV as well as to the individual RV structural proteins in  -  133  -  12 34 I  -••  LJ.  E2  Fig.32 Radioimmunoprecipitation of RV structural proteins expressed in COS cells. COS cells were transfected with pCMV5-24S (Honman and Gillam, 1989), labeled with S]-methionine 35 [ and lysed. RV structural proteins were recovered from cell lysates with mouse anti-RV antibodies pre-bound to Sepharose 4B-protein A beads as previously described (Hobman and Gifiam, 1989) and separated by SDS-PAGE. Sera were from mice immunized with El protein (Lane 2), VLPs (Lane 3), RV (Lane 4) or from pre-immune mice (Lane 1). The positions of RV structural proteins are indicated.  -  134  -  Table 5. Comparison of antibody titres of mouse sera from mice immunized with different RV antigens. ELISA titre to RV proteina titre”  HAl titrec  immunogen  C  E2  El  El  <10  10  10  <2  <8  RV  40  80  160  16  32  VLP  320  40  80  8  16  alndividual RV structural proteins (C, E2 and El) were purified from SF9 cells infected with baculovirus recombinants expressing each RV structural protein (unpublished data). bExpressed as the reciprocal of the highest antibody dilution that show 50% reduction in plaque formation. cExpressed as the reciprocal of the highest antibody dilution that inhibited hemagglutination.  -  135  -  CI)Ifl  xl,000  50  40 ± E2 30 El 20  1  0 0  2  4  6  8  10  12  14  16  18  protein concentration (jig/mi)  Fig.33 Lymphoproliferation responses of mice immunized with VLPs. Lymphocytes from VLP immunized mice (2.5x 10 /well) were incubated with different concentrations of proteins El (, 5 E2 (+) or C (*) at 37°C for 5 days before addition of [ H1-thymidine (1 jiCi/well). All assays 3 were performed in triplicate and results are expressed as mean values. Using UV-inactivated RV (l0 PFU/ml), lymphoproliferation responses in the presence and absence of RV antigen were 23,000 and 2,000cpm respectively.  -  136  -  a dose-dependent manner (Fig.33).  3.4.7. Summary and Discussion Noninfectious VLPs containing three structural proteins were expressed in a BHK cell line (BHK 24S) by using an inducible promoter. These VLPs were found to resemble RV virions in terms of their size, morphology and some biological activities. In immunoblotting studies, VLPs were found to bind similarly to native RV virions with 10 of a panel of 12 RV-specific murine monoclonal antibodies. Immunization of mice with VLPs induced specific antibody responses against RV structural proteins as well as VN and HAT antibodies. After immunization of mice with VLPs, in vitro challenge of isolated lymphocytes with inactivated RV and individual RV structural proteins stimulated proliferation. The assembly of RV vision involves at least two major steps: encapsidation and envelopment of nucleocapsid. In RV, encapsidation occur in the cytoplasm as newly synthesized capsid protein interacts with genomic RNA to form icosahedral nucleocapsids. The packaging of genomic RNA into the nucleocapsid is believed to be a specific event as the 40S genomic RNA but not the 24S subgenomic RNA is packaged into RV vision (Oker-Blom et al., 1984). Recently, a stretch of 31 nucleotides on the 5’ end of the RV genome has been identified as responsible for the binding of the genomic RNA to the capsid protein in vitro (Liu et al., unpublished data). Employing reverse transcription combined with the polymerase chain reaction (Saiki et al., 1988), we failed to detect any RV-specific RNA in the pseudovirion secreted from stable BHK-24S cells (data not shown), suggesting that capsid proteins can interact with each other and form a nucleocapsid-like structure. The VLPs were found to have a higher ELISA titre with a C-specific mAb (Table 4) and to elicit stronger anti-C antibody response in mice than those from RV (Table  -  137  -  5), implying that the VLPs contain more C protein than RV either due to its relative amount or conformational exposure in the particles. Although the pseudovirions do not contain RV-specific RNA, we cannot rule out the possibility that they might package some cellular RNAs or even DNAs into the nucleocapsid. Incorporation of nucleocapsid into the membrane envelope to form virus particles is a yet poorly understood event in virus assembly. We found that in BHK-C and BHK-E2E1 cells, no RV proteins were released into the medium (Fig.28A). In BHK-24S cells, all three structural proteins were present in the medium as the result of subviral particle formation and egress (Fig.28A). These data strongly suggest that the interaction between glycoproteins and the nucleocapsid is the driving force for the assembly and release of the VLPs. This interaction has been defined to occur between the cytoplasmic tail of glycoprotein El and the capsid protein. Deletion or substituton of the cytoplasmic domain of RY El abolished the delivery of the VLPs into the medium (Hobman et al., unpublished data). Besides being a useful tool to study RV assembly, BHK-24S cells in which VLPs are steadily assembled and released can be used as a potential source for mass production of rubella antigens at low cost under inducing conditions. BHK-24S cells continuously produce VLPs for up to 5 days without cell lysis when 30 jiM ZnSO 4 is present in the medium and up to one month in DME/F12 (GIBCO) medium. VLPs can be harvested daily from the medium, which is replaced with fresh medium after harvesting. Depending on the methods used to quantitate the yields of the VLPs and RV from culture medium, the yield of VLPs was found to be two-fold higher than RV in an HA assay, five times higher in ELISA assays using human sera, and more than ten times higher by protein quantitation using silver staining after gel electrophoresis (data  -  138  -  not shown). It has been proposed that immunogenicity can be achieved by presenting antigens on a polyvalent particle structure. This concept led to the development of chimeric virus (Clarke et al., 1987; Michel et al., 1988; Li et al., 1993), virus-like particles (Griffiths et aL, 1993) or immunostimulating complexes (Takahashi et al., 1990), in which multiple copies of antigen are integrated in a particulate form. These particles have been found to induce both humoral and cellmediated immune responses, including VN antibodies (Michel et al., 1988; Griffiths et al., 1993; Li et al., 1993), T helper cells or cytotoxic T lymphocytes (Takahashi et al., 1990; Griffiths et al., 1993; Li et al., 1993) in animals. In our case, the VLPs were found to be significantly more active than the soluble El protein in inducing antibody responses in mice, especially for the production of VN and HAl activity (Table 5). The VLPs also evoked cell-mediated immune response to RV and RV structural proteins. This is believed to be important in providing protective immunity against RV infection. Preliminary results have shown that CD4 T cells may be the major effector in cell- mediated immune responses elicited by the VLPs in mice, whereas CD8 T cells may be also involved (data not shown). A study of the phenotype of the effector cells in proliferation assays is in progress. The VLPs are composed of all three structural proteins of RV, which make them similar to RV regarding antigen-presentation. These studies suggest that the noninfectious but highly immunogenic VLPs may serve as a candidate for safe vaccine development.  -  139  -  4. SUMMARY and PERSPECTIVES In the Togavirus family, RV bears a striking similarity to the prototype aiphaviruses (SFV and SIV) in terms of genomic organization and strategy for gene expression. However, RV research has fallen far behind that of aiphaviruses due to the fact that RV has a much slower replication kinetics and limited cytopathological effects, and there has been limited success in producing large numbers of monoclonal antibodies directed to RV structural proteins (C, E2). In recent years, with the aid of recombinant DNA technology and mammalian expression systems, much progress has been made in studies on the expression, processing and biological functions of RV structural proteins. In this study, I have attempted to employ various mammalian expression systems (COS cell transient expression, vaccinia recombinant virus, stably transformed cells and RV-infected cells), combined with recombinant DNA techniques (including site-directed mutagenesis), to explore the structure/function relationship of RV structural proteins. The role of N-linked glycosylation of RV El and E2 has been studied with respect to their biological function in RV maturation. RV El is the dominant surface molecule of the RV virion and the major target for human immune surveillance. The influence of N-linked glycosylation on antigenicity and immunogenicity of RV El has been investigated by expressing El glycosylation mutant proteins via vaccinia recombinants and by analyzing the immunoreactivity of deglycosylated El protein from RV virions treated with glycosidase to remove carbohydrates on El. It appears that all three N-linked glycosylation sites on El are required to maintain an optimal protein configuration for the exposure of epitopes determining biological functions (i.e. neutralization and hemagglutination). Deletion of any of the N-linked glycosylation sites on El results in reduction of antibody production and this effect seems to be additive. Carbohydrates  -  140  -  on El, important for the hemagglutination activity of the virion, however, are not involved in the constitution of the epitopes recognized by monoclonal antibodies used in this study. Unlike El, the biological function of E2 is not clear except that it is believed that in RV infected cells, E2 serves as a carrier to deliver El from a post-ER, pre-Golgi compartment to the Golgi complex. The role of N-linked glycosylation on intracellular transport and processing of RV E2 has been analyzed by transient expression of E2 glycosylation mutant proteins in COS cells. N-linked glycosylation at all three sites is essential for transport competence of E2. Deletion of any glycosylation site on E2 leads to a significant reduction in rate of glycan processing, lower stability and retention of mutant proteins in the ER. A conm-ion effect of deleting glycosylation sites between El and E2 is the formation of aberrant disulfide bonds. Amino acid sequences predicted from cDNA sequences indicate that both El and E2 are rich in cysteine residues. The implication of this is that proper folding and correct disulfide bonding may be a slow process, sensitive to modulation by structural alteration or environmental influence. Iritra- and intermolecular disulfide bonds have been observed in wildtype, as well as mutant El and E2 when they are expressed separately (Fig.9, Fig.23). It appears that deletion of glycosylation sites adjacent to a cysteine residue (E 11, E2G2 and E2G3) has a more profound deleterious effect. Therefore, it is conceivable that the addition of carbohydrate contributes to the proper folding and subsequent correct disulfide bonding, which may be critical for the biological functions of El and E2. Although the role of N-linked glycosylation on the functions of El and E2 has been studied in some detail, the effect of glycosylation during RV replication and infection is still largely unknown. Deglycosylated RV virions lost hemagglutination activity (Table 3) while their  -  141  -  infectivity has not been determined. Recently, Dr. T.K. Frey and co-workers have succeeded in the construction of RV genome-length cDNA clones. Transfection of cells with RNA transcribed from one of these clones results in the production of virus that preserved the genetic and phenotypic characteristics of the parental virus from which the cDNA clone was derived. Such an infectious cDNA clone will facilitate the evaluation of site-specific effect of glycosylation in viral assembly and infectivity (Wang et al., 1994). Due to the fact that RV infectious cDNA was not available during this study, an alternative approach was taken to elucidate the structure/function relationship of RV structural proteins during virus assembly. Tunicamycin, BFA and monensin were used to inhibit or to induce an altered processing and transport of RV glycoprotein in cDNA-transfected cells and RV infected cells. Protein processing and transport as well as virus assembly and release were analyzed. BFA and monensin dramatically reduce the assembly of intracellular infectious viions while tunicamycin completely inhibit virus assembly. The effect of tunicamycin, BFA and monensin on virus infectivity parallels that of the disruption of distribution of El and E2 in an intact Golgi complex, which points out the possibility that probably a stable association of El and E2 with the Golgi structure may be essential for efficient assembly of RV. This is in a good agreement with the fact that RV El and E2 are found to be targeted to the Golgi complex (Hobman et al., 1993) with a limited amount expressed at the cell surface (Hobman et al., 1990). In RV-infected cells, the released virus has a slightly higher virus titre than that of intracellular virus (even during the early stage of virus life cycle), and is found to contain 0-linked glycans as well as complex-type of N-linked sugars on E2. The intracellular viruses has not been purified to homogeneity or distinguished from unassembled RV structural proteins due to technical  -  142  -  difficulties, therefore, the detailed oligosaccharide structures on El and E2 of intracellular virions are not known. It is possible, however, that the difference in the status of glycan maturation between extra- and intracellular viruses may contribute to their infectivity. In addition, further studies are necessary to define the mechanism of retention of RV glycoprotein in the Golgi apparatus, and the importance of Golgi-specific modifications (i.e. 0-linked glycosylation) on virus assembly. Three stably transformed cell lines expressing RV structural proteins have been constructed. RV structural proteins are found to assemble into virus-like particles in the Golgi complex prior to their release from the cells. The assembly of VLPs is found to be independent