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Nuclear entry of the parvovirus minute virus of mice Cohen, Sarah 2010

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Nuclear entry of the parvovirus minute virus of mice by Sarah Cohen  B.Sc., University of British Columbia, 2005  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES (Zoology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  December, 2010  © Sarah Cohen, 2010  Abstract In order to promote infection, viruses must target their genomes to specific compartments within the host cell. I have used the parvovirus minute virus of mice (MVM) as a model to study the trafficking of non-enveloped viruses. Parvoviruses are single-stranded DNA viruses which replicate in the nucleus of the cell. Most viruses that replicate in the nucleus transport their genomes through nuclear pore complexes, large protein assemblies that mediate nucleocytoplasmic transport. However, previous studies have shown that MVM can induce disruption of the nuclear membranes, called the nuclear envelope (NE). This led to the hypothesis that MVM enters the nucleus by an unusual mechanism: disruption of the NE and entry through the resulting breaks.  The objectives of this thesis were to: (1) characterize the effect of MVM on the NE, (2) define the molecular mechanism used by MVM to induce NE disruption, (3) determine the role of NE disruption in the MVM replication cycle, and (4) identify host proteins involved in MVM infection. I found that MVM causes small, transient disruptions of the NE early during infection. I tested the hypothesis that viral enzymatic activity is necessary for MVM-induced NE disruption and found that this was not the case. Next I tested the hypothesis that MVM hijacks a cellular program for NE breakdown, and found that MVM utilizes apoptotic proteases called caspases to facilitate these disruptions. Caspase inhibition prevents NE disruption in MVM-infected cells, reduces viral gene expression, and prevents entry of MVM capsids into the nucleus. I propose that NE disruption involving caspases facilitates parvovirus genome delivery into the nucleus. NE disruption also alters the compartmentalization of host proteins, which may be favorable for the virus.  ii  I have shown that MVM uses a novel nuclear entry strategy, unlike those previously described for any virus or cellular protein. It will be of great interest to determine whether this strategy is shared by other viruses. Parvoviruses are not considered a serious threat as human pathogens. However, they may prove useful as vectors for gene therapy. An understanding of the basic biology of parvoviruses could help in the development of parvovirus-based therapeutics.  iii  Preface Some of the work presented in this thesis has been a collaborative effort. During the course of my PhD, I worked closely with two postdoctoral fellows, Dr. Ali Bezhad and Dr. Alexandra Marr. Dr. Ali Bezhad assisted with optimizing the preparation of MVM-infected cells for the electron microscopy experiments described in Chapter 3. Studies on the involvement of the MVM phospholipase A2 in MVM-induced nuclear envelope disruption were performed together with Dr. Alexandra Marr, who also assisted with optimizing immunofluorescence microscopy of the nuclear lamina. Steven Huang, a co-op student in the lab, assisted with time-course experiments visualizing MVM and lamin A/C by immunofluorescence microscopy, shown in Fig. 3-7.  In addition, during my PhD I have supervised a graduate student, Pierre Garcin, and an exchange student, Sanne Terpstra. Pierre Garcin performed the Western blots for nuclear lamins shown in Fig. 4-6. Sanne Terpstra assisted with optimizing the immunoprecipitation of MVM discussed in Chapter 6. The identification of MVM-binding partners (Chapter 6) is a project in collaboration with Dr. Leonard Foster, and the mass spectrometry was performed by Isabelle Kelly in his lab.  I designed all the experiments described in this thesis together with my supervisor Dr. Nelly Panté. I analyzed the data, and wrote the first drafts of the manuscripts presented in Chapters 3-5, which were then revised together with Dr. Panté. The review articles excerpted in Chapters 1-2 were written together with Shelly Au, who also created Fig. 11/7-1, and revised together with Dr. Panté.  iv  Details of the publications arising from work presented in this thesis are as follows: •  Cohen, S.*, Au, S.*, and Panté, N. How viruses access the nucleus. Biochimica et Biophysica Acta – Molecular Cell Research. In press. (Chapter 1).  •  Au, S.*, Cohen, S.*, and Panté, N. (2010). Microinjection of Xenopus laevis oocytes as a system for studying nuclear transport of viruses. Methods 51(11): 114-120. (Chapter 2).  •  Cohen, S., Behzad, A.R., Carroll, J.B., and Panté, N. (2006). Parvoviral nuclear import: bypassing the host nuclear transport machinery. Journal of General Virology 87: 3209-3213. (Chapter 3).  •  Cohen, S., Marr, A.K., Garcin, P., and Panté, N. Nuclear envelope disruption involving host caspases plays a role in the parvovirus replication cycle. Submitted. (Chapters 4 and 5).  * Equal contribution.  The research presented in this thesis was approved by the UBC Animal Care Committee (Certificate A07-0298) and the UBC Bio-Safety Committee (Certificate B10-0057).  v  Table of Contents Abstract ...............................................................................................................ii Preface................................................................................................................iv Table of Contents...............................................................................................vi List of Tables.......................................................................................................x List of Figures ....................................................................................................xi List of Abbreviations .......................................................................................xiii Acknowledgments ............................................................................................xv Dedication .......................................................................................................xvii 1. Introduction .....................................................................................................1 1.1 Virus entry and trafficking........................................................................................ 1 1.2 Nuclear import......................................................................................................... 2 1.3 Nuclear entry of viruses .......................................................................................... 4 1.3.1 Nuclear entry during mitosis .......................................................................................... 7 1.3.2 Genome release in the cytoplasm, followed by entry through the nuclear pore complex ................................................................................................................................................ 8 1.3.3 Genome release at the cytoplasmic side of the nuclear pore complex ....................... 11 1.3.4 Nuclear entry of intact capsids through the nuclear pore complex, followed by genome release .................................................................................................................................. 14 1.3.5 Summary...................................................................................................................... 16  1.4 Parvoviruses ......................................................................................................... 17 1.4.1 Introduction to parvoviruses......................................................................................... 17 1.4.2 The parvovirus replication cycle .................................................................................. 20 1.4.3 Parvoviral nuclear entry ............................................................................................... 23 1.4.4 Nuclear import of parvovirus capsid proteins .............................................................. 25  1.5 Nuclear envelope breakdown................................................................................ 26 1.5.1 Mitotic nuclear envelope breakdown ........................................................................... 26 1.5.2 Apoptotic nuclear envelope breakdown....................................................................... 27  1.6 Objectives ............................................................................................................. 28 1.6.1 Aim 1: To characterize the effect of MVM on the host nuclear envelope .................... 29 1.6.2 Aim 2: To determine the molecular mechanism of MVM-induced nuclear envelope disruption .............................................................................................................................. 29  vi  1.6.3 Aim 3: To determine the role of MVM-induced nuclear envelope disruption in the viral replication cycle .................................................................................................................... 30 1.6.4 Aim 4: To identify host proteins that are post-translationally modified in MVM-infected cells ....................................................................................................................................... 30 1.6.5 Aim 5: To identify host proteins that interact with the MVM capsid ............................. 31  2. Materials and Methods .................................................................................32 2.1 Antibodies ............................................................................................................. 32 2.2 Purification of MVM ............................................................................................... 32 2.3 Xenopus oocyte microinjection ............................................................................. 34 2.4 Tissue culture, transfection and infection.............................................................. 37 2.5 Electron microscopy.............................................................................................. 38 2.5.1 Negative staining and electron microscopy of purified virus ....................................... 38 2.5.2 Embedding and electron microscopy of microinjected Xenopus oocytes ................... 38 2.5.3 Embedding and electron microscopy of MVM-infected fibroblast cells ....................... 39 2.5.4 Immunogold electron microscopy of MVM-infected fibroblast cells............................. 40  2.6 Fluorescence microscopy ..................................................................................... 41 2.6.1 Immunofluorescence microscopy of MVM-infected cells............................................. 41 2.6.2 Semipermeabilized cell assay for nuclear envelope disruption................................... 42 2.6.3 TUNEL assay............................................................................................................... 42  2.7 Enzymatic activity assays ..................................................................................... 43 2.7.1 Phospholipase A2 activity of MVM .............................................................................. 43 2.7.2 Colorimetric assay for caspase-3 activity .................................................................... 43 2.7.3 FLICA assay for caspase-3 activity ............................................................................. 43  2.8 Western blot and 2D-DIGE ................................................................................... 44 2.8.1 Western blot................................................................................................................. 44 2.8.2 Two-dimensional differential in gel electrophoresis..................................................... 44  2.9 Immunoprecipitation of MVM and mass spectrometry .......................................... 45  3. Effect of MVM on the Host Nuclear Envelope.............................................46 3.1 Introduction ........................................................................................................... 46 3.2 Results .................................................................................................................. 47 3.2.1 Microinjection of MVM into Xenopus oocytes causes disruption of the nuclear envelope ............................................................................................................................... 47 3.2.2 Other parvoviruses also disrupt the nuclear envelope ................................................ 47 3.2.3 MVM disrupts the nuclear envelope and alters the nuclear morphology of infected fibroblast cells ....................................................................................................................... 49  vii  3.2.4 Timing of nuclear envelope disruption is consistent with entry of MVM into the nucleus .............................................................................................................................................. 55 3.2.5 MVM disrupts the nuclear lamina of infected fibroblast cells....................................... 57  3.3 Discussion............................................................................................................. 59  4. Mechanism of MVM-Induced Nuclear Envelope Disruption ......................62 4.1 Introduction ........................................................................................................... 62 4.2 Results .................................................................................................................. 63 4.2.1 MVM-induced nuclear envelope disruption does not require viral phospholipase A2 activity ................................................................................................................................... 63 4.2.2 Establishment of a digitonin-permeabilized cell assay to identify inhibitors of MVMinduced nuclear envelope disruption .................................................................................... 