of genomic RNA but is strictly dependent upon co-expression of all three structural proteins. The VLPs resemble RV virions in terms of size, buoyant density, morphological appearance and protein composition. These observations point out the future direction in studying the structure/function relationship and protein-protein interaction during RV assembly. Stable cell lines expressing RV structural proteins in different combinations under an inducible promotor, or with molecular modifications, will be a useful system. The non-infectious VLPs have been found to bind similarly to native RV virions with RV specific monoclonal antibodies as well as human sera. Humoral (including viral neutralizing and hemagglutination inhibition) and cell-mediated immune responses have been detected in mice immunized with the VLPs. Therefore, the VLPs may serve as a convenient source of RV antigen for serodiagnostic assays and a potential candidate for vaccine development. The VLPs contains all three RV structural proteins and closely resemble RV regarding antigen presentation, having the advantage over synthetic peptide or subunit vaccines containing single species of RV  -  143  -  structural proteins. However, it should be noted that since it has been suggested that molecular  mimicry of E2 (Yoon et al., 1992) and C (Karounos et al., 1993) may lead to autoimmune tissue damage, such epitopes should be deleted from the VLPs for safer vaccine development.  -144-  REFERENCES Abernathy, E.S., Wang, C.Y., and Frey, T.K. (1990). Effect of antiviral antibody on maintenance of long term RV persistent infection in Vero cells. J. Virol. 64:5183-5187. Adams, G.A., and Rose, J.K. (1985). Structure requirements of a membrane-spanning domain for protein anchoring and cell surface transport. Cell 41:1007-1015. Alexander, S. and Elder, J.H. (1984). Carbohydrate dramatically influence immune reactivity of antiserum to viral glycoprotein antigens. Science 226:1328-1330. Allchatib, G., Shen, S.-H., Briedis, D., Richardson, C., Massie, B., Weinberg, R., Smith, D., Taylor, J., Paoletti, E., and Roder, J. (1994). Functional analysis of N-linked glycosylation mutants of the measles virus fusion protein synthesized by recombinant vaccinia virus vectors. J. Virol. 68:1522-1531. Andersson, S., Davis, D.L., Dahlback, H., Jornvall, H. and Russell, D.W. (1989). Cloning, structure and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J. Biol. Chem. 264:8222-8229. Assad, F., and Ljungars-Esteves, K. (1985). Rubella-world impact. Rev. Inf. Dis. 7:S29-36. Bardeletti, G., Tektoff, I., Gautheron, D. (1979). Rubella virus maturation and production in two host cell system. Intervirology 11:98-103. Baron, M. and Forsell, K. (1991). Oligomerization of the structural proteins of rubella virus. Virology 185:811-819. Benjouad, A., Gluckman, J.-C., Rochat, H., Montagnier, L., and Bahraoui, E. (1992). Influence of carbohydrate moieties on the immunodeficiency virus type 1 recombinant gpl6O. J. Virol. 66:2473-2483. Bowden, D.T. and Westaway, E.G. (1984). Rubella virus: structural proteins and nonstructural proteins. J. Gen. Virol. 65:933-943. Bowden, D.T. and Westaway, E.G. (1985). Changes in glycosylation of rubella virus envelope proteins during maturation. 3. Gen. Virol. 66:20 1-206. Brown, D.T., and Smith, J.F. (1975). Morphology of BHK-21 cells infected with Sindbis virus temperature-sensitive mutants in complementation group D and E. J. Virol 15:1262-1266. Buimovici-Klein, E., and Cooper, L.Z. (1985). Cell-mediated immune response in rubella infection. Rev. Infect. Dis. 7:S123-S128.  -  145  -  Cash, P. (1982). Inhibition of LaCrose virus replication by monensin, a monovalent ionophore. J. Gen. Virol. 59:193-196. Caust, J., Dyall-Smith, M.L., Lazdins, I., and Holmes, I.H. (1987). Glycosylation, an important modifier of rotavirus antigenicity. Arch. Virol. 96:123-134. Chantler, J.K., Ford, D.K. and Tingle, A.J. (1982). Persistent rubella virus infection and rubellaassociated arthritis. Lancet 1:1323-1325. Chaye, H.H., Chong, P., Tripet, B., Brush, B. and Gillam, S. (1992a). Localization of the virus neutralizing and hemagglutinin epitopes of El glycoprotein of rubella virus. Virol. 189:483-492. Chaye, H.H., Mauracher, C.A., Tingle, A.J. and Gilam, S. (1992b). Cellular and humoral immune responses to rubella virus structural proteins El, E2 and C. J. din. Microb. 30:23232329. Chaye, H.H., Ou, D., Chong, P., and Gillam S. (1993). Human T and B cell epitopes of El glycoprotein of rubella virus. J. Clin. Immunol. 13:93-100. Chen, S., Matsuoka, Y. and Compans, R.W. (1991). Assembly and polarized release of Punta Toro virus and effect of brefeldin A. J. Virol. 65:1427-1439. Clarke, B.E., Newton, S.E., Carroll, A.R., Francis, M.J., Appleyard, G., Syred, A.D., Highfield, P.E., Rowlands, D.J., and Brown, F. (1987). Improved immunogenicity of a peptide epitope to hepatitis B core protein. Nature (London) 330:381-384. Clarke, D.M., Loo, T.W., Hui, I., Chong, P. and Gillam, S. (1987). Nucleotide sequence and in vitro expression of rubella virus 24S subgenomic mRNA encoding the structural proteins El, E2 and C. Nucl. Acids Res. 15:3041-3057. Clarke, D.M., Loo, T.W., McDonald, H. and Gillam, S. (1988). Expression of rubella virus cDNA coding for the structural proteins. Gene 65:23-30. Cohen, G.H., Dietzschold, B., Ponce de Leon, M., Long, D., Golub, E., Varrichio, A., Periera, L. and Eisenberg, R.J. (1984). Localization of discontinuous epitopes of herpes simplex virus glycoprotein D: Use of a non-denaturing “native gel” system of polyacrylamide gel electrophoresis coupled with western blotting. J. Virol. 49:102-108. Collins, P.L., and Mottet, G., (1992). Oligomerization and post-translational processing of glycoprotein G of human respiratory syncytial virus: Altered 0-glycosylation in the presence of brefeldin A. J. Gen. Virol. 73:849-863. Cooper, L.Z. and Buimovici-Klein, E. (1985). Rubella. In Fields, B.N. (ed) “Field’s Virology”, NY: Raven Press, pplOOS-1O2O.  -  146  -  Davis, N.L., Peuce, D.F., Meyer, W.J., Schmaljohn, A.L., and Johnston, R.E. (1987). Alternative forms of a strain-specific neutralizing antigenic site on the Sindbis virus E2 glycoprotein. Virology 161:101-108. Dedera, D., Gu, R., and Ratner, L. (1992). Role of asparagine-linked glycosylation in human immunodeficiency virus type 1 transmembrane envelope protein function. Virology 187:377-382. deMazancourt, A. and Perricaudet, M. (1989). Expression of rubella virus cDNA encoding the El structural protein. Biochimie 71:681-685. Deshpande, K., Fried, V.A., Ando, M., and Webster, R.G. (1987). Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence. Proc. Nati. Acad. Sci. USA. 84:36-40. Dominguez, G., Wang, C-Y, and Frey, T.K. (1990). Sequence of the genome RNA of rubella virus: Evidence for genetic rearrangement during Togavirus evolution. Virology 177:225-238. Doms, R.W., Russ, D., and Yewdell, J.W. (1989). Brefeldin A redistributes resident and itinerant Golgi proteins to the endoplasmic reticulum. J. Cell Biol. 109:61-72. Dorsett, P.H., Miller, D.C., Green, K.Y. and Byrd, F.I. (1985). Structure and function of the rubella virus proteins. Rev. Inf. Dis. 7:S250-S 156.  Dubois-Dalq, M., Holmes, K.V., and Rentier, B. (1984). Assembly of paramyxoviridae. In Assembly of enveloped RNA viruses. Vienna:Springer-Verlag. pp44-65. Dunphy, W.G. and Rothman. J.E. (1985). Compartmental organization of the Golgi stack. Cell 42:13-21. Earl, P.L., Moss, B., and Doms, R.W. (1991). Folding, interaction with GRP78-BiP, assembly, and transport of the human immunodeficiency virus type I envelope proteins. J. Virol. 65:20472055. Elbein, A.D. (1987). Inhibitors of the biosynthesis and processing of N-linked oligosaccharide chains. Annu. Rev. Biochem. 56:497-534. Elder, J.H., McGee, J. S., and Alexander, 5. (1986). Carbohydrate side chains of Rauscher leukemia virus envelope glycoproteins are not required to elicit a neutralizing antibody response. J. Virol. 57:340-342. Fenouillet, E., Gluckman, J.C., and Bahraoui, E. (1990). Role of N-linked glycans of envelope glycoproteins in infectivity of human immunodeficiency virus type 1. J. Virol. 64:2841-2848.  -  147  -  Francki, R.J.B., Fauquet, M., Knudson, D.L., and Brown, F. (eds). (1991). Classification and nomenclature of viruses. In Fifth report of the International Committee on Taxonomy of Viruses. Arch. Virol. (sup.2). Frey, T.K., Man, I.D., Hemphill, M.L. and Dominguez, G. (1986). Molecular cloning and sequencing of the region of rubella virus genome encoding for glycoprotein El. Virol. 154:228232. Frey, T.K. and Man, I.D. (1988). Sequence of the region codifying virion proteins C and E2 and the carboxy terminus of the non-structural proteins of rubella virus: comparison to aiphavirus. Gene 62:85-99. Frey, T.K. (1994). Molecular biology of rubella virus. Arch. Virol. Fujiwara, T., Oda, K., Yokota, S., Takatsuki, A and Ikehara, Y. (1988). Brefeldin A causes disassembly of the Golgi complex and accumulation of secretory proteins in the endoplasmic reticulum. J. Biol. Chem. 263, 18545-18552. Fukuda, A., Hisshiyama, M., Umino, Y. and Suguira, A. (1987). Immunocytochemical focus assay for potency determination of measles-mumps-rubella trivalent vaccine. J. Virol. Methods 15:279-284.  Gaedigk-Nitschko, K., and Schlesinger, M.J. (1991). The Sindbis virus 6K protein can be detected in viions and is acylated with fatty acids. Virology 183:206-214. Gallagher, P., Henneberry, J., Wilson, I., Sambrook, J. and Gething, M. (1988). Addition of carbohydrate site chains at novel sites on influenza virus hemagglutinin can modulate the folding, transport, and activity of the molecule. J. Cell Biol. 107:2059-2073. Gallagher, P., Henneberry, J., Sambrook, J. and Gething, M. (1992). Glycosylation requirements for intracellular transport and function of the hemagglutinin of influenza virus. J. Virol. 66:7 1367145. Gorman, C.M., Moffat, L.F. and Howard, B.H. (1982). Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2:1044-1051. Gorman, W.L., Pridgen, C., and Portner, A. (1991). Glycosylation of the hemagglutinin neuraminidase glycoprotein of human parainfluenza virus type 1 affects its function but not its antigenic properties. Virology 183:83-90. Green, K.Y. and Dorsett, P.H. (1986). Rubella virus antigen: localization of epitopes involved in hemagglutination and neutralization by using monoclonal antibodies. J. Virol. 57:803-898.  -  148  -  Griffiths, J.C., Harris, S.J., Layton, G.T., Berrie, E.L., French, T.J., Burns, N.R., Adams, S.E., and Kingsman, A.J. (1993). Hybrid human immunodeficiency virus gag particles as an antigen carrier system: Induction of cytotoxic T-cell and humoral responses by a gag:V3 fusion. J. Virol. 67:3191-3198. Guan, J-L., Cao, H., and Rose, J.K. (1988). Cell-surface expression of a membrane-anchored form of the human chorionic gonadotropin ct-subunit. J. Biol. Chem. 253:5306-5311. Haggerty, S., Dempsey, M.P., Bukrinsky, M.I., Guo, L., and Stevenson, M. (1991). Posttranslational modification within the FIIV envelope glycoprotein which restrict virus assembly and CD4-dependent infection. Aids Res. & Human Retroviruses. 7:501-510. Hakinii, J. and Atkinson, P.H. (1982). Glycosylation of intracellular Sindbis virus glycoproteins. Biochemistry 21:2140-2195. Hansen, J.-E., Clausen, H., Nielsen, C., Teglbrg, L.S., Hansen, L.L., Nielsen, C.M., Dabeisteen, E., Mathiesen, L., Hakomori, S.-I., and Nielsen, J.O. (1990). Inhibition of human immunodeficiency virus (HIV) infection in vitro by anticarbohydrate monoclonal antibodies: peripheral glycosylation of HIV envelope glycoprotein gpl2O may be a target for virus neutralization. J. Virol. 64:2833-2840. Helenius, A., Marsh, M. and White, J. (1982). Inhibition of Semliki Forest virus penetration by lysomotrophic weak bases. J. Gen. Virol. 58:47-61. Hobman, T.C., Shukin, R. and Gillam, S. (1988). Translocation of rubella virus glycoprotein El into the endoplasmic reticulum. J. Virol. 62:4259-4264. Hobman, T.C. and Gillam, S. (1989). In vitro and in vivo expression of rubella virus glycoprotein E2: The signal peptide is contained in the C-terminal region of capsid protein. Virology 173:241250. Hobman, T.C., Lundstrom, M.L. and Gilam, S. (1990). Processing and intracellular transport of rubella virus structural proteins in COS cells. Virology 178:122-133. Hobman, T.C., Qiu, Z.Y., Chaye, H.H. and Gillam, S. (1991). Analysis of rubella virus El glycosylation mutants expressed in COS cells. Virology 181:768-772. Hobman T.C., Woodward, L., and Farquhar, M.G. (1992). The rubella virus El glycoprotein is arrested in a novel post-ER, pre-Golgi compartment. J. Cell Biol. 118:792-781. Hobman, T.C., Woodward, L., and Farquhar, M.G. (1993). The rubella virus E2 and El spike glycoproteins are targeted to the Golgi complex. I. Cell Biol. 121: 269-281.  -  149  -  Hobman, T.C., Seto, N.O., and Gillam, S. (1994). Expression of soluble forms of rubella virus glycoproteins in mammalian cells. Virus Res. 31:277-289. Holmes, L.H., Work, M.C. and Warbruton, M.F. (1969). Is rubella an arbovirus? II Ultrastructure, morphology, and development. Virology 37:15-25. Holmes, K.V., Dollers, E.W., and Sturman, L.S. (1981). Tunicamycin resistant glycosylation of a coronavirus glycoprotein: demonstration of a novel type of viral glycoprotein. Virology 115:334-344. Ho-Terry, L. and Cohen, A. (1984). The role of hemagglutination and immunological reactivity of rubella virus. Arch. Virol. 79:139-146. Ho-Terry, L. and Cohen, A. (1985). Rubella virus hemagglutinin: association with single virion glycoprotein. Arch. Virol. 84:207-215. Horzinek, M.C. (1981). In Non-arthropod-bome Togaviruses. (T.W. Tinsley and F. Brown, ed), Academic Press, London, United Kingdom, pp39-44. Hsieh, P. and Robbins P.W. (1984). Regulation of asparagine-linked oligosaccharide processing. J. Biol. Chem. 259:2375-2382. ,  Hurtley, S.M., Bole, D.G., Hoover-Litty, H., Helenius, A., and Copeland, C.S. (1989). Interaction of misfolded influenza hemagglutinin with binding protein (bip) J. Cell Biol. 108:2 1 17-2 126. Hurtley, S.M. and Helenius, A. (1989). Protein oligomerization in the endoplasmic reticulum. Ann. Rev. Cell Biol. 5:277-307. Ilonen, J. and Salmi, A. (1986). Comparison of HLA-Dwl and HLA-Dw2 positive adherent cells in antigen presentation to heterozygous T-cell lines: a low rubella antigen-specific response associated with HLA-Dw2. Hum. Immunol. 