67 4.2.3 Caspases are involved in MVM-induced nuclear envelope disruption ........................ 67 4.2.4 Caspases are involved in MVM-induced lamin cleavage in infected cells .................. 73 4.2.5 Caspase-3 is not activated above basal levels in MVM-infected cells, but is relocalized .............................................................................................................................................. 76 4.2.6 Outer nuclear membrane proteins are involved in MVM-induced nuclear envelope disruption .............................................................................................................................. 81  4.3 Discussion............................................................................................................. 83  5. Role of Nuclear Envelope Disruption in the MVM Replication Cycle .......85 5.1 Introduction ........................................................................................................... 85 5.2 Results .................................................................................................................. 85 5.2.1 Nuclear envelope disruption is not due to induction of apoptosis ............................... 85 5.2.2 Nuclear envelope disruption is important for the replication cycle of MVM at a stage prior to gene expression ....................................................................................................... 86 5.2.3 MVM-induced nuclear envelope disruption mediates nuclear entry of the capsid ...... 89 5.2.4 MVM-induced nuclear envelope disruption alters the compartmentalization of cellular proteins ................................................................................................................................. 93  5.3 Discussion............................................................................................................. 93  6. Proteomic Approaches for Studying MVM Infection .................................98 6.1 Introduction ........................................................................................................... 98 6.2 Results .................................................................................................................. 99 6.2.1 Post-translational modifications in cells infected with MVM ........................................ 99 6.2.2 Putative binding partners of the MVM capsid ............................................................ 103  6.3 Discussion........................................................................................................... 107  7. Conclusion ..................................................................................................110 7.1 Effect of MVM on the host nuclear envelope ...................................................... 110 viii  7.2 Mechanism of MVM-induced nuclear envelope disruption.................................. 112 7.2.1 The role of caspases in MVM-induced nuclear envelope disruption ......................... 112 7.2.2 The role of outer nuclear membrane proteins in MVM-induced nuclear envelope disruption ............................................................................................................................ 115  7.3 The role of MVM-induced nuclear envelope disruption in the viral replication cycle .................................................................................................................................. 116 7.3.1 MVM-induced nuclear envelope disruption can mediate nuclear entry of the MVM capsid.................................................................................................................................. 117 7.3.2 Can the nuclear pore complex also mediate nuclear entry of parvoviruses?............ 118 7.3.3 Nuclear entry of MVM compared with other viruses.................................................. 120 7.3.4 Other possible functions of nuclear envelope disruption in the MVM replication cycle ............................................................................................................................................ 121  7.4 Post-translational modifications in MVM-infected cells ....................................... 121 7.5 Putative binding partners of MVM: significance and future directions................. 123 7.5.1 Proteins involved in vesicular trafficking and nuclear envelope integrity................... 123 7.5.2 Cell surface proteins .................................................................................................. 125 7.5.3 Cytoskeleton-associated proteins.............................................................................. 125 7.5.4 Regulators of protein-turnover ................................................................................... 126 7.5.5 Proteins involved in translation .................................................................................. 127 7.5.6 Proteins involved in metabolism ................................................................................ 128  7.6 Concluding remarks ............................................................................................ 128  Bibliography ....................................................................................................131  ix  List of Tables Table 6-1: Post-translational modifications in MVM infected cells………………………101 Table 6-2: Putative binding partners of the MVM capsid: proteins with a MVM/mock ratio of >1 in two rounds of mass spectrometry………………………………………………….105 Table 6-3: Putative binding partners of the MVM capsid: proteins with a MVM/mock ratio of >1.5 in one round of mass spectrometry………………………………………………...106  x  List of Figures Figure 1-1: How viruses access the nucleus…………………………………………………6 Figure 1-2: Transcription and translation of the MVM genome…………………………...19 Figure 1-3: The MVM replication cycle………………………………………………………21 Figure 2-1: Purified MVM……………………………………………………………………..35 Figure 3-1: MVM induces disruption of the NE in microinjected Xenopus oocytes…….48 Figure 3-2: The parvoviruses H1 and CPV also induce NE disruption…………………..50 Figure 3-3: MVM infection causes alterations in nuclear morphology……………………51 Figure 3-4: MVM infection causes alterations in nuclear morphology……………………53 Figure 3-5: MVM induces disruption of the NE in infected fibroblast cells………………54 Figure 3-6: Immunogold localization of MVM capsids at two hours post infection……..56 Figure 3-7: MVM infection causes disruption of the nuclear lamina……………………...58 Figure 3-8: MVM-induced disruption of the nuclear lamina is transient………………….60 Figure 4-1: Characterization of the PLA2 activity of MVM………………………………...64 Figure 4-2: MVM-induced NE disruption does not involve viral PLA2 activity…………..66 Figure 4-3: MVM-induced NE disruption in semipermeabilized cells depends on caspases………………………………………………………………………………………...68 Figure 4-4: Caspase inhibitors prevent MVM-induced NE disruption in microinjected Xenopus oocytes……………………………………………………………………………….70 Figure 4-5: Caspase inhibitors prevent nuclear lamina disruption in MVM-infected cells………………………………………………………………………………………………72 Figure 4-6: B-type lamins, but not A/C-type lamins, are cleaved in a caspase-dependent manner in MVM-infected cells……………………………………………………...…………74 Figure 4-7: Caspase-3 is not activated above basal levels in MVM-infected cells (enzymatic assay)………………………………………………………………………………77 Figure 4-8: Caspase-3 is not activated above basal levels in MVM-infected cells (FLICA)…………………………………………………………………………………………..79 Figure 4-9: Basally activated caspase-3 is relocalized in MVM-infected cells…………..80 Figure 4-10: MVM does not induce NE disruption in cells with mislocalized ONM proteins…………………………………………………………………………………………..82 Figure 5-1: MVM does not cause apoptosis until 48 hours after infection……………….87 Figure 5-2: Time course of NS1 expression in MVM-infected cells………………………88  xi  Figure 5-3: Caspase-mediated NE disruption is important for the parvovirus replication cycle……………………………………………………………………………………………...90 Figure 5-4: Inhibition of caspase-mediated NE disruption prevents nuclear entry of MVM……………………………………………………………………………………………..92 Figure 5-5: MVM-induced NE disruption alters the compartmentalization of cellular proteins at two hours post infection…………………………………………………………..94 Figure 5-6: The compartmentalization of host proteins is restored at 24 hours post infection………………………………………………………………………………………….95 Figure 6-1: Post-translational modifications to host proteins in MVM-infected cells at two hours post infection…………………………………………………………………………...100 Figure 6-2: Post-translational modifications to host proteins in MVM-infected cells at 24 hours post infection…………………………………………………………………………...102 Figure 6-3: IP of MVM and putative cellular binding partners……………………….…..104 Figure 7-1: How viruses access the nucleus – revised…………………………………..111 Figure 7-2: Model of MVM-induced NE disruption………………………………………..114  xii  List of Abbreviations 2D-DIGE: 2-dimensional differential in gel electrophoresis AAV: adeno-associated virus AcMNPV: Autographa californica multiple-capsid nucleopolyhedrovirus APAR: autonomous parvovirus-associated replication BC: basic cluster cNLS: classical nuclear localization sequence CCT: chaperonin containing TCP-1 CDK1: cyclin-dependent kinase 1 CPV: canine parvovirus DMEM: Dulbecco’s modified Eagle’s medium eIF: eukaryotic initiation factor EM: electron microscopy ER: endoplasmic reticulum FAM-VAD-fmk: carboxyfluorescein-Val-Ala-Asp-fluoromethylketone FBS: fetal bovine serum FLICA: fluorochrome inihibitor of caspase FPV: feline panleukopenia virus G3BP: galectin-3-binding protein GDP: guanosine triphosphate GFP: green fluorescent protein GTP: guanosine diphosphate HBV: hepatitis B virus HIV-1: human immunodeficiency virus 1 Hsp: heat shock protein HSV-1: herpes simplex virus 1 IF: immunofluorescence INM: inner nuclear membrane IP: immunoprecipitation KASH: Klarsicht/Anc-1/Syne-1 homology LAP: lamina-associated protein LINC: linker of the nucleoskeleton and cytoskeleton LSB: low salt buffer  xiii  MALDI-ToF: matrix assisted laser desorption/ionization time-of-flight MBS: modified Barth’s saline MEM: minimum essential medium MOI: multiplicity of infection MLV: murine leukemia virus mRNA: messenger RNA MTOC: mictrotubule organizing centre MVM: minute virus of mice NE: nuclear envelope NEBD: nuclear envelope breakdown NLM: nuclear localization motif NLS: nuclear localization sequence NP: nucleoprotein NPC: nuclear pore complex ONM: outer nuclear membrane ORF: open reading frame PBS: phosphate-buffered saline PIC: pre-integration complex PKC: protein kinase C PLA2: phospholipase A2 RCC1: regulator of chromosome condensation SDS: sodium dodecyl sulfate SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis Sra1: specifically Rac1-associated protein STS: staurosporine SUN: Sad1p/Unc-84 homology SV40: simian virus 40 TEM: transmission electron microscope TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling vRNP: viral ribonucleoprotein WGA: wheat-germ agglutinin zDEVD-fmk: benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone zVAD-fmk: benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone zVEID-fmk: benzyloxycarbonyl-Val-Glu-Ile-Asp-fluoromethylketone  xiv  Acknowledgments First of all, I would like to thank my supervisor, Dr. Nelly Panté, for all of her support and encouragement. The opportunity to join your lab as an undergraduate inspired me to pursue a career in research, and instilled in me a love for bench work, especially electron microscopy. Thank you also for helping me to develop a range of skills which include writing, presentation, and mentoring.  I would also like to thank my committee members, Dr. François Jean, Dr. Linda Matsuuchi, Dr. Lacey Samuels and Dr. David Theilmann, for their insights and helpful discussion, as well as for personal support and invaluable career advice!  Financial support for this work has been provided by the Canadian Institutes of Health Research, the Michael Smith Foundation for Health Research, and the Natural Sciences and Engineering Research Council of Canada.  Thanks to our collaborators, who have provided ideas, tools and reagents: Dr. Leonard Foster (University of British Columbia), Dr. Michael Kann (University of Bordeaux), Dr. Ulrike Kutay (ETH Zurich), Dr. Gergely Lukacs (McGill University), Dr. Kyle Roux (University of Florida), Dr. Peter Tattersall (Yale University School of Medicine), and Dr. Matti Vuento (University of Jyväskylä). Thanks also to the staff of the UBC Bioimaging Facility and Flow Cytometry Facility.  My time in the Panté lab has been greatly enriched by interaction with many other lab members, past and present. Thanks to Dr. Ali Bezhad and Dr. Jeffrey Carroll, who started the MVM project. Thanks to Maria Acevedo for making me laugh; Shelly Au for xv  writing three papers with me, for her mad power point skills, and for being the lab social coordinator; Nikta Fay for always asking ‘why’; Pierre Garcin, Stephen Huang, and Dr. Alexandra Marr for all their hard work and helpful discussion on the MVM project; Andrea Mattenly for persevering with the purification of MVM; Sanne Terpstra for her excellent hands and calm presence; Lindsay Weaver for being a joy to work with; Wei Wu for introducing me to the culinary delight of jellyfish; Dr. Winco Wu for patiently answering many questions; and Dr. Lixin Zhou for her amazing positive spirit.  I would also like to thank my family: my mother, Leni Gelten, for instilling in me a love of biology at a very early age, during investigations of tidal pools; my father, Nechemjah Cohen, for making a dedicated effort to understand what it is I do; and my brother, Shamai Cohen – it has been fun watching you grow into a thoughtful and mature person.  Last but not least, thanks to my husband, Graham Diering, for immense amounts of support. You are my rock, my partner in everything, my very best sounding board. I look forward to seeing where this journey will take us…  xvi  Dedication  For my grandparents, especially Dr. Philip Cohen, who is passionate about scientific research and told me no other career could compare.  