17:94-101. Johnson, D.C. and Schlesinger, M.J. (1980). Vesicular stomatitis virus and sindbis virus glycoprotein transport to the cell surface is inhibited by ionophores. Virology 143:407-424. Kalkinnen, N., Oker-Blom, C. and Pettersson, R.F. (1984). Three genes code for rubella virus structural proteins El, E2a, E2b and C. J. Gen. Virol. 65:1549-1577. Kaluza, G., Rott, R., and Schward, R. (1980). Carbohydrate-induced conformation changes of Semliki Forest virus glycoproteins determine antigenicity. Virology 102:286-299. Karounos, D.G., Wolinsky, J.S., and Thomas, J.W. (1993). Monoclonal antibody to rubella virus capsid protein recognizes a f3-cell antigen. J. Immunol. 150:3080-3085.  -  150  -  Katow, S. and Sugiura, A. (1985). Antibody response to individual rubella virus proteins in congenital and other rubella virus infection. J. Clin. Microbiol. 21:449-45 1. Katow, S. and Sugiura, A. (1988). Low pH-induced conformational changes of rubella virus envelope proteins. J. Gen. Virol. 69:2797-2807. Kayman, S., Kopelman, R., Projan, S., Kinney, D.M., and Pinter, A. (1991). Mutational analysis of N-linked glycosylation sites of Friend Murine leukemia virus envelope protein. J. Virol. 65:5323-5332. Kelly, R.B. (1985). Pathways of protein secretion in eukaryotes. Science 230:25-32. Kennedy, S.I.T. (1974). The effect of enzymes on structural and biological properties of Semliki Forest virus. J. Gen. Virol. 23:129-136. Kerr, C.L., and Pennington, T.H. (1984). The effect of monensin on viral production and protein secretion in pseudorabies virus infected cells. J. Gen. Virol. 65:1033-1041. Klenk, H.-D. (1990). Influence of glycosylation on antigenicity of viral proteins, in ‘immunochemistry of Viruses” Vol. 2, (M.H.V. Van Regenmortel and A.R. Neurath, Eds.) Elsevier, Amsterdam/New York, pp2S-37. Kornfeld, R. and Komfeld, S. (1985). Assembly of asparagine-linked oligosaccharides. Ann. Rev. Biochem. 54:631-664. Kuismanen, H., Hedman, K., Saraste, J., and Petterson, R.F. (1982). Uukuniemi virus maturation: accumulation of virus particles and virus antigen in the Golgi complex. Mol. Cell Biol. 2:14441458. Kuismanen, H., Bang, B., Hurme, M., and Petterson, R.F. (1984). Uukuniemi virus maturation: immunofluorescence microscopy with monoclonal glycoprotein-specific antibodies. J. Virol. 51:137-146. Kuismanen, H., Saraste, J., and Petterson, R.F. (1985). Effect of monensin on the assembly of Uukuniemi virus in the Golgi complex. J. Virol. 55:813-822. Kunda, A., Jabbar, M.A., and Nayak, D.P. (1991). Cell surface transport, oligomerization, and endocytosis of chimeric type II glycoproteins: role of cytoplasmic and anchor domains. Mol. Cel. Biol. 11:2675-2685. Laemmli, U.K. (1970). Cleavage of structural proteins during assemble of the head of bacteriophage T4. Nature (London) 227:680-685.  -  151  -  Lambert, D.M., (1988). Role of oligosaccharides in the structure and function of respiratory syncytial virus glycoproteins. Virology 164:458-466. Leavitt, R., Schlesinger, S., and Kornfeld, S. (1977). Tunicamycin inhibits glycosylation and multiplication of Sindbis and vesicular stomatitis viruses. J. Virol. 21:375-385. Lewis, M.J. and Peiham, H.R. (1989). A human homologue of the yeast HDEL receptor. Nature (London) 348:162-163. Li, Y., Luo, L., Rasool, N., and Kang, C.Y. (1993). Glycosylation is necessary for the correct folding of human immunodeficiency virus gpl2O in CD4 binding. J. Virol. 67:584-588. Li, S., Polonis, V., Isobe, H., Zaghouani, A., Guinea, R., Moran, T., Bona, C., and Palese, P. (1993). Chimeric influenza virus induces neutralizing antibodies and cytotoxic T cells against human immunodeficiency virus type I. J. Virol. 67:6659-6666. Liebhaber, H. (1970). Measurement of rubella antibody by haemagglutination I: Variables affecting rubella haemagglutination. J. Immunol. 104:818-825. Lippincott-Schwartz, J., Bonofacino, J.S., Yuan, L.C., and Klausner, R.D. (1988). degradation from the endoplasmic reticulum: Disposing of newly synthesized proteins. Cell 54:209-220. Lippincott-Schwartz, J., Yuan, L.C., Bonofacino, J.S. and Klausner, R.D. (1989). Rapid redistribution of Golgi protein into the endoplasmic reticulum in cells treated with brefeldin A: Evidence for membrane cycling from Golgi to ER. Cell 56:801-8 13. Lis, H. and Sharon, N. (1984). In Biology of Carbohydrates (V. Ginsburg and P. Robbins ed.), Academic Press, New York, ppl-34. Loo, T.W., MacDonald, I., Clarke, D., Trudel, M., Tingle, A.J., and Gillam, 5. (1986). Detection of antibodies to individual proteins of rubella virus. J. Virol. Methods 13:149-159. Lovett, A.E., McCarthy, M., and Wolinsky, J.S. (1993). Mapping cell-mediated immunodominant domains of the rubella virus structural proteins using recombinant proteins and synthetic peptides. J. Gen. Virol. 74:445-452. Lovett, A.E., Hahn, C.S., Rice, C.M., Frey, T.K., and Wolinsky, J.S. (1993). Rubella virusspecific cytotoxic T-lymphocyte responses: Identification of the capsid as a target of major histocompatibiity complex class I-restricted lysis and definition of two epitopes. J. Virol. 67:5849-5858. Long, L., Portetelle, D., Ghysdael, J., Gonze, M., Burny, A. and Meulemans, G. (1986). Monoclonal antibodies to hemagglutinin-neuraminidase and fusion glycoprotein of Newcastle disease virus: relationship between glycosylation and reactivity. J. Virol. 57:1198-1202.  -  152  -  Lozzi, L., Rustiei, M., Corti, M., Cusi, M.G., Valensin, P.E., Bracci, L., Santucci, A., Soldani, P., Spreafico, A. and Neri, P. (1990). Structure of rubella El glycoprotein epitopes established by multiple peptide synthesis. Arch. Virol. 110:271-276. Lundstrom, M., Mauracher, C.A. and Tingle, A.J. (1991). Characterization of carbohydrates linked to rubella virus glycoprotein E2. J. Gen. Virol. 72:834-850. Machamer, C.E., Florkiewicz, R.Z. and Rose, J.K. (1985). A single N-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatic virus G protein to the cell surface. Mol. Cell Biol. 5:3074-3083. Machamer, C.E. and Rose, J.K. (1987). A specific transmembrane domain of a coronavirus El glycoprotein is required for its retention in the Golgi region. J. Cell Biol. 105:1205-1214. Machamer, C.E. and Rose, J.K. (1988a). Influence of new glycosylation sites on expression of the vesicular stomatic virus G protein at the plasma membrane. J. Biol. Chem. 263:5948-5954. Machamer, C.E., and Rose, J.K. (1988b). Vesicular stomatic virus G proteins with altered glycosylation sites display temperature-sensitive intracellular transport and are subject to abelTant intermolecular disulfide bonding. J. Biol. Chem. 263:5955-5960. Machamer, C.E., Mentone, S.A., Rose, J.K. and Farquhar, M.G. (1990). The El glycoprotein of an avian coronavirus is targeted to the cis Golgi complex. Proc. Natl. Acad. Sci. USA. 87:69446948. Machamer, C.E., Grim, M.G., Esquela, A., Chung, S.W., Rolls, M., Ryan, K., and Swift, A.M. (1993). Retention of a cis Golgi protein requires polar residues on one face of a predicted alphahelix in the TM domain. Mol. Biol. Cell. 4:695-704. Mackett, M., Smith, G.L., and Moss, B. (1985). The construction and characterization of vaccinia virus recombinants expressing foreign genes. In tt DNA Cloning Vol. 2 (D.M.Glover eds.). IRL Press, Oxford! Washington DC. Mackett, M., Smith, G.L. and Moss, B. (1990). General method for production and selection of infectious vaccinia virus recombinants expressing foreign genes. J. Virol. 