xvii  1. Introduction* 1.1 Virus entry and trafficking In order to establish a productive infection, viruses must overcome multiple barriers within the host cell. These barriers include the plasma membrane and underlying cell cortex, an extremely dense cytoplasm through which molecular traffic is highly restricted, and any other membranes that must be crossed in order to access the sites of viral replication or assembly. How different viruses accomplish these feats depends to a large degree on the size and structure of the virus. Viruses consist of an RNA or DNA genome surrounded by either multiple copies of capsid proteins (non-enveloped viruses) or both capsid proteins and a lipid bilayer membrane (enveloped viruses). The size of animal viruses ranges from approximately 25 nm to over 300 nm.  Viruses first attach to the host cell through interactions between viral membrane proteins (enveloped viruses) or three-dimensional structures on the capsid (non-enveloped viruses) and cell surface receptors; viruses are then internalized either by direct fusion of the viral envelope with the plasma membrane, or via one of the cell’s many endocytic pathways (Marsh & Helenius, 2006, Smith & Helenius, 2004). If entry is by endocytosis, then the virus escapes from the endocytic compartment to the cytosol. The escape strategy depends on the type of virus. For enveloped viruses, this involves fusion of the viral envelope with endosomal membranes. For non-enveloped viruses the endosomal escape process is less well understood, but can involve lysis of the endosomal membrane employing lytic peptides (Smith & Helenius, 2004). The released viral capsid *  A version of part of this chapter has been published: • Cohen, S., Au, S., and Panté, N. How viruses access the nucleus. Biochimica et Biophysica Acta – Molecular Cell Research. In press.  1  or nucleoprotein complex then traverses the cytoplasm, often by associating with cellular motor proteins which traffic along various cytoskeleton components (Radtke et al., 2006). After reaching the cellular compartment where viral replication occurs, the capsid or nucleoprotein complex disassembles and releases the viral genome. After using the cellular machinery for genome synthesis and production of new viral proteins, progeny virions are assembled, and then released from the cell. Release is usually through budding at the plasma membrane or into the endoplasmic reticulum (ER) followed by exocytosis for enveloped viruses; for non-enveloped viruses, it is generally thought that virions are released during cell lysis, although some viruses may also be released by exocytosis (Marsh & Helenius, 2006).  Many viruses, including most DNA viruses and some RNA viruses, depend on nuclear proteins for replication; therefore, their viral genome must enter the nucleus of the host cell (Greber & Fornerod, 2005, Whittaker et al., 2000). Although there are numerous benefits, entry into the nucleus also poses a serious challenge for these viruses, since the nuclear envelope (NE) acts as a barrier between the cytoplasm and the nucleus, and transport of molecules into and out of the nucleus is tightly regulated. Because many viruses make use of the host nuclear transport machinery during infection, the principles of cellular nuclear transport will briefly be described before discussion of the diverse strategies used by viruses to deliver their genomes into the host nucleus.  1.2 Nuclear import The NE consists of an inner nuclear membrane (INM) and an outer nuclear membrane (ONM) separated by the perinuclear space, a regular gap of about 30-50 nm. Embedded in these membranes are the nuclear pore complexes (NPCs) - large protein complexes that act as passageways for the transport of molecules into and out of the nucleus. The  2  NPC is composed of multiple copies of 30 different proteins, called nucleoporins, arranged in an octagonal structure that is 120 nm in diameter and has a molecular mass of 125 megadaltons (reviewed by Brohawn et al., 2009). In addition to the NPCs, a major feature of the NE is the nuclear lamina, a thin (20-30 nm) protein layer that is closely associated with both the INM and the underlying chromatin. The nuclear lamina is composed primarily of A- and B-type lamins, members of the intermediate filament protein family. During cell division of many eukaryotes, the nuclear lamina and NE are temporarily disassembled to allow the partitioning of chromosomes between daughter cells.  Two general mechanisms have been described for nuclear import: passive diffusion and facilitated translocation. Passive diffusion is for ions and molecules smaller than 9 nm in diameter or proteins smaller than 40 kDa, whereas facilitated nuclear import can accommodate the transport of molecules with diameters of up to 39 nm (Panté & Kann, 2002). The facilitated nuclear import mechanism requires a signal residing on the imported molecule (or cargo), and cytoplasmic receptors (called nuclear import receptors, importins, or karyopherins) that recognize the signal and mediate the translocation of the cargo through the NPC (reviewed by Cook et al., 2007, Lange et al., 2007). Although there are many nuclear import signals, the first identified and most studied signal consists of one or two short stretches of basic amino acids, called the classical nuclear localization sequences (cNLSs). The nuclear import of cNLS-bearing proteins is mediated by a heterodimer import receptor consisting of importin α and importin β. The driving force behind nuclear import is a gradient of the GTPase Ran across the NE. RanGTP abounds inside the nucleus, while RanGDP predominates within the cytoplasm (Kalab et al., 2002). This is due to the presence of the guanine  3  nucleotide exchange factor, regulator of chromosome condensation 1 (RCC1), on chromatin (reviewed by Hadjebi et al., 2008). Cytoplasmic RanGDP favors the binding of importins to cargo, and nuclear RanGTP interacts with importins, leading to the dissociation and subsequent release of the cargo from the importins into the nucleus (Gorlich et al., 1996, Izaurralde et al., 1997).  An emerging picture is that different transport routes or pathways exist. In other words, different classes of molecules have different types of NLSs, which are recognized by different importins (with at least 28 different importins in humans). These, in turn, are recognized by different nucleoporins. Despite significant progress in identifying NLSs and their receptors (many of which have been crystallized and their structure solved, reviewed by Cook et al., 2007) and in characterizing the basis of the recognition of these molecules, the precise mechanism used by molecules to cross the NPC remains unknown. Several models have been proposed in recent years speculating on the mechanism for the translocation of molecules through the NPC (Peters, 2009). Because viral capsids are among the largest cargos that translocate through the NPC, studies on nuclear import of viruses might provide important information that can be used to test the several proposed models for NPC translocation.  1.3 Nuclear entry of viruses The general current understanding is that viruses deliver their genome into the nucleus of their host cells by using the machinery developed by the cells for the nuclear import of proteins (i.e., NPCs, NLSs, importins, GTP, and Ran). Because the size and structure of viruses vary enormously (for example, herpes simplex virus is 120 nm in diameter, Roizman et al., 2007, but parvoviruses are 18-26 nm in diameter, Parrish & Berns, 2007) and because there are several nuclear import pathways, each virus has developed a  4  unique strategy to deliver its genome into the nucleus. Two of the main factors affecting the nuclear entry strategy of a given virus are the size of the capsid, and the cellular location of genome release. As indicated in Fig. 1-1, four general strategies have been identified, based on where in the cell uncoating of the viral genome occurs: 1) Some retroviruses gain access to the nucleus during mitosis, when the NE is temporarily disassembled. 2) Some viruses, such as influenza and human immunodeficiency virus 1 (HIV-1), undergo extensive disassembly in the cytoplasm. The cytoplasmic released components contain NLSs and are thereby able to cross the NPC using the host transport machinery. 3) Some viral capsids use importins or viral proteins to attach to the cytoplasmic side of the NPC. Interaction with the NPC is then used as a cue for disassembly, and the viral genome is released into the nucleus, often as a complex with viral proteins. Viruses that use this strategy include herpesviruses and adenoviruses. 4) Some viral capsids, such as those of hepatitis B virus (HBV) and some baculoviruses, are small enough to cross the NPC intact. Genome release then occurs at the nuclear side of the NPC or inside the nucleus. Although much progress has been made in characterizing the general nuclear entry strategies of different viruses, many of the molecular details remain obscure. The study of viral nuclear entry is complicated by the fact that viral proteins may enter the nucleus multiple times during the virus life-cycle: both as part of an incoming capsid or nucleoprotein, and perhaps also as a newly synthesized protein if assembly of progeny virions occurs in the nucleus. Thus, identification of NLSs and host factors involved in a particular viral nuclear import step can be challenging. Post-translational modifications such as phosphorylation of viral proteins can also play an important role in the exposure of NLSs. Viral transport into the nucleus and genome release are part of an intricate 5  Figure 1-1: How viruses access the nucleus. (1) The MLV PIC gains access to the nucleus during mitosis, when the NE is temporarily disassembled. (2) Influenza A virus undergoes extensive disassembly in the cytoplasm. The cytoplasmic released vRNPs contain NLSs and are thereby able to cross the NPC using the host transport machinery. (3) HSV-1 capsids use importins to attach to the cytoplasmic side of the NPC. Interaction with the NPC then triggers the release of the viral genome, which then enters the nucleus through the NPC. (4) Capsids of the baculovirus AcMNPV cross the NPC intact. Genome release presumably occurs inside the nucleus. (Modified from a figure originally published by Cohen et al., 2011. Copyright Elsevier).  6  dance between the virus and host cell, many details of which remain to be elucidated. In the following sections, the four general strategies of nuclear import of viral genomes are discussed, with particular emphasis on the best-studied viruses.  1.3.1 Nuclear entry during mitosis Some viruses, such as the retrovirus murine leukemia virus (MLV), can only access the nucleus of a host cell during mitosis, when the NE is temporarily disassembled (reviewed by Goff, 2007, Suzuki & Craigie, 2007). Retroviruses are RNA viruses which reverse transcribe their RNA genomes into DNA; the DNA is then integrated into the host genome, where it serves as a template for the synthesis of new RNA genomes. Retroviruses may enter the cell either by direct fusion of the viral envelope at the cell surface, or by fusion after internalization using an endocytic route (Goff, 2007). Fusion results in the release of the viral nucleoprotein core particle into the cytoplasm. This is followed by a poorly understood uncoating step and the formation of the reverse transcription complex, which for MLV includes the viral RNA genome, reverse transcriptase, integrase and the capsid protein (Fassati & Goff, 1999). Reverse transcription of RNA to DNA produces the pre-integration complex (PIC), which must then enter the nucleus to integrate into the host genome.  The PIC of MLV is too large to enter the nucleus through the NPC by passive diffusion. Several lines of evidence indicate that MLV must wait for NE disassembly in order for the PIC to enter the nucleus. Most retroviruses can only infect actively dividing cells. For MLV, it is thought that the barrier to infection of non-dividing cells is the inability of the PIC to access the nucleus. When the cell cycle is arrested at the G1-S transition, MLV PICs are present in the cytoplasm, but DNA integration is blocked; if the cell cycle is then allowed to progress to metaphase, the PICs enter the nucleus and integration  7  occurs (Roe et al., 1993). The viral proteins p12 (an MLV-specific cleavage product of the Gag polyprotein) and capsid protein are likely involved in the inclusion of the PIC within the reforming nucleus after mitosis (Yuan et al., 2002, Yuan et al., 1999, Yueh & Goff, 2003, Yueh et al., 2006).  1.3.2 Genome release in the cytoplasm, followed by entry through the nuclear pore complex While similar in many ways to MLV, lentiviruses such as HIV-1 are able to infect terminally differentiated cells in the absence of cell division. HIV-1 entry into cells is similar to the process described above for MLV, although the composition of the resulting PIC is somewhat different. While the MLV PIC includes reverse transcriptase, integrase and the capsid protein, the HIV-1 PIC is composed of reverse transcriptase, integrase, matrix protein, and the accessory protein Vpr, with the capsid protein largely dissociating prior to nuclear entry (reviewed by Suzuki & Craigie, 2007). It is generally agreed that the HIV-1 PIC enters the nucleus by active transport through the NPC, but the molecular mechanism remains poorly understood.  Every component of the HIV-1 PIC has been suggested to participate in mediating its nuclear entry (Suzuki & Craigie, 2007). The matrix protein contains NLS-like sequences which can target fusion proteins to the nucleus (Bukrinsky et al., 1993, Haffar et al., 2000, von Schwedler et al., 1994), although this effect may be due to nuclear retention mediated by DNA binding rather than facilitated import (Hearps et al., 2008). Vpr contains an atypical NLS (Jenkins et al., 1998, Karni et al., 1998), and can also interact directly with nucleoporins (Jenkins et al., 1998, Popov et al., 1998). Integrase contains several putative NLSs (Bouyac-Bertoia et al., 2001, Gallay et al., 1997, Ikeda et al., 2004), can mediate importin α/β-dependent nuclear import of plasmid DNA (Hearps and  8  Jans, 2006), and interacts directly with the nucleoporin Nup153 (Woodward et al., 2009). In addition, a 99-bp triple-strand DNA structure in the centre of the viral DNA called the central polypurine tract or central DNA flap has also been suggested to participate in nuclear entry of the PIC (Arhel et al., 2006, Zennou et al., 2000). Interestingly, none of these viral components seems to be absolutely necessary or sufficient for nuclear entry of the PIC (Riviere et al., 2010, Yamashita & Emerman, 2005), suggesting that many highly redundant viral components are involved in nuclear transport of the HIV-1 PIC.  