49:857-864. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982). Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Marr, L.D., Sanchez, A. and Frey, T.K. (1991). Efficient in vitro translocation and processing of the rubella virus structural proteins in the presence of microsomes. Virology 180:400-405. Marr, L.D., Wang, C.-Y., and Frey, T.K. (1994). Expression of the rubella virus nonstructural protein ORF and demonstration of proteolytic processing. Virology 198:586-592.  -  153  -  Martin, R., Marquardt, P., O’Shea, S., Borkenstein, M., and Kreth, H.W. (1989). Virus-specific and autoreactive T-cell lines isolated from cerebrospinal fluid of a patient with chronic rubella panencephalitis. J. Neuroimmunol. 23:1-10. Massalski, A., Coulter-Mackie, M., knobler, R.L., Buchmeier, M.J. and Dales, S. (1982). In vivo and in vitro models of demyelinating diseases V. Comparison of the assembly of mouse hepatitis virus, strain JHM, in two murine cell lines. Intervirology 18:135-146. Matthews, R.E.F. (1982). Classification and nomenclature of viruses. Third report of the international committee on taxonomy of viruses. DKC 17:1-199. Matzuk, M.M. and Boime, I. (1988). The role of the asparagine-linked oligosaccharide of the alpha-subunit in the secretion and assembly of human chorionic gonadotropin. J. Cell Biol. 106:1049-1059. Mauracher, C.A., Gillam, S., Shukin, R. and Tingle, A.J. (1991). pH dependant shift of rubella virus capsid protein. Virology 181:773-777. Mayer, T., Tamura, T., Falk, M., and Niemann, H. (1988). Intracellular accumulation of Punta Toro virus glycoproteins expressed from cloned cDNA. Virology 167:251-260. Mayer, T., Tamura, T., Fallc, M., and Niemann, H. (1988). Membrane integration and intracellular transport of the coronavirus glycoprotein expressed from cloned cDNA. Virology 167:251-260. Maynell, L.A., Kirkegaard, K., and Klymkowsky, M.W. (1992). Inhibition of poliovirus RNA synthesis by brefeldin A. J. Virol. 66:1985-1994. McCarthy, M., Lovett, A., Kerman, R.H., Overstreet, A. and Wolinsky, J.S. (1993). Immunodominant T-cell epitopes of rubella virus structural proteins defined by synthetic peptides. J. ViroL 67:673-681. McDonald, H., Hobman, T.C. and Gillam, S. (1991). The influence of capsid protein cleavage on the processing of E2 and El glycoproteins of rubella virus. Virology 183:52-60. Michel, M.-L., Mancini, M., Sobczack, E., Favier, V., Guetard, D., Bahraqui, E.D., and Tollias, P. (1988). Induction of anti-human immunodeficiency virus (HIV) neutralizing antibodies in rabbits immunized with recombinant HIV-hepatitis B surface antigen particles. Proc. Nati. Acad. Sci. USA 85:7957-7961. Misumi, Y., Miki, K., Takatsuki, A., Tamura, G. and Ikehara, Y. (1986). Novel blockade by brefeldin A of intracellular transport of secretory proteins in cultures of rat hepatocytes. J. Biol. Chem. 261:11398-11403.  -  154  -  Mitchell, L.A., Zhang, T., Ho, M., Decarie, D., Zrein, M., Lacroix, M. and Tingle, A.J. (1992). Characterization of rubella specific antibody responses using a new synthetic peptide based enzyme linked immunosorbent assay. J. Clin. Microbiol. 30:1841-1847. Mitchell, L.A., Decarie, D., Tingle, A.J., Zrein, M., and Lacroix, M. (1993). Identification of immunoreactive regions of rubella virus El and E2 envelope proteins by using synthetic peptides. Virus Res. 29:33-57. Mollenhauer, H.H., Orr, D.J., Rowe, L.D. (1990). Alteration of intracellular traffic by monensin: mechanism, specificity and relationship to toxicity. Biochimica et Biophysica Acta. 1031:225-246. Moreman, K.W. and Touster, 0. (1988). Mannosidases in mammalian glycoprotein processing. In Protein transfer and organelle biosynthesis. (R.C. Das and P.W. Robins ed.) pp2O9-24O. Academic Press, San Diego. Montreuil, J., Bouquelet, S., Debray, H., Fournet, B., Spik, G. and Strecker, G. Glycoproteins. (1986). In Carbohydrate analysis. (M.F. Chaplin and J.F. Kennedy ed.) ppl43-204.. IRL Press, Oxford-Washington DC. Morrison, T., Ward, L.J., and Semerjian, A. (1985). Intracellular processing of Newcastle disease virus fusion glycoprotein. J. Virol. 53:851-857. Murphy, F.A., Halonen, P.E. and Harrison, A.K. (1968). Electronmicroscopy of the development of rubella virus in BHK-21 cells. J. Virol. 2:1223-1227. Nagaya, T., Nakamura, T., Tokino, T. (1987). The model of hepatitis B virus DNA integration in chromosomes of human hepatocellular carcinoma. Gene Dev. 1:773-782. Nakhasi, H.L., Meyer, B.C. and Liu, T-Y (1986). Rubella virus cDNA: Sequence and expression of El envelope protein. J. Biol. Chem. 261:16616-16621. Nakhasi, H.L., Thomas, D., Zheng, D. and Liu, T-Y (1989). Nucleotide sequence of capsid, E2 and El genes of rubella virus vaccine strains RA27/3. Nucl. Acids Res. 17:4393-4397. Nath, A. and Wolinsky, J.S. (1990). Antibody response to RV structural proteins in multiple sclerosis. Annals of Neurology 27:533-536. Newcomb, W.W., Tobin, G.J., McGowan, J.J., Brown J.C. (1982). In vitro assembly of vesicular stomatitis virus skeletons. J. Virol. 41:1055-1062. Ng, D.P., Randall, R.E., and Lamb, R.A. (1989). Intracellular maturation and transport of the SV5 type II glycoprotein hemagglutinin-neuraminidase: Specific and transient association with GRP78Bip in the endoplasmic reticulum and extensive internalization from the cell surface. J. Cell Biol. 109:3273-3289.  -  155  -  Ng, D.P., Hieber, S.W. and Lamb, R.A. (1990). Different roles of individual N-linked oligosaccharide chains in folding, assembly and transport of the simian virus 5 hemagglutinin neuraminidase. Mol. Cell. Biol. 10:1989-2001. Niemann, H., Boschek, B., Evans, D., Rosing, M., Tamura, T., and Klenk, H.-D. (1982). Posttranslational glycosylation of coronavirus glycoprotein El: inhibition by monensin. EMBO J. 1:1499-1504. Oda, K., Fujiwara, T. and Ikehara, Y. (1990). Brefeldin A arrests the intracellular transport of viral envelop proteins in primary cultured rat hepatocytes and HepG2 cells. Biochem. J. 265:16 1167. Oker-Blom, C., Kalkinnen, N., Kaariainen, L. and Pettersson, R.F. (1983). Rubella virus contains one capsid protein and three envelope glycoproteins El, E2a and E2b. J. Virol. 46:964-973. Oker-Blom, C., Ulmanen, I., Kaariainen, L. and Pettersson, R.F. (1984a). Rubella virus 40S genome RNA specifies a 24S subgenomic mRNA that codes for a precursor to structural proteins. J. Virol. 49:403-408. Oker-Blom, C. (1984b). The gene order for rubella virus structural proteins is N11 -C-E2-E12 COOH. J. Virol. 