Which host factors are involved in nuclear entry of the HIV-1 PIC is also not clear. Members of the importin α family (Gallay et al., 1997, Gallay et al., 1996, Vodicka et al., 1998), importin β (Popov et al., 1998), importin 7 (Ao et al., 2007, Fassati et al., 2003, Zaitseva et al., 2009), and transportin 3 (Brass et al., 2008, Christ et al., 2008) have all been shown to be involved in the nuclear import of either individual viral proteins or of the PIC. The nucleoporins Nup98, Nup358 and Nup153 have also been implicated as factors involved in HIV-1 infection (Brass et al., 2008, Konig et al., 2008). A recent study showed that a single point mutation in the HIV-1 capsid protein could change the nuclear transport requirements of the virus (Lee et al., 2010). Wild-type HIV-1 was sensitive to Nup153 depletion, whereas HIV-1 with an N74D mutation in the capsid protein was more sensitive to Nup155 depletion, indicating that HIV-1 may be flexible in its use of host nuclear transport pathways (Lee et al., 2010). Thus, the flexibility of HIV-1 in its use of viral and host proteins has made the nuclear entry mechanism of this virus extremely challenging to unravel.  Of the viruses that release their genomes in the cytoplasm prior to nuclear entry, the nuclear import of influenza A virus is probably the best studied. The influenza A virus is an enveloped virus, containing a segmented genome consisting of eight single-stranded 9  negative-sense RNAs. While most RNA viruses replicate in the cytoplasm, influenza replication takes place in the nucleus, likely due to the requirement for cellular splicing machinery present there (reviewed by Engelhardt & Fodor, 2006). Each of the eight RNA segments is separately packed with several copies of the structural nucleoprotein (NP) and a single copy of a trimeric viral RNA polymerase into a viral ribonucleoprotein complex (vRNP) (Palese & Shaw, 2007). In this complex, NP forms a core around which the RNA is helically wrapped (Baudin et al., 1994). The influenza A virus is internalized into cells via the endocytic pathway using either clathrin- or caveolae-dependent mechanisms (Nunes-Correia et al., 2004, Roy et al., 2000, Sieczkarski & Whittaker, 2002). The acidic environment of the endosome then triggers the viral fusion machinery, resulting in fusion of the viral and endosomal membranes (Skehel & Wiley, 2000) and disassociation of the viral matrix protein M1 from the vRNPs (Martin & Helenius, 1991). This allows the vRNPs to be released into the cytoplasm. Each vRNP has a diameter of about 15 nm and a length between 50 and 100 nm (Compans et al., 1972). Thus, vRNPs are too large to enter the nucleus by passive diffusion, and must rather use facilitated translocation.  It is thought that NP mediates nuclear import of the vRNPs. NP contains at least two NLSs: NLS1, also termed the nonclassical or unconventional NLS, spanning residues 113 at the N terminus (Neumann et al., 1997, Wang et al., 1997), and NLS2, also termed the classical bipartite NLS, spanning residues 198-216 (Weber et al., 1998). While both NLS1 and NLS2 can contribute to nuclear import of the vRNPs, studies have indicated that NLS1 is a more potent mediator of nuclear import than NLS2 (Cros et al., 2005, Ozawa et al., 2007, Wu et al., 2007). This difference might be due to the location of NLS2 in the intact vRNP. Consistent with its role in nuclear import, NP binds to a number of human importins α, both in vitro and in vivo (Melen et al., 2003, O'Neill & Palese, 10  1995, Wang et al., 1997). Thus, it is thought that vRNPs are transported into the nucleus using the classical importin α/importin β pathway.  1.3.3 Genome release at the cytoplasmic side of the nuclear pore complex Herpesviruses and adenoviruses are among the largest and most complex of the viruses that replicate in the nucleus. While each virus has its unique cell entry and disassembly strategy, their capsids - which are released in the cytoplasm during cell entry - attach to the cytoplasmic side of the NPC. However, with diameters of 120 nm (for herpesvirus) and 90 nm (for adenovirus), these capsids are too large to cross the NPC intact; therefore each virus has developed a unique strategy to deliver its genome into the nucleus.  Herpesviruses are enveloped viruses with an icosahedral capsid containing the viral double-stranded DNA, and a proteinaceous layer (called the tegument) between the capsid and the envelope (Roizman et al., 2007). The best-characterized herpesvirus in terms of nuclear import is the human herpes simplex virus 1 (HSV-1). HSV-1 enters host cells by fusing its envelope with cellular membranes; either with the plasma membrane (which is thought to be the primary entry pathway) or endosomal membranes after internalization by endocytosis (Nicola et al., 2005). The capsid with its surrounding tegument is then released into the peripheral cytoplasm. The tegument-capsid structure is then transported along microtubules to the NPC (Dohner et al., 2002, Sodeik et al., 1997).  Electron microscopy (EM) studies have demonstrated that HSV-1 binds to the cytoplasmic side of the NPC at a distance of ~50 nm away from the NPC (Ojala et al., 2000, Shahin et al., 2006, Sodeik et al., 1997). Thus, the capsids are speculated to bind  11  to the cytoplasmic filaments of the NPC. NPC-binding of the HSV-1 capsid is importin βdependent and requires the small GTPase Ran (Ojala et al., 2000). The viral proteins that mediate the association to the NPC via binding to importin β have not been identified, although tegument proteins have been implicated (Copeland et al., 2009, Ojala et al., 2000). After binding to the NPC, the HSV-1 capsid releases its DNA into the cell nucleus through the NPC. This process leaves intact capsids devoid of the DNA associated with the NPC (Ojala et al., 2000, Sodeik et al., 1997). Very little is known about the mechanisms of DNA release from the HSV capsid, and its transport through the NPC. It is thought that Nup214 provides the cue that triggers DNA release from the capsid (Pasdeloup et al., 2009). In response, DNA release occurs through the capsid portal, a ring structure formed by 12 copies of the tegument protein pUL6 located at a unique capsid vertex (Cardone et al., 2007, Newcomb et al., 2001, Trus et al., 2004), in a process that requires cytosol and energy (Ojala et al., 2000) as well as cleavage of the tegument protein Vp1/2 (Jovasevic et al., 2008).  The adenovirus capsid has also evolved a nuclear import mechanism in which its genome is released at the cytoplasmic side of the NPC. In contrast to HSV-1, however, the adenovirus capsid completely disassembles at the cytoplasmic side of the NPC. Adenoviruses are non-enveloped viruses composed of an icosahedral capsid surrounding an inner nucleoprotein core formed by the double-stranded DNA genome and several copies of four viral core proteins (Berk, 2007). A distinct structural feature of adenoviruses is the fibers projecting from the vertices of the capsid. Adenoviruses enter their host cells by receptor-mediated endocytosis and escape the endosome using a capsid component with membrane-lytic activity (Maier et al., 2010, Wiethoff et al., 2005). Both the cellular internalization and the acid environment of the endosome trigger virion disassembly (Greber et al., 1993, Nakano et al., 2000), which continues in the cytosol. 12  By the time it is delivered to the NPC via microtubule- and dynein mediated motility (Kelkar et al., 2004, Suomalainen et al., 1999), the virion has shed its fibers and several capsid-stabilizing proteins, and some of the remaining viral proteins have been proteolytically processed (Greber et al., 1996, Greber et al., 1993). Upon binding to the cytoplasmic face of the NPC (Greber et al., 1997, Wisnivesky et al., 1999), adenovirus capsids undergo complete disassembly resulting in the subsequent nuclear import of the viral genome and capsid proteins through the NPC (Trotman et al., 2001).  Binding of the adenovirus capsid to the NPC is through Nup214 (Trotman et al., 2001), which is located at the base of the NPC cytoplasmic filaments. Thereby, adenovirus is able to dock closer to the centre of the NPC than HSV-1. Strikingly, neither cytosol nor importins α or β are required for binding of the adenovirus capsid to isolated NE (Trotman et al., 2001). Capsid disassembly is blocked by antibodies against Nup214 (Trotman et al., 2001), implying that NPC binding is a cue for final capsid disassembly. Capsid disassembly and nuclear import of the viral genome requires cellular factors, including nuclear import receptors, heat shock protein 70 (Hsp70) and histone H1 (Saphire et al., 2000, Trotman et al., 2001). The adenoviral core proteins have also been implicated in viral DNA nuclear import (Wodrich et al., 2006). Consistent with this idea, protein VII, the most abundant core protein and the most tightly associated with the viral DNA, has been shown to contain NLSs (Lee et al., 2003, Wodrich et al., 2006) and to bind in vitro to several nuclear import receptors including importin α, importin β, importin 7 and transportin (Wodrich et al., 2006). Taken together these data have led to the current model for nuclear import of the adenoviral genome, which states that after docking to the NPC through Nup214, the adenovirus capsid recruits cellular factors (including histone H1, importin β, importin 7 and Hsp70), which triggers the  13  disassembly/conformational changes required for transportin to bind to protein VII and import the viral DNA into the nucleus through the NPC (Hindley et al., 2007).  1.3.4 Nuclear entry of intact capsids through the nuclear pore complex, followed by genome release HBV and baculovirus are among the viruses with capsids small enough to cross the NPC intact. HBV is an enveloped virus with a total diameter of 42 to 47 nm, containing a capsid with a single copy of the partially double-stranded DNA genome (3 kb) (Seeger et al., 2007). The HBV capsid, which is the component that must pass through the NPC, is composed of 240 copies of a single type of protein (the core protein, 21 kDa) arranged into an icosahedral capsid of 36 nm in diameter (Vanlandschoot et al., 2003). A minor population of capsids with a diameter of 32 nm and composed of 180 copies of core protein also exists. Due to the lack of tissue-culture cell lines that can be infected with HBV, the mechanism of HBV cell entry remains uncertain. Analogy with duck HBV suggests that the capsid is released in the cytoplasm after fusion of the viral envelope with a cellular membrane (Glebe & Urban, 2007) and is transported along microtubules towards the nucleus (Kann et al., 2007, Rabe et al., 2006).  The HBV capsid binds to the NPC in a phosphorylation- and importin α and β-dependent manner (Kann et al., 1999). Phosphorylation of the C-terminus of the core protein is important to expose two cNLSs (Eckhardt et al., 1991, Kann et al., 1999, Yeh et al., 1990). Following the nuclear import of phosphorylated recombinant capsids after their injection into Xenopus laevis oocytes by EM it was demonstrated that the capsid not only binds to the NPC, but is able to cross the NPC without disassembly (Panté & Kann, 2002). Capsids are, however, not released into the nucleus but get arrested within the NPC nuclear basket - a filamentous structure that extends from the NPC into the nucleus  14  - suggesting that uncoating of the viral genome occurs at the nuclear side of the NPC (Panté & Kann, 2002). More recently, HBV capsids were shown to bind to Nup153 (Schmitz et al., 2010), which resides in the nuclear basket.  In comparison to HBV, the baculovirus capsid is not arrested at the nuclear basket. Baculoviruses are rod-shaped (30-60 x 250-300 nm), enveloped viruses with circular double-stranded DNA genomes ranging in size from 90 to 180 kbp (Friesen, 2007). Baculoviruses are unique compared to other viruses because they have two infectious forms: budded virions comprising a single virion enveloped by a plasma-derived membrane, which is involved in cell to cell transmission, and occlusion-derived virions comprising enveloped virions embedded within a crystalline matrix of protein, which are involved in initial host infection when released into the environment upon the death of the host (Friesen, 2007). Although both the occlusion-derived and budded forms contain rod-shaped capsids enclosed within envelopes of different origins, it is the capsid which eventually gets released into the cytoplasm, is propelled through the cytoplasm by virusinduced actin-polymerization (Charlton & Volkman, 1993, Lanier et al., 1996, Lanier & Volkman, 1998) and delivers the genome into the nucleus by a mechanism that is largely unknown.  EM studies have detected intact capsids of several types of baculoviruses at the cytoplasmic side of the NPC and inside the nucleus of cells from larvae inoculated with nucleopolyhedrovirus (Granados, 1978, Granados & Lawler, 1981, Raghow & Grace, 1974). However, the capsids seen in the nucleus might be newly assembled capsids produced during infection. To clarify this issue, a study followed the infection pathway of the baculovirus Autographa californica multiple-capsid nucleopolyhedrovirus (AcMNPV) in tissue-culture cells arrested at the G1/S phase of the cell cycle, and detected 15  AcMNPV capsids at the cytoplasmic side of the NPCs and inside the nucleus (van Loo et al., 2001). Injection of purified AcMNPV capsids into Xenopus oocytes, a cell system in which the virus does not replicate, also reveals capsids docking at the NPCs and inside the nucleus (S. Au and N. Panté, unpublished results), suggesting that the intact AcMNPV capsid enters the nucleus through the NPC. The viral and cellular proteins or receptors that are involved in the initial binding steps of the capsid to the NPC are largely unknown. Similarly, the viral and cellular components triggering genome release and capsid disassembly remain to be determined.  1.3.5 Summary Based on the nuclear entry mechanisms described above, it appears that viruses have developed four general strategies to gain access to the host nucleus (Fig. 1-1). Some viruses, such as the retrovirus MLV, can only enter the nucleus during mitosis, when the barrier of the NE is temporarily absent. This strategy has the disadvantage of restricting the virus to infection of dividing cells. Other viruses, such as HIV-1 and influenza A, undergo extensive disassembly in the cytoplasm. This is likely because the structure of the influenza virion is such that the component released upon envelope fusion is a compact vRNP; similarly, it seems that the retroviral PIC must form in the cytoplasm. The consequence is that for both these viruses, the resulting nucleoprotein complex is small enough to traverse the NPC without further disassembly. In contrast, for herpesviruses and adenoviruses, the viral component released to the cytoplasm is a large, relatively stable icosahedral capsid. Since the capsid is too large to traverse the NPC, docking occurs at the cytoplasmic face. In both cases, interaction with the NPC is used as a cue to trigger genome release. However, in the case of HSV-1 this involves ejection of the genome from an intact capsid, while the adenovirus capsid disassembles completely. Lastly, viruses such as HBV and baculoviruses have capsids small enough  16  to traverse the NPC intact. This strategy has the advantage that viral genomes are protected from detection and degradation in the cytoplasm. However, having entered the nucleus intact, disassembly must occur in the nucleus. For HBV, binding at the nuclear face of the NPC serves as a cue. Host factors involved in triggering disassembly of baculoviruses are currently unknown, but docking at the nuclear face of the NPC does not seem to occur.  Evidently, much progress has been made in understanding how viruses gain access to the host nucleus. However, many molecular details, such as which viral NLSs are exposed at different times during infection, which viral protein interacts with cellular components, and which host transport factors are involved in each step, remain to be elucidated. And for many viruses, nuclear entry mechanisms have not been studied at all. With the exception of adenoviruses, all the viruses described above are enveloped viruses. The nuclear entry mechanisms of non-enveloped viruses are much less understood. Therefore, this thesis will examine the nuclear entry of a non-enveloped virus: the parvovirus minute virus of mice (MVM). Unlike adenoviruses, parvoviruses are small enough to cross the NPC intact. Thus, it is of significant interest to determine whether parvoviruses use a nuclear entry mechanism similar to that of HBV or baculovirus, or rather use a different mechanism entirely.  1.4 Parvoviruses 1.4.1 Introduction to parvoviruses Parvoviruses are non-enveloped, icosahedral, single stranded DNA viruses. At 18-26 nm in diameter, they are among the smallest DNA animal viruses (Cotmore & Tattersall, 2006). Parvoviruses are divided into the dependoviruses, which require co-infection with  17  an unrelated helper virus (adenovirus or herpesvirus), and the autonomous parvoviruses, which include MVM. Parvoviruses can infect a large range of host organisms, including mammals and insects; they are responsible for a broad array of serious diseases in animals (Bloom & Kerr, 2006). However, parvoviruses may also cause persistent inapparent infections, indicating that they are not necessarily linked to disease (Bloom & Kerr, 2006). Several human parvoviruses have been implicated in disease. The pathogenic human parvovirus, B19, generally causes a mild childhood rash known as fifth disease, although B19 infection can also result in transient aplastic crisis in patients with underlying hemolytic disorders, and can cause hydrops fetalis, resulting in fetal death of 2-10% of maternal infections (Kerr & Modrow, 2006). In addition, a novel parvovirus named human bocavirus 1 was recently isolated from the respiratory secretions of patients presenting with pneumonia (Allander et al., 2005). Several other human bocaviruses have since been identified, and have been associated with both respiratory and gastrointestinal illness; however, these viruses are often found in association with other potential pathogens, making their role in disease unclear (Chow & Esper, 2009).  Parvoviruses have a genome of ~5 kb, containing two open reading frames (ORFs). The “left” (3’) ORF codes for two non-structural proteins, NS1 and NS2, while the “right” (5’) ORF codes for the capsid proteins (Fig. 1-2). NS1 is involved in viral replication and gene expression, while NS2 is involved in particle assembly and virion egress from the nucleus (Nuesch, 2006). The parvovirus capsid is made up of three proteins, VP1 (80-86 kDa), VP2 (64-75 kDa) and VP3 (60-62 kDa). VP1 and VP2 result from alternate splice variants, and are identical except for an ~140 amino acid unique portion at the N terminus of VP1, termed the VP1 unique portion; VP2 is proteolytically cleaved to VP3 in  18  Figure 1-2: Transcription and translation of the MVM genome. Transcripts are shown relative to the genome diagrammed above, with the positions of promoters indicated by arrows. Introns are indicated by black lines, and the transcrips are colour coded to match the proteins they encode, shown below. Known functional domains are indicated above some of the proteins. PLA2, phospholipase A2; BC, basic cluster.  19  intact virions during infection of host cells (Fig. 1-2) (Cotmore & Tattersall, 1987). The MVM icosahedrally symmetrical capsid is made up of 60 protein subunits, of which approximately nine are VP1, the rest being made up of a mixture of VP2 and VP3 (Cotmore & Tattersall, 1987).  1.4.2 The parvovirus replication cycle Infection by parvoviruses involves multiple steps and cellular trafficking processes (Fig. 1-3). The first barrier the virus must overcome to gain entry to the cell is the plasma membrane. Parvoviruses are thought to require receptor-mediated endocytosis for cell infection (Fig. 1-3, step 1) (reviewed by Harbison et al., 2008). A number of cell-surface receptors have been identified for parvoviruses. For example, canine parvovirus (CPV) and feline panleukopenia virus (FPV) both use the transferrin receptor to infect cells, while the human parvovirus B19 requires the globoside erythrocyte P antigen (Harbison et al., 2008). Cell surface receptors and binding molecules identified for various adenoassociated viruses (AAVs) include heparin sulfate proteoglycan, αVβ5 integrin, fibroblast growth factor receptor 1, platelet-derived growth factor receptor, and 37/67-kDa laminin receptor, as well as α2-3 and α2-6 sialic acid (reviewed by Vihinen-Ranta et al., 2004). Although the protein receptor for MVM has not been identified, it was shown that MVM binds several specific sialic acid motifs (Nam et al., 2006).  After binding to receptors, parvoviruses are taken up via clathrin-mediated endocytosis (Parker & Parrish, 2000). It has recently been shown that porcine parvovirus can also enter cells via macropinocytosis (Boisvert et al., 2010), and it will be interesting to see whether this is the case for other parvoviruses as well. Following endocytosis, virions slowly escape from endocytic compartments to the cytosol (Fig. 1-3, step 2); injection of antibodies against CPV capsid into the cytosol up to 8 hours post infection could block  20  Figure 1-3: The MVM replication cycle. (1) Attachment of the MVM capsid to an unknown cell surface receptor is followed by clathrin-mediated endocytosis and (2) escape from endosomes. (3) Virions then traffic along microtubules toward the microtubule organizing centre (MTOC), and (4) enter the nucleus via an unknown mechanism. (5) The single-stranded DNA genome is converted to a double-stranded form, (6) which then serves as a template for transcription. (7) Viral non-structural genes are expressed, and NS1 then trans-activates expression of the capsid proteins and (8) initiates viral DNA replication. (9) Capsid proteins are translated in the cytoplasm, where they form trimers which are then transported to the nucleus through the NPC. (10) Progeny virions are assembled in the nucleus and (11) virions are exported from the nucleus through the NPC. Virions are then released from the cell by cell lysis (12), or possibly also by regulated secretion (13).  21  viral replication (Vihinen-Ranta et al., 2002, Vihinen-Ranta et al., 2000). It has been shown that the mechanism of parvoviral escape from endosomes likely involves a viral phospholipase. Parvoviruses contain a conserved phospholipase A2 (PLA2) motif in the VP1 unique portion (Zadori et al., 2001). PLA2 mutant MVM virions are not viable, but can be rescued by three different treatments that disrupt endosomes: coinfection with wild type MVM, co-infection with endosomolytic adenovirus, and polyethyleneimineinduced endosome rupture (Farr et al., 2005). These results suggest that the parvoviral PLA2 functions in endosomal escape.  Following release into the cytoplasm, parvoviruses are transported to the perinuclear region (Fig. 1-3, step 3) (Seisenberger et al., 2001). Perinuclear accumulation of CPV is microtubule-dependent (Suikkanen et al., 2003a, Suikkanen et al., 2003b, Vihinen-Ranta et al., 2000). In addition, CPV capsids can be precipitated from infected cells along with dynein, a minus-end directed microtubule motor protein (Suikkanen et al., 2003a). Thus, parvoviruses probably associate with dynein, directly or indirectly, in order to be transported along microtubules toward the nucleus.  After perinuclear accumulation, the parvovirus genome is transported to the nucleus by a largely undefined mechanism (Fig. 1-3, step 4; see section 1.4.3 below). It is thought that entry of the cell into S-phase promotes conversion of the single-stranded viral genome into duplex molecules, which can serve as a template for transcription and replication (Fig. 1-3, step 5) (Weitzman, 2006). The first genes to be expressed are the viral non-structural genes (Fig. 1-3, step 6/7); NS1 then trans-activates expression of the capsid proteins, and initiates viral DNA replication (Fig. 1-3, step 7/8) (Nuesch, 2006). MVM capsid proteins VP1 and VP2 are translated in the cytoplasm. In the cytoplasm, they form two types of trimers (VP2 only trimers, or trimers composed of 2 copies of VP2 22  and one copy of VP1) which are then transported to the nucleus through the NPC (Fig. 1-3, step 9) (Lombardo et al., 2000, Riolobos et al., 2006). The assembly of progeny virions takes place in the nucleus (Fig. 1-3, step 10), and virions are exported from the nucleus through the NPC using a nuclear export signal at the VP2 N-terminal domain (Fig. 1-3, step 11) (Maroto et al., 2004). Eventually, infection results in cell lysis, and progeny virions are released into the extracellular milieu (Fig. 1-3, step 12). However, recent evidence indicates that MVM progeny virions traffic from the nucleus to the cell periphery in modified endosomal or lysosomal vesicles, challenging the idea that parvovirus egress is a passive process (Fig. 1-3, step 13) (Bar et al., 2008).  Throughout the parvovirus replication cycle, a series of conformational transitions occurs in the capsid: the VP2 N-terminus is cleaved, resulting in VP3; the VP1 unique portion becomes externalized; and the genome becomes externalized (Cotmore et al., 1999, Farr et al., 2005, Mani et al., 2006, Ros et al., 2006). However, the function of these transitions and the point during infection at which they occur remain incompletely understood.  1.4.3 Parvoviral nuclear entry Several lines of evidence indicate that the parvoviral genome enters the nucleus in association with an intact capsid after escape from endosomes. Multiple studies using immunofluorescence, or green fluorescent protein (GFP)- or fluorophore-conjugated virions have detected parvoviral capsid proteins in the nucleus of infected cells (Bartlett et al., 2000, Lux et al., 2005, Mani et al., 2006, Seisenberger et al., 2001, Vihinen-Ranta et al., 1998). In addition, microinjection of antibodies against the capsid of the human parvovirus adeno-associated virus 2 (AAV2) into the nucleus can inhibit productive infection of tissue-culture cells (Sonntag et al., 2006). Lastly, immunogold EM has  23  revealed apparently intact capsids of canine parvovirus in the nucleus of cells infected in the presence of cyclohexamide, which prevents the synthesis of new capsid proteins (Suikkanen et al., 2003a). However, an alternative has also been suggested. Several studies have noted that only very small amounts of capsid protein are detected in the nucleus, causing the authors to suggest either that nuclear transport of parvoviruses is very inefficient or that the genome dissociates from the capsid prior to nuclear entry (Lux et al., 2005, Mani et al., 2006).  