5 1:354-358. Olden, K., Parent, J.B., and White, S.L. (1982). Carbohydrate moieties of glycoproteins. A re evaluation of their function. Biochem. Biophys. Acta 6650:209-232. Ou, D., Chong, P., Tripet, B. and Gillam S. (1992a). Analysis of T and B cell epitopes of capsid protein of rubella virus by using synthetic peptides. J. Virol. 66:1674-168 1. Ou, D., Chong, P., Choi, Y., McVeigh, P., Jefferies, W.A., Koloitits, G., Tingle, A.J. and Gillam, S. (1992b). Identification of T cell epitopes on E2 protein of rubella virus, as recognized by human T cell lines and clones. J. Virol. 66:6788-6793. Ou, D., Chong, P., McVeish, P., Jefferies, W.A. and Gillam, S. (1 992c). Characterization of the specificity and genetic restriction of human CD4+ cytotoxic T cell clones reactive to capsid antigen of rubella virus. Virology 191:680-686. Ou, D., Chong, P., Tingle, A.J. and Gillam, 5. (1993). Mapping T cell epitopes of rubella virus structural proteins El, E2 and C recognized by T cell lines and clones derived from infected and immunized populations. J. Med. Virol. 40:175-183. Palmiter, R.D., Behringer, R.R., Quaife, C.J., Maxwell, F., Maxwell, I.H and Brinster, R.L. (1987). Cell lineage ablation in transgenic mice by cell-specific expression of toxic gene. Cell 50:435-443.  -  156  -  Partanen, P., Seppanen, H., Suni, J., and Vaheri, A. (1985). Selective reactivity of antibodies to human inimunoglobulins G, M, and A with rubella virus proteins. J .Clin. Microbiol. 2 1:800-802. Payment, P., Ajdukovic, D., and Pavilanis, V., (1975). Le virus de la rubeole. I. Morphologie et proteines structurales. Can. J. Microbiol. 21:703-709. Pettersson, R.F., Gahmberg, N., Kuismanen, E., Kaariainen, L., Ronnholm, R., Saraste, J. (1988). Bunyavirus membrane glycoproteins as models for Golgi-specific proteins. Mol. Cell Biol. 6:6596. Pfeffer, S.R., and Rothman, J.E. (1987). Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annu. Rev. Biochem. 56:829-852. Pique, C., Pham, D., Tursz, T., and Dokhelar, M.-C. (1992). Human T-cell leukemia virus type 1 envelope protein maturation process: Requirement for syncytium formation. J. Virol. 66:906913. Pitta, A.M., Rose, J.K., and Machamer, C.E. (1989). A single-amino-acid substitution eliminates the stringent carbohydrate requirement for intracellular transport of a viral glycoprotein. J. Virol. 63:3801-3809. Plummer, T.H., Elder, J.H., Alexander, S., Phelan, A.W., and Tarentino, A.L. (1984). Demonstration of peptide N-glucosides F activity in endo-3-N-acetyl-glucosaminidase F preparations. J. Biol. Chem. 259: 10700-10704. Pollack, L. and Atkinson, P.H. (1983). Correlation of glycosylation forms with position in amino acid sequence. J. Cell Biol. 97:293-298. Porterfield, J.S., Casals, J., Chumakov, M.P., Gaidamovich, S.Y., Hannoun, C., Holmes, I.H., Horzinek, M.C., Mussgay, M., Oker-Blom, N., Russel, P.K. and Trent, D.W. (1978). Togaviridae. Intervirology 9:128-145. Qiu, Z., McDonald, L.H., Chen, J., Hobman, T.C. and Gillam, S.G. (1994). Mutational analysis of the arginine residues in the E2-E1 junction region on the proteolytic processing of the polyprotein precursor of rubella virus. Virology 200:821-825. Roberts, C., Garten, W., and Klenk, H.-D. (1993). The role of conserved glycosylation in the maturation and transport of the influenza hemagglutinin. J. Virol. 67:3048-3060. Rose, J.K., and Doms, R.W. (1988). Regulation of protein export from the endoplasmic reticulum. Annu. Rev. Cell Biol. 4:257-288. Rose, J.K., and Bergmann, A.E. (1983). Altered cytoplasmic domains affect intracellular transport of the vesicular stomatitis virus glycoprotein. Cell 34:513-524.  -  157  -  Rottirer, P.J. and Rose, J.K. (1987). Coronavirus El glycoprotein expressed from cloned cDNA localizes in the Golgi region. J. Virol. 61:2042-2045. Sailci, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., Erlich, H.A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491. Salonen, E.M., Hovi, T., Neurman, 0., Vesikari, T., and Vaheri, A. (1985). Kinetics of specific IgA, IgD, IgE, IgG and 1gM antibody responses in rubella. J. Med. Virol. 16:1-9. Sanchez, A., and Frey, T.K. (1991). Vaccinia-vectored expression of rubella virus structural proteins and characterization of the El and E2 glycosidic linkages. Virology 183:636-646. Saraste, J., von Bonsdorff, C.-H., Hashimoto, K., Kerane, S., and Kaariainen, L. (1980). Reversible transport defect of virus membrane glycoproteins in Sindbis virus mutant infected cells. Cell Biol. mt. Rep. 4:279-286. Schlesinger, S., Gottlieb, C., Feil, P., Gelb, N., and Kornfeld, S. (1975). Growth of enveloped RNA viruses in a line of Chinese hamster ovary cells with deficient N-acetyl glucosaminyltransferase activity. J. Virol. 17:239-246. Schlesinger, S. and Schlesinger, M.J. (1990). Replication of togaviridae and flaviridae. In Fields, B.N. and Knipe, D.M. (eds) Fields Virology, New York: Raven Press, pp.687-711. Schmidt, N.J., Lennette, E.H., Gee, P.S., and Dannis, J. (1968). Physical and immunological properties of rubella antigens J. Immunol. 100:851-857. Seppanen, H., Huhtala, M.-L., Vaheri, A., Summers, M.D. and Oker-Blom, C. (1991). Diagnostic potential of baculovirus-expressed rubella virus envelope proteins. J. Clin. Microbiol. 29:18771882. Singer, S.J., Maher, P.A., and Yaffe, M.P. (1987). On the transfer of integral membrane proteins into membranes. Prot. Nail. Acad. Sci. USA. 84:1960-1964. Singh, V.K., Tingle, A.J. and Schulzer, M. (1986). Rubella associated arthritis II. Relationship between circulating immune complex levels and joint manifestations. Ann. Rheum. Dis. 45:115119. Skehel, J.J., Stevens, D.J., Daniels, R.S., Douglas, A.R., Knossow, M., and Wilson, l.A. (1984). A carbohydrate site-chain on hemagglutinin of Hong Kong influenza viruses inhibits recognition by a monoclonal antibody. Proc. Nati. Acad. Sci. USA. 81, 1779-1783.  -  158  -  Smith, J.F., and Brown, D.T. (1977). Envelopment of Sindbis virus: synthesis and organization of proteins in cells infected with wild-type and maturation-defective mutants. J. Virol. 22:662678. Sodora, D.L., Cohen, G.H., Muggeridre, M.I., and Eisenberg, R.J. (1991). Absence of asparagine linked oligosaccharides from glycoprotein D of herpes simplex virus type I results in a structurally altered but biologically active protein. J. Virol. 65:4424-4431. Stephens, E.B., and Compans, R.W. (1988). Assembly of animal virus at cell membranes. Ann. Rev. Microbiol. 42:489-5 16. Stokes, A., Mims, C.A., and Grahams, R. (1986). Subclass distribution of IgG and IgA responses for rubella virus in man. J. Med. Microbiol. 21:283-285. Strauss, E.G., Rice, C.M. and Strauss, J.H. (1984). Complete nucleotide sequence of the genomic RNA of Sindbis virus. Virology 133:92-110. Suomalainen, M., Garoff, H., and Baron, M. (1990). The E2 signal sequence of rubella virus remains part of the capsid protein and confers membrane association in vitro. J. Virol. 64:55005509. Suomalainen, M., Liljstrom, P., and Garoff, H. (1992). Spike protein-nucleocapsid interaction drive the budding of alphaviruses. J. Virol. 