At only ~26 nm in diameter (Llamas-Saiz, 1997), parvovirus capsids are small enough to enter the nucleus intact through the NPC, and it has been assumed that this is how the parvovirus genome accesses the nucleus. However, we recently proposed an alternative mechanism for nuclear import of parvoviruses. We have shown that after microinjection into Xenopus oocytes, MVM induces disruptions of the NE which support nuclear import of proteins in a manner that is independent of the NPC (Cohen & Panté, 2005). We have also shown that MVM disrupts the nuclear membranes of purified rat liver nuclei (Cohen & Panté, 2005). Based on these results, we proposed that MVM enters the nucleus using a unique mechanism that is independent of the host nuclear import machinery: instead of crossing the NPC, MVM disrupts the NE and enters the nucleus through the resulting breaks. Consistent with this mechanism, Hansen and colleagues previously found that AAV can enter purified intact nuclei in the absence of nuclear import receptors and other cytoplasmic factors required for NPC-mediated import (Hansen et al., 2001). In addition, blocking the NPCs with the lectin wheat germ agglutinin (WGA) had no effect on the uptake of AAV into purified nuclei (Hansen et al., 2001). These studies suggest that parvoviruses can bypass the host nuclear transport machinery during entry to the nucleus. However, the molecular mechanism by which MVM disrupts the NE remains  24  unclear, and it is also not clear whether an NPC-mediated nuclear transport pathway exists for parvoviruses as well.  1.4.4 Nuclear import of parvovirus capsid proteins Although little is known about parvoviral nuclear import, some work has been done on the nuclear import of individual capsid proteins. It has been shown that nuclear import of VP2 is mediated by an unconventional nuclear localization motif (NLM) (Lombardo et al., 2000). While classical NLSs consist of a short monopartite or bipartite sequence of basic amino acids (Lange et al., 2007), nuclear import can also be mediated by non-classical NLSs which do not fit a consensus sequence and bind different families of import receptors. Nuclear import of VP2 is mediated by a three-dimensional NLM on the basic face of an amphipathic beta-strand (Lombardo et al., 2000). However, this NLM is buried within the capsid of assembled virions (Agbandje-McKenna et al., 1998, Lombardo et al., 2000). Thus, while it may mediate nuclear import of nascent VP2 molecules prior to capsid assembly in the nucleus, the NLM of VP2 is unlikely to mediate nuclear import of intact capsids during onset of infection.  In addition to the NLM, VP1 has several other putative NLSs. Four putative NLSs were identified in the VP1 unique portion based on sequence analysis (Lombardo et al., 2002). These were termed basic cluster 1-4 (BC1-BC4) (Lombardo et al., 2002). By creating deletions and site-directed mutations in VP1, it was determined that the NLM and BC1 constitute major nuclear localization domains, while BC2 functions as a weak NLS, and BC3 and BC4 do not display nuclear transport activity (Lombardo et al., 2002). Like the NLM, the VP1 unique portion is also buried within assembled nascent virions (Cotmore et al., 1999). However, there is increasing evidence that the VP1 unique portion becomes externalized when virions are exposed to acidification in endosomes  25  during the onset of infection (Cotmore et al., 1999, Farr et al., 2005, Mani et al., 2006). Thus, BC1 and/or BC2 could potentially mediate nuclear import of capsids during the onset of infection.  1.5 Nuclear envelope breakdown We have shown that microinjection of MVM into Xenopus oocytes results in disruption of the NE (Cohen & Panté, 2005). This disruption may be caused directly by a viral enzyme, such as the capsid-tethered PLA2. However, it is also possible that MVM induces NE disruption by triggering a cellular program for NE breakdown (NEBD). There are two situations in which NEBD normally occurs: cell division and apoptosis (programmed cell death).  1.5.1 Mitotic nuclear envelope breakdown During cell division, chromosomes must be partitioned between daughter cells. In some higher eukaryotes this is achieved through ‘open mitosis.’ In open mitosis, the NE is completely disassembled and removed from chromatin; this allows the cytoplasmic mitotic spindle to access chromosomes. In contrast, during the ‘closed-mitosis’ of yeast the NE remains intact and the mitotic spindle forms inside the nucleus. Intermediate forms where the NE partially opens up near to centrosomes also exist, for example in Caenorhabditis elegans early embryos (reviewed by Guttinger et al., 2009).  The NEBD of open mitosis proceeds via several steps: NPCs disassemble, the nuclear lamina depolymerizes, INM proteins detach from chromatin and ONM proteins retract into the ER (Guttinger et al., 2009). In addition, dynein and microtubules are involved in clearing the nuclear membranes from chromatin (Beaudouin et al., 2002, Salina et al., 2002). Many of these events are controlled by the activation of mitotic kinases. Of the  26  kinases involved, the role of cyclin-dependent kinase 1 (CDK1) is currently the best understood. Multiple nucleoporins undergo phosphorylation at CDK1 sites during mitosis, which is thought to contribute to NPC disassembly (Guttinger et al., 2009). In addition, phosphorylation of lamins by CDK1 leads to lamina depolymerization (Heald & McKeon, 1990, Peter et al., 1990). Lastly, the INM proteins lamina-associated protein 2α (LAP2α), LAP2β and lamin B receptor are all targets of CDK1, suggesting that this kinase may also play a role in the release of INM proteins from chromatin and lamins (Guttinger et al., 2009). Protein kinase C (PKC) also plays a role in mitotic NEBD; lamin B is a target of PKCβ2, and inhibition of PKC results in G2 arrest (Thompson & Fields, 1996). In addition, aurora A and polo-like kinase 1 have also been implicated in NEBD (Guttinger et al., 2009).  1.5.2 Apoptotic nuclear envelope breakdown Apoptosis, or programmed cell death, occurs either developmentally or in response to stress such as DNA damage. One of the hallmarks of apoptosis is NEBD. Like mitotic NEBD, the dismantling of the NE during apoptosis involves kinases. For example, lamin B is phosphorylated by PKCδ, resulting in lamina depolymerization (Cross et al., 2000). However, unlike mitotic NEBD, during apoptosis nucleoporins, nuclear lamins, and INM proteins are all cleaved by apoptotic proteases (reviewed by Fahrenkrog, 2006). Lastly, analogous to the requirement of mitotic NEBD for dynein and microtubules, apoptotic NEBD requires myosin and actin to generate the force required for nuclear disintegration (Croft et al., 2005).  Apoptosis is carried out by a family of cysteine proteases called caspases. Caspases are divided into two categories, initiators and executioners. While both types are constitutively present in the cytoplasm as latent precursors called zymogens or  27  procaspases, they are activated by different mechanisms. Initiator caspases exist as monomeric zymogens, which require dimerization to become active; cleavage of initiator caspases is thought to stabilize the dimer without being necessary for formation of an active site (reviewed by Pop & Salvesen, 2009). In contrast, executioner caspase zymogens are already dimers, and cleavage is thought to be the activating event (Pop & Salvesen, 2009). There are two main apoptotic pathways: the extrinsic pathway, initiated during development or by immune cells, activates initiator caspase-8 or -10, while the intrinsic pathway, initiated in response to cell stress such as DNA or mitochondrial damage, activates initiator caspase-9 (Pop & Salvesen, 2009). Both of these pathways lead to activation of executioner caspases, which include caspase-3, -6, and -7; these executioner caspases then go on to cleave a wide variety of targets (Pop & Salvesen, 2009). The executioner caspases implicated in the cleavage of NE and NE-associated proteins are caspase-3 and -6. Some of the targets of these caspases include the nucleoporins Nup153 (which connects the NPC to the nuclear lamina), Nup214, and Nup358; the A- and B-type lamins; and the INM proteins LAP2α and LAP2β (Fahrenkrog, 2006).  1.6 Objectives Much progress has been made in characterizing the mechanisms of viral entry into host cells, as well as early trafficking events. However, how different viruses gain access to the host nucleus, where genome replication often occurs, is less well understood. In particular, less is known about trafficking and nuclear entry of non-enveloped viruses. The research described in this thesis examines the nuclear entry of a model nonenveloped virus, the parvovirus MVM, as well as using unbiased proteomic approaches to identify cellular factors involved in the viral replication cycle. Our previous observations suggested that MVM may use an unconventional nuclear entry  28  mechanism, involving disruption of the host NE and entry through the resulting disruptions. Based on this observation, the following questions were asked: 1) What is the effect of MVM on the host NE? 2) How does MVM induce disruption of the host NE? 3) What is the role of MVM-induced NE disruption in the viral replication cycle? 4) Which host proteins undergo post-translational modification in MVM-infected cells? 5) Which host proteins interact with the MVM capsid? A combination of approaches including electron and fluorescence microscopy, enzymatic assays, and proteomics was used to address these questions.  1.6.1 Aim 1: To characterize the effect of MVM on the host nuclear envelope We previously observed that microinjection of MVM into Xenopus oocytes resulted in disruption of the NE (Cohen & Panté, 2005). The research described in Chapter 3, entitled Effect of MVM on the host nuclear envelope, aimed to test the hypothesis that MVM induces disruption of the NE in infected cells. The ability of other parvoviruses to induce NE disruption was also examined. In addition, conventional EM, immunogold EM, and immunofluorescence (IF) microscopy were used to characterize the effect of MVM on the host nuclear membranes and nuclear lamina in infected mouse fibroblast cells.  1.6.2 Aim 2: To determine the molecular mechanism of MVM-induced nuclear envelope disruption I next investigated the molecular mechanism of MVM-induced NE disruption. I first tested the hypothesis that viral PLA2 activity, which is the only enzymatic activity exhibited by the MVM capsid, is required for MVM-mediated NE disruption. When this hypothesis turned out to be false, I used a semipermeabilized cell assay to test the  29  hypothesis that host enzymes are necessary for MVM-mediated NE disruption. After finding that the apoptotic protease caspase-3 was involved, a variety of microscopic and biochemical assays were used to characterize the role of caspase-3 in disruption of the NE by MVM. In addition the hypothesis that host ONM proteins are involved in MVMinduced NE disruption was tested. This work is described in Chapter 4, entitled Mechanism of MVM-induced nuclear envelope disruption.  1.6.3 Aim 3: To determine the role of MVM-induced nuclear envelope disruption in the viral replication cycle With the knowledge of caspase involvement in MVM-induced NE disruption described in Chapter 4, it was possible to examine the role of NE disruption in the MVM replication cycle. Pharmacological inhibition of caspase-3 was used to test whether NE disruption is in fact necessary for viral replication, and to test the hypothesis that NE disruption facilitates nuclear entry of MVM. In addition, the effect of MVM infection on the compartmentalization of cellular proteins was examined. This work is described in Chapter 5, entitled Role of nuclear envelope disruption in the MVM replication cycle.  1.6.4 Aim 4: To identify host proteins that are post-translationally modified in MVM-infected cells Many viruses induce post-translational modification of a multitude of cellular proteins during infection of host cells. These modifications include but are not restricted to cleavage, phosphorylation, and ubiquitination. In addition, host proteins may be upregulated or downregulated in response to infection. A comprehensive study of posttranslational modifications in cells infected with parvovirus has not been carried out. Two-dimensional differential in gel electrophoresis (2D-DIGE) analysis was performed in an attempt to identify host proteins that are modified in MVM-infected cells. Based on the  30  results described in Chapter 3, we expected to observe differences in the amount or modification state of nuclear lamins and perhaps nucleoporins or other proteins associated with the NE. We also hoped to observe differences in other proteins pertaining to virus trafficking or replication, for example components of the cytoskeleton, proteins involved in cell signaling, or transcription factors. Chapter 6, entitled Proteomic approaches for studying MVM infection, describes the results of this line of enquiry.  1.6.5 Aim 5: To identify host proteins that interact with the MVM capsid Lastly, in addition to the candidate approach to identifying host proteins involved in MVM-induced NE disruption described in Chapter 4, we also sought to use an unbiased approach. Thus, immunoprecipitation of MVM capsids followed by mass spectrometry was used to identify putative binding partners of MVM. Using this approach, we hoped to identify NE or signaling proteins involved in induction of NE disruption through direct interaction with the MVM capsid. Other anticipated binding partners included a cell surface receptor or co-receptor, cytoskeleton-associated motor proteins, and signaling proteins such as kinases or phosphatases, as well as transcription factors. This work is also described in Chapter 6, entitled Proteomic approaches for studying MVM infection.  31  2. Materials and Methods* 2.1 Antibodies Unless otherwise specified, the antibody used to detect intact MVM capsids was a mixture of antibodies from three mouse hybridoma clones (D4H1, D10E10 and EllF3, provided by Dr. P. Tattersall, Yale University School of Medicine). To detect NS1 a mouse monoclonal antibody specific for the C-terminus of MVM NS1 was used (provided by Dr. P. Tattersall, Yale University School of Medicine).  Commercial primary antibodies used were for lamin A/C (rabbit polyclonal, Santa Cruz Biotechnology, sc-20681), lamin B (goat polyclonal, Santa Cruz Biotechnology, sc6216), and cleaved caspase-3 (rabbit polyclonal, Cell Signaling Technology, 9661).  Fluorophore-conjugated secondary antibodies were from Invitrogen. Goat anti-mouse secondary antibody conjugated to 10-nm gold was from Ted Pella.  2.2 Purification of MVM A protocol based on those described by Tattersall et al. (1976) and Williams et al. (2004) was used to purify the parvovirus MVM. LA9 mouse fibroblast cells (Littlefield, 1964) were grown in suspension in minimum essential medium (MEM) supplemented with 5% fetal bovine serum (FBS) and 1% penicillin/streptomycin, in an environment of 5% CO2 at 37°C. Cells were maintained at 2 x 105 cells/ml, and grown in suspension for at least 2 weeks prior to infection. Approximately 200 ml of cells were then infected with MVM at a  *  A version of part of this chapter has been published: • Au, S., Cohen, S., and Panté, N. (2010). Microinjection of Xenopus laevis oocytes as a system for studying nuclear transport of viruses. Methods 51(11): 114-120.  32  multiplicity of infection (MOI) of 1 x 10-3 pfu/cell. The cells were diluted daily to maintain the concentration at 2 x 105 cells/ml, resulting in about 2 L final volume. Cells were monitored for cell death and cessation of growth, which usually occurred five days after infection, and then harvested immediately.  MVM was purified from the infected cells rather than the culture medium. Care was taken to perform all steps of the purification with chilled, sterile buffers, at 4°C or on ice to avoid proteolytic activity. Infected cells were pelleted by centrifugation at 1,600 x g at 4°C and washed with 100 ml TNE buffer (50 mM Tris, 150 mM NaCl, 0.5 mM EDTA, pH 8.7). Cells were pelleted once more, and the pellet was resuspended in 20 ml TE. Cells were lysed by 3-4 brief (5 seconds) bursts of gentle sonication on a low setting, with tubes placed on ice between bursts. Cell debris was pelleted by centrifugation at 17,800 x g for 30 minutes at 4°C. Debris was discarded, and virus particles were precipitated from the supernatant by adding CaCl2 to a final concentration of 25 mM and incubating on ice for 30 minutes. Precipitated virus was pelleted by centrifugation at 3,100 x g for 25 minutes, and pellets were resuspended in 20 ml T20E buffer (50 mM Tris, 20mM EDTA, pH 8.7), again with 3-4 bursts of gentle sonication. This suspension was cleared once more by centrifugation at 12,300 x g for 10 minutes, and the supernatant was loaded onto a continuous CsCl gradient created by loading four Beckman ultraclear centrifuge tubes (14 mm x 89 mm) with 5 ml CsCl (0.53 g/ml in TE), followed by a sucrose cushion of 0.75 ml 1 M sucrose in TE, followed by 5 ml virus-containing supernatant. Gradients were centrifuged at 96,800 x g for 20 hours. The next day, 3 bands were visible. A single band of full (genome containing) capsids was apparent near the bottom of the tube, and a doublet of empty capsids appeared about 1 cm above. The bands were removed from the centrifuge tubes with a syringe, and dialysed against TE 4 times for at least 4 hours each, in order to remove the CsCl. 33  To confirm the purity and the integrity of the virus, a 10 µl drop of the viral solution was used to prepare EM grids by negative staining (see section 2.5.1). Successful virus preparations yielded round particles of 26 nm in diameter. Full capsids from the bottom band of the gradient excluded the uranyl acetate stain (Fig. 2-1a), while empty capsids from the top two bands did not (Fig. 2-1b). Purity of the virus preparation was also assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining, which yielded three distinct bands corresponding to the three parvoviral capsid proteins - VP1 (83 kDa), VP2 (64 kDa), and VP3 (60 kDa). The virus titer was determined by plaque assay according to the protocol described by Tattersall (1972). Virus was used immediately, or aliquoted and stored frozen at -80°C for later use. A successful purification typically yielded several ml of virus at 1x108-1x109 pfu/ml.  2.3 Xenopus oocyte microinjection In order to isolate oocytes, a female Xenopus frog was narcotized by immersion in a solution of 300 mg/L Tricaine methane sulfonate solution (MS222; 3-Aminobenzoic acid ethyl ester, Sigma Aldrich) buffered to pH 7.5 with about 600 mg/L sodium bicarbonate for 30-45 minutes. Once the frog was narcotized, a 1-cm long incision through the skin was made with surgical scissors at about 1 cm above the leg fold of the frog and slightly offset from the ventral middle line of the stomach. The muscle under the skin was then cut (about 6 mm-long incision), and a small portion of the ovary (1-2 cm) was pulled out with sterilized forceps and removed with a pair of sterilized scissors. The oocytes were placed on a Petri dish containing modified Barth’s saline (MBS: 88 mM NaCl, 1 mM KCl, 0.82 mM MgSO4, 10 mM Hepes, 0.33 mM Ca(NO3), 0.41 mM CaCl, pH 7.5), the incision was sutured with sterilized medical surgical thread, and the frog was placed in fresh water for recovery.  34  Figure 2-1: Purified MVM. Electron micrographs of parvovirus full capsids (a) and empty capsids (b) that have been prepared for visualization by negative staining as described in section 2.5.1. Scale bar, 100 nm. (Figure originally published by Au et al., 2010. Copyright Elsevier).  35  Oocytes were then de-folliculated in a 50 ml conical tube containing 20 ml collagenase solution (5 mg/ml collagenase in calcium-free MBS) and placed on a shaker for approximately 40 minutes at 100 rpm. When the oocytes were sufficiently de-folliculated, oocytes were washed three times in MBS. Stage VI oocytes with a distinct white rim separating the black animal hemisphere and the creamy colored vegetal hemisphere were selected and transferred into a multiwell dish (Nunc, 10 µl well volume) for microinjection. Purified viral capsids were mixed with 1% bromophenol blue in a 10:1 ratio to aid in the visualization of the microinjection. Injection needles made by pulling a 6.6 µl Drummond micropipette with the Inject+Matic Puller and calibrated to 50 nl were used to aspirate the viral solution. For consistent cytoplasmic injection, the needle was inserted into the white rim separating both hemispheres at a 45 degree angle. A volume of 50-100 nl of the viral solution was injected into the cytoplasm of each oocyte. Injected oocytes were placed in a small Petri dish filled with MBS, and incubated at room temperature for different time points. After this incubation time, the injected oocytes were transferred into a solution of 2% glutaraldehyde (Ted Pella) in MBS and fixed overnight at 4oC.  In various experiments, Xenopus oocytes were mock injected with 50 nl PBS or injected with 50 nl MVM (1 x 108 pfu/ml, approximately equal to 6 x 1010 genomes/ml), H1 (2 x 109 pfu/ml, courtesy of Dr. M. Kann, University of Bordeaux), CPV (1 x 1013 particles/ml, courtesy of Dr. M. Vuento, University of Jyväskylä), or H42R-MVM (6 x 1010 genomes/ml, courtesy of Dr. P. Tattersall, Yale University School of Medicine). For the PLA2-inhibition experiment, MVM particles were incubated with 10 µM manoalide (Alexis Biochemicals) for 1 hour at room temperature with agitation prior to microinjection. Where indicated 50 mM of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Aspfluoromethylketone (zVAD-fmk), benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone 36  (zDEVD-fmk) or benzyloxycarbonyl-Val-Glu-Ile-Asp-fluoromethylketone (zVEID-fmk) (Tocris Biosciences) were included in the MVM solution to be injected, resulting in an intracellular inhibitor concentration of approximately 200 µM. Oocytes were then incubated at room temperature for 30 minutes to two hours, as indicated in the figure legends.  2.4 Tissue culture, transfection and infection Adherent LA9 mouse fibroblast cells (Littlefield, 1964), HeLa cells stably expressing a fusion of GFP to lamina-associated polypeptide 2β (GFP-LAP2β, courtesy of Dr. U. Kutay, ETH Zurich), and MC3T3 cells inducibly expressing SS–HA–Sun1L–KDEL (courtesy of Dr. K. Roux, University of Florida) were maintained at 5% CO2 and 37°C in complete Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. To induce expression of SS–HA– Sun1L–KDEL, doxycycline (1 µg/ml, Clontech) was added to the medium for 48 hours.  For transient expression of tandem GFP, LA9 cells were grown in monolayers on poly-Llysine-coated coverslips and transfected using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions, 48 hours prior to infection with MVM.  For infection, LA9 cells were grown in monolayers on poly-L-lysine-coated coverslips or in 10 cm dishes, and then mock infected or infected with MVM at a MOI of 4 pfu/cell in DMEM supplemented with 1% FBS. Cells were incubated for one hour at 4°C to allow the virus to bind at the cell surface. The medium was then replaced, and cells were incubated at 37°C as indicated in the figure legends. Where indicated caspase inhibitors zVAD-fmk, zDEVD-fmk or zVEID-fmk (Tocris Biosciences) were included in the medium for one hour prior to infection and during the infection steps.  37  2.5 Electron microscopy 2.5.1 Negative staining and electron microscopy of purified virus To evaluate the purity and integrity of virus preparations, a 10 µl drop of purified virus was placed on top of a parlodion/carbon coated copper EM grid that had been glow discharged for 30 seconds. After 5 minutes, the grid was washed four times in 10 µl drops of TE, and then negatively stained in a 10 µl drop of 2% uranyl acetate for 1 minute. The uranyl acetate was wicked away with filter paper, and the grid was visualized under a Hitachi H7600 transmission electron microscope (TEM).  2.5.2 Embedding and electron microscopy of microinjected Xenopus oocytes Fixed oocytes prepared as described in section 2.3 were washed three times in MBS, for 5 minutes each. Oocytes were placed into a small Petri dish filled with low salt buffer (LSB: 1 mM KCl, 0.5 mM MgCl2, 10 mM HEPES, pH 7.5) and the black animal hemisphere (which contains the nucleus) was dissected using dissecting tweezers. At this point, the success of the microinjection was evaluated through the presence of a light blue color (from the bromophenol blue) in the cytosol. The protocol was followed only for successfully injected oocytes.  After dissection, the vegetal hemisphere was discarded and the animal hemisphere was fixed once more with 2% glutaraldehyde in LSB for 1 hour at room temperature. After this fixation, samples were washed three times in LSB, for 5 minutes each, and embedded in 2% low-melting agarose. The agarose-embedded samples were transferred to glass vials and post-fixed with 1% OsO4 in LSB for 1 hour. Samples were washed three times with LSB, for 5 minutes each, and then sequentially dehydrated in  38  50%, 70%, and 90% ethanol for 20 minutes each, followed by two 15 minute-long incubations in 100% ethanol. Dehydration was completed by placing the samples in 100% acetone for 15 minutes. Fixed and dehydrated samples were sequentially infiltrated by incubation in a mixture of Epon 812 resin (Fluka) and acetone at a 1:1 ratio for 1 hour, followed by incubation in a mixture of Epon and acetone at a 2:1 ratio for 2 hours. The samples were finally incubated in Epon for 8 hours. Epon-infiltrated samples were placed into flat embedding molds (Ted Pella) containing pure Epon and allowed to polymerize at 60oC for two days.  After polymerization, 50-nm thick sections were obtained from the Epon-embedded oocytes using an ultramicrotome. Sections were collected on parlodion/carbon-coated EM grids. Grids were stained with 2% uranyl-acetate for 15 minutes, followed by four washes with distilled water, and then stained with 2% lead citrate for 5 minutes, followed by another four washes with distilled water. Samples were then visualized under a Hitachi H7600 TEM. Quantification of the proportion of NE damage was performed using ImageJ software (National Institutes of Health).  2.5.3 Embedding and electron microscopy of MVM-infected fibroblast cells Monolayers of LA9 cells were grown on Aclar film (Pelco) and infected with MVM as described in section 2.4. After infection cells were fixed and embedded according to a modification of the protocol described by Fricker et al. (1997). The cells were first washed twice with PBS and then fixed with 2% glutaraldehyde in PBS for 1 hour at room temperature. After this fixation, samples were washed three times in PBS, for 5 minutes each, and then post-fixed with 1% OsO4 in PBS for 1 hour. This step was followed by five washes with distilled water, for 5 minutes each. In order to enhance the contrast of the nuclear membranes, cells were then stained overnight with 0.5% uranyl acetate.  39  Samples were washed three times with PBS, for 5 minutes each, and then sequentially dehydrated in 50%, 70%, and 90% ethanol for 10 minutes each, followed by two 10 minute-long incubations in 100% ethanol. Dehydration was completed by two incubations in 100% acetone for 10 minutes each. Fixed and dehydrated samples were sequentially infiltrated by incubation in a mixture of Epon 812 resin (Fluka) and acetone at a 1:1 ratio for 2 hours, followed by incubation in a mixture of Epon and acetone at a 2:1 ratio overnight. The samples were finally incubated in Epon for 8 hours. Eponinfiltrated samples were placed onto gelatin capsules (Ted Pella) filled with pure Epon, cell-side down, and allowed to polymerize at 60oC for 2 days. Aclar was removed from the capsules, and the resulting samples were then sectioned, stained and visualized as described in section 2.5.2.  2.5.4 Immunogold electron microscopy of MVM-infected fibroblast cells Monolayers of LA9 cells were grown in 10 cm dishes and infected with MVM as described in section 2.4. Cells were fixed with 4% paraformaldehyde in PBS for 1 hour, washed twice with PBS, and harvested by scraping. The resulting cell pellets were embedded in 2% low-melting agarose and sequentially dehydrated in 50% and 70% ethanol for 20 minutes each, followed by 100% ethanol twice for 10 minutes each. The samples were then infiltrated by incubation in a mixture of LR white acrylic resin (Sigma Aldrich) and ethanol at a ratio of 2:1 for 1 hour, followed by incubation in pure LR white overnight. The next day, samples were infiltrated again in pure LR white for 2 hours. Next, the samples were placed in gelatin capsules (Ted Pella) containing pure LR white. The capsules were capped closed and exposed to ultraviolet light (365 nm) for 2 days. Once the LR white polymerized, 50-nm thick sections were obtained with an ultramicrotome and collected on parlodion/carbon-coated EM grids, which were then immunogold labeled.  