66:4737-4747. Swift, A.M. and Machamer, C.E. (1991). A Golgi retention signal in a membrane-spaning domain of coronavirus El protein. J. Cell Biol. 115:19-30. Takahashi, H., Takeshita, T., Morein, B., Putney, S., Germain, R.N., and Berzofsky, J.A. (1990). Induction of CD8 cytotoxic T cells by immunization with purified HIV- 1 envelope proteins in IS COMs. Nature (London) 344:873-875. Tarentino, A.L. and Maley, F. (1974). Purification and properties of an endo-f3-Nacetylglucosaminidase from Streptomyces griseus. 3. Biol. Chem. 249:811-817. Takkinen, K. (1986). Complete nucleotide sequence coding for the aiphavirus nonstructural genes of Semliki Forest virus. Nucl. Acids Res. 14:5667-5682. Tang, X.L., Tregear, G.W., White, D.O. and Jackson, D.C. (1988). Minimum requirement for immunogenic and antigenic activities of homologs of a synthetic peptide of Influenza virus hemagglutinin. J. Virol. 62:4745-475 1. Tardieu, M., Grospierre, B., Durandy, A. and Griscelli, C. (1980). Circulating immune complexes containing rubella antigen in late onset rubella syndrome. J. Paediat. 97: 370-373.  -  159  -  Tartakoff, A. and Vassalli, P. (1983). Lectin-binding sites as markers of Golgi subcompartment: proximal-to-distal maturation of oligosaccharides. J. Cell Biol. 97:1243-1248. Terry, G.M., Ho-Terry, L., Londesborough, P. and Rees, K.R. (1988). Localization of the rubella El epitopes. Arch. Virol. 98:189-197. Terry, G.M., Ho-Terry, L., Londesborough, P. and Rees, K.R. (1989). A bio-engineered rubella El antigen. Arch. Virol. 104:63-75. Tikoo, S.K., Parker, M.D., van den Hurk, J.V., Kowaiski, J., Zamb, T.J., Babiuk, L.A. (1993). Role of N-linked glycans in antigenicity, processing, and cells surface expression of bovine herpesvirus 1 glycoprotein gIV. J. Virol. 67:726-733. Tooze, J., Tooze, S.A., and Warren, G. (1984). Replication of coronavirus MHV-A59 in sac cells: determination of the first site of budding of progeny virions. Eur. J. Cell Biol. 37:203-212. Tooze, J., and Tooze, S.A. (1985). Infection of At20 murine pituitary tumour cells by mouse hepatitis virus strain A59: virus budding is restricted to the Golgi region. Eur. J. Cell Biol. 33:281-293. Tooze, J., Tooze, S.A., and Fuller, S.D. (1987). Sorting of progeny coronavirus from condensed secretory protein at the exit from the trans-Golgi network of AtT2O cells. J. Cell Biol. 105:12151226. Towbin, H., Staehelin, Y., and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. Umemoto, J., Bhavanandan, V.P., and Davidson, E.A., (1977). Purification and properties of an endo-cL-N-acetyl-D-galactosaminidase from Diplococcus pneumoniae. J. Biol. Chem. 252, 860986 14. Vaananen, P. and Kaariainen, L. (1980). Fusion and hemolysis of erythrocytes by three togaviruses: Semliki Forest virus, Sindbis virus and Rubella virus. J. Gen. Virol 46:467-475. Vaheri, A. and Hovi, T. (1972). Structural proteins and subunits of rubella virus. J. Virol 9:10-16. van Drunen Little-van den Hurk, S., Parker, M., Fitzpatrick, D.R., Zamb, T.J., van den Hurk, J.V., Campos, M., Harland, R., and Babiuk, L.A. (1991). Expression of bovine herpesvirus 1 glycoprotein GIV by recombinant baculovirus and analysis of its immunogenic properties. I. Virol. 65:263-271.  -  160  -  Vesikari, T. and Buimovici-Klein, E. (1974). Lymphocyte response to rubella antigen and phytohemagglutinin after administration of the RA27/3 strain of live attenuated rubella vaccine. Infect. Immun. 11:748-758. Vidal, S., Mottet, G., Kolakofsky, D., and Roux, L. (1989). Addition of high-mannose sugars must proceed disulfide bond formation for proper folding of Sendai virus glycoproteins. J. Virol. 63:892-900. Vidgren, G., Takkinen, K., Kalkkinen, N., Kaariainen, L. and Pettersson, R.F. (1987) Nucleotide sequence of the genes coding for the membrane glycoproteins El and E2 of rubella virus. J. Gen. Virol. 68:2347-2357. Virtenen, J., Ekblom, P., and Laurila, P. (1980). Subcellular compartmentalization of saccharide moieties in cultured normal and malignant cells. J. Cell Biol. 85:429-434. von Bonsdorff, C.H. and Vaheri, A. (1969). Growth of rubella virus in BHK-2l cells: Electron microscopy of morphogenesis. J. Gen. Virol. 5:47-5 1. Von Heijne, G. (1984). How signal sequences maintain cleavage specificity. J. Mol. Biol. 173:243-251. Wang, C.-Y., Dominguez, G. and Frey, T.K. (1994). Construction of rubella virus genome-length cDNA clones and synthesis of infectious RNA transcripts. J. Virol. 68:3550-3557. Waxham, M.N. and Wolinsky, J.S. (1983). Immunochemical identification of rubella virus hemagglutinin. Virology 126:194-203. Waxham, M.N. and Wolinsky, J.S. (1985a). Detailed immunologic analysis of the structural polypeptides of rubella virus using monoclonal antibodies. Virology 143:153-156. Waxham, M.N. and Wolinsky, J.S. (1985b). A model of the structural organization of rubella virions. Rev. Inf. Dis. 7:S 133-S 139. Whealy, M.E., Card, J.P., Meade, R.P., Robbins, A.K., and Enquist, L.W. (1991). Effect of Brefeldin A on alphaherpesvirus membrane protein glycosylation and virus egress. J. Virol. 65, 1066-1081. Wickner, W.T., and Lodish, H.F. (1985). Multiple mechanisms of protein insertion into and across membranes. Science 230:400-407. Wolinsky, J.S. (1990). Rubella. In Fields, B.N. and Knipe, D.M. (eds) Virology. New York: Raven Press, pp. 8 15-839.  -  161  -  Wolinsky, J.S., McCarthy, M., Allen-Cannady, 0., Moore, W.T., Jin, R., Cao, S.N., Lovett, A. and Simmons, D. (1991). Monoclonal antibody defined epitope map of expressed rubella virus protein domains. J. Virol. 65:3986-3994. Wolinsky, J.S., Sukholutsky, E., Moore, W.T., Lovett, A., McCarthy, M. and Adame, B. (1993). An antibody and synthetic peptide defined rubella virus El glycoprotein neutralizing domain. J.Virol. 67:961-968. Wright, K.E., Salvato, M.S. and Buchmeier, M.J. (1989). Neutralizing epitopes of lymphocyte choriomeningitis virus are conformational and require both glycosylation and disulfide bonds for expression. Virology 17 1:417-426. Yang, D.C. et al., (1993). unpublished results. Yoon, J.W., Choi, D-S., Liang, H-C., Baek, H-S., Ko, I-Y., Jun, H.S. and Gillam, S. (1991). Induction of an organ specific autoimmune disease, lymphocytic hypophysis in hamsters by recombinant rubella virus glycoprotein and prevention of disease by neonatal thymectomy. J. Virol. 66: 1210-1214. Zhang, T., Mauracher, C.A., Mitchell, L.A. and Tingle, A.J. (1991). Detection of rubella virusspecific immunoglobulin G (IgG), 1gM and IgA antibodies by immunoblot assays. J.Clin. Microbiol. 30:824-830. Zheng, D.X., Dickens, L., Liu, T-Y and Nakhasi, H.L. (1989). Nucleotide sequence of the 24S subgenomic mRNA of vaccine strain of rubella virus comparison with wild type strain M33. Gene 82:343-349. Ziola, B., Lund, G., Meurman, 0. and Salmi, A. (1983). Circulating immune complexes in patients with acute measles or rubella virus infections. Infect. Immun. 41:578-583.  -  162  -  


Citation Scheme:


Citations by CSL (citeproc-js)

Usage Statistics



Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            async >
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:


Related Items