40  For immunogold labeling, grids containing the EM sections were floated (with sections face down) on three 20 µl drops of 2% bovine serum albumin (BSA) in PBS for 10 minutes on each drop. Grids were then floated on drops of primary antibody for intact MVM capsids diluted 1:5 in 2% BSA in PBS and incubated at 4°C overnight. After antibody incubation, grids were washed by floating them on a series of six drops of 2% BSA in PBS, for 5 minutes on each drop. Grids were then placed on drops of the appropriate gold-conjugated secondary antibody diluted 1:50 with 2% BSA in PBS for 1 hour. Grids were then washed by sequentially floating them on six drops of 0.5% BSA in PBS, followed by flotation on six drops of PBS, for 5 minutes on each drop. Grids were fixed with 1% glutaraldehyde in PBS for 5 minutes. Finally, grids were washed in distilled water and stained with 2% uranyl acetate for 5 minutes, followed by four brief washes with distilled water, and 2% lead citrate for one minute, followed by another four brief washes with distilled water. Samples were then visualized using a Hitachi H7600 TEM.  2.6 Fluorescence microscopy 2.6.1 Immunofluorescence microscopy of MVM-infected cells Cells were grown and infected on coverslips as described in section 2.4. For lamin-A/C immunostaining cells were first permeabilized (0.2% Triton X-100, 30 seconds), then fixed (3% paraformaldehyde, 3 minutes), blocked (2% BSA, 20 minutes), and labeled with primary antibodies for intact MVM capsids (1:100) and lamin A/C (1:200). For caspase-3 immunostaining, cells were fixed (3% paraformaldehyde, 10 minutes), then permeabilized (ice-cold methanol, 10 minutes at -20°C), blocked (0.3% Triton X-100, 5% normal goat serum, 1 hour), and labeled with primary antibodies for MVM (1:100) and cleaved caspase-3 (1:25). For MVM immunostaining of cells transfected with tandem GFP and NS1 immunostaining, cells were fixed (3% paraformaldehyde, 10 minutes),  41  then permeabilized (0.2% Triton X-100, 5 minutes), blocked (1% BSA, 1 hour), and labeled with a primary antibody for MVM (1:100) or NS1 (1:300). All primary antibodies were followed by appropriate fluorescently labeled secondary antibodies (Invitrogen). Coverslips were mounted using Prolong Gold Antifade with DAPI (Invitrogen), and visualized using a Zeiss Axioplan 2 fluorescence microscope (lamin A/C and NS1 experiments) or an Olympus Fluoview FV1000 laser scanning microscope (caspase-3 and tandem GFP experiments). Quantification of the cells expressing NS1 was performed using ImageJ software (National Institutes of Health).  2.6.2 Semipermeabilized cell assay for nuclear envelope disruption Evenly spread GFP-LAP2β HeLa cells grown in µ-slide 8 well dishes (Ibidi, ibiTreat) were washed in permeabilization buffer (PB: 20 mM Hepes KOH, pH 7.4, 110 mM KoAc, 5 mM MgOAc, 0.5 mM EGTA and 250 mM sucrose), permeabilized in PB containing 15 µg/ml digitonin for 3 minutes, and then washed three times in PB for 2 minutes. Permeabilized cells were then incubated or mock incubated with MVM at a MOI of 4 pfu/cell in transport buffer (TB: 20 mM Hepes KOH, pH 7.3, 110 mM KoAc, 5 mM NaOAc, 1 mM EGTA and 2 mM DTT) containing 155 kDa TRITC-labeled dextran (250 µg/ml, Sigma-Aldrich) and visualized immediately on an Olympus Fluoview FV1000 laser scanning microscope at 37°C. Where indicated the caspase inhibitors zVAD-fmk (200 µM), zDEVD-fmk (50 µM) or zVEID-fmk (50 µM) (Tocris Biosciences) were included in the last washing step and the incubation/visualization step.  2.6.3 TUNEL assay LA9 cells were grown and infected as described in section 2.4 and then fixed and labeled for double-stranded DNA breaks using a terminal deoxynucleotidyl transferase  42  dUTP nick end labeling (TUNEL) kit (Roche), according to the manufacturer’s instructions.  2.7 Enzymatic activity assays 2.7.1 Phospholipase A2 activity of MVM The PLA2 activity of MVM particles was assayed using a PLA2 Activity Kit (Cayman Chemical), according to the manufacturer’s instructions. For the PLA2-inhibition assay, 5 µg of MVM particles were incubated with 10 µM manoalide (Alexis Biochemicals) for one hour at room temperature with agitation before PLA2 activity was measured. To determine PLA2 activity under high pH conditions, 5 µg of MVM particles were exposed to 0.6 M NaOH for 5 minutes at room temperature and then where applicable incubated with 10 µM manoalide for 1 hour at room temperature with agitation before enzyme activity was determined.  2.7.2 Colorimetric assay for caspase-3 activity The caspase-3 activity of 50 µg of lysate from cells that were mock infected or infected with MVM for two hours as described in section 2.4, or treated with 1 µM staurosporine (STS, Alexis Biochemicals) for 6 hours, was measured using a colorimetric Caspase-3 Cellular Assay Kit (Biomol), according to the manufacturer’s instructions.  2.7.3 FLICA assay for caspase-3 activity LA9 cells were grown in suspension as described in section 2.2. Cells (2 x 106 cells/condition) were then mock infected or infected with MVM at an MOI of 4 for two hours at 37°C, or treated with 1 µM STS for 6 hours at 37°C. They were then labeled with carboxyfluorescein-Val-Ala-Asp-fluoromethylketone (FAM-VAD-fmk, Immunochemistry Technologies) and fixed according to the manufacturer’s instructions.  43  Flow cytometry to detect caspase activity was performed by Dr. Lixin Zhou and Andy Johnson (UBC Flow Cytometry Facility).  2.8 Western blot and 2D-DIGE 2.8.1 Western blot LA9 cells were grown and infected as described in section 2.4 and then lysed in PBS containing 0.5% NP40 and protease inhibitor mixture (Roche Applied Science) on ice for 30 minutes. Lysates were cleared by centrifugation at 16 000 x g for 5 minutes at 4°C. The supernatants were mixed with Laemmli sample buffer and aliquots with equal amounts of protein were loaded on a SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose membrane, and proteins present in the lysates were detected by Western blot using antibodies for lamin A/C (1:200) or lamin B (1:200).  2.8.2 Two-dimensional differential in gel electrophoresis Monolayers of LA9 cells were mock infected or infected with MVM at an MOI of 2 as described in section 2.4. Cells were collected by scraping, snap frozen, and sent to Applied Biomics Inc. for 2D-DIGE analysis. Briefly, cell lysates were prepared, protein samples from mock- and MVM-infected cells were labeled with fluorophores of different colors (green for mock-infected cells and red for MVM-infected cells), and samples were loaded onto the same SDS-polyacrylamide gel. Proteins were resolved in two dimensions using isoelectric focusing in the first dimension and molecular weight in the second. Spots selected for identification were excised from the gel and identified by matrix assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry.  44  2.9 Immunoprecipitation of MVM and mass spectrometry For immunoprecipitation (IP) of MVM, antibody for intact MVM capsids (clone D4H1) was first coupled to NHS-activated sepharose beads (GE Healthcare) according to the manufacturer’s instructions. For optimal coupling, 250 µg of antibody were coupled to 250 µl of sepharose beads. For IP of MVM, 30 µl of beads were incubated or mock incubated with 1 µg MVM, overnight at 4°C on a rocker. Beads were then washed three times with lysis buffer (150 mM NaCl, 50 mM Tris, 1 mM EDTA, 1% NP40, pH 7.2). Meanwhile, LA9 cells were lysed in lysis buffer containing protease inhibitor mixture (Roche Applied Science) and pre-cleared by overnight incubation at 4°C with 30 µl inactivated sepharose beads per 1 mg of lysate (in the absence of antibody). Next, the MVM- and mock-incubated beads were each incubated with 5 mg of pre-cleared lysate, overnight at 4°C. This was followed by four washes with lysis buffer and two washes with PBS. The beads were dried, and bound protein was eluted by incubation with 30 µl of Laemmli sample buffer at 96°C for 5 minutes.  Samples were then given to Isabelle Kelly (Foster Lab, Dept. of Biochemistry and Molecular Biology) for analysis by mass spectrometry. Samples were loaded into a SDSpolyacrylamide gel, and the protein bands were cut and ingel digested with trypsin. The peptides were extracted from the gel and isotopically labeled. A search was performed against the IPI mouse protein database and the Swiss Prot Virus protein database.  45  3. Effect of MVM on the Host Nuclear Envelope* 3.1 Introduction The nuclear entry mechanism of parvoviruses – and non-enveloped viruses in general – is currently not well understood. We previously attempted to study this problem by microinjecting the parvovirus MVM into the cytoplasm of Xenopus oocytes followed by visualization using transmission EM (Cohen & Panté, 2005). Microinjection of Xenopus oocytes is a very good system for studying nuclear transport, because the oocytes yield nuclear membranes with an extremely high density of NPCs. Both membranes and NPCs are very well preserved for EM. In addition, this system has been used successfully to study the nuclear import of other viruses. As described in section 1.3.4, microinjection of recombinant HBV capsids into the cytoplasm of Xenopus oocytes revealed that these capsids can cross the NPC intact (Rabe et al., 2003). In contrast, we did not observe MVM capsids associated with or traversing the NPCs of microinjected oocytes. Instead, we found that MVM caused disruption of the NE in a time- and concentration- dependent manner. Microinjection experiments in which the NPCs were blocked with the lectin wheat germ agglutinin (WGA) showed that NE disruption induced by MVM was independent of the NPC. To address the question of whether this effect of MVM was specific to the NE we incubated purified organelles with MVM. Visualization by EM revealed that MVM did not affect all intracellular membranes. Based on these results, we proposed that MVM may enter the nucleus using a unique mechanism that is independent of the NPC and involves disruption of the NE and entry through the resulting breaks. In this chapter I continue to test the hypothesis that autonomous *  A version of part of this chapter has been published: • Cohen, S., Behzad, A.R., Carroll, J.B., and Panté, N. (2006). Parvoviral nuclear import: bypassing the host nuclear transport machinery. Journal of General Virology 87: 32093213.  46  parvoviruses disrupt the NE. I investigated the ability of parvoviruses other than MVM to induce NE disruption. I also tested the hypothesis that MVM disrupts the host NE and nuclear lamina of infected fibroblast cells, which is more physiologically relevant than microinjected Xenopus oocytes.  3.2 Results 3.2.1 Microinjection of MVM into Xenopus oocytes causes disruption of the nuclear envelope In order to confirm our previous results, Xenopus oocytes were mock injected or injected with MVM, and their NEs were examined by transmission EM. As before, MVM capsids were not observed in transit through NPCs. Instead, we observed that MVM caused disruption of the NE (Fig. 3-1). Mock-injected oocytes yielded NEs with intact membranes. However, in MVM-injected oocytes disruption of the NE was observed as early as 30 minutes post injection. At this time, small disruptions of about 100 nm in diameter were observed only in the ONM. By one hour post injection, the size of the ONM disruptions had increased, and tiny disruptions of the INM were also occasionally present. At two hours post injection, the ONM disruptions had reached a maximum diameter of about 200 nm, and INM disruptions were often associated with the ONM disruptions. These are results are consistent with those we have previously published (Cohen & Panté, 2005).  3.2.2 Other parvoviruses also disrupt the nuclear envelope We next set out to examine whether this ability to disrupt the NE is particular to MVM, or rather whether it is a feature common to multiple species of parvovirus. To test this, the autonomous parvoviruses H1, which infects rats, and CPV, which infects dogs, were microinjected into Xenopus oocytes. The NEs of injected oocytes were visualized by 47  Figure 3-1: MVM induces disruption of the NE in microinjected Xenopus oocytes. Views of NE cross-sections with adjacent cytoplasm (c) and nucleus (n) from Xenopus oocytes that have been mock injected or injected with MVM. Two representative NE views are shown for each condition. After injection oocytes were incubated for 30 minutes, one hour or two hours at room temperature and processed for embedding and thin section EM. Brackets indicate disruptions in the NE caused by MVM. Scale bar, 100 nm.  48  transmission EM at four hours post injection, in order to maximize the ability to detect NE disruption. While mock-injected oocytes yielded intact NEs, disruption of the NE was observed in both H1- and CPV-injected oocytes (Fig. 3-2). It appeared that the NE disruptions induced by CPV were less severe, and more often restricted to the ONM, than those induced by MVM or H1. However, the different viruses were each purified and quantified in a different laboratory, by slightly different methods, making a quantitative comparison difficult. Nevertheless, these results indicate that the ability to induce disruption of the host NE is a feature common to multiple species of parvovirus.  3.2.3 MVM disrupts the nuclear envelope and alters the nuclear morphology of infected fibroblast cells After observing that MVM caused disruption of the NE when it was microinjected into Xenopus oocytes, we went on to investigate whether the same effect occurred in MVMinfected mouse fibroblast cells. EM analysis revealed that MVM infection was associated with dramatic alterations in nuclear morphology (Fig. 3-3). While the nuclei of mockinfected cells were round with smoothly delineated borders, the nuclei of MVM-infected cells became increasingly irregular in shape with time post infection. Most micrographs of infected cells contained multiple amorphous invaginations of the NE. This irregularity was apparent as early as one hour post infection, but became even more dramatic at two and four hours post infection. In addition to changes in nuclear shape, we observed alterations in chromatin structure after infection with MVM. While in mock-infected cells densely staining chromatin was limited to a small area (the nucleolus), in MVM-infected cells densely staining chromatin was more abundant, and was present throughout the nucleus at four hours post infection (Fig. 3-3).  49  Figure 3-2: The parvoviruses H1 and CPV also induce NE disruption. Views of NE cross-sections with adjacent cytoplasm (c) and nucleus (n) from Xenopus oocytes that have been mock injected or injected with H1 or CPV. Two representative NE views are shown for each condition. After injection oocytes were incubated for four hours at room temperature and processed for embedding and thin section EM. Brackets indicate disruptions in the NE caused by MVM. Scale bar, 100 nm.  50  Figure 3-3: MVM infection causes alterations in nuclear morphology. LA9 cells were mock infected or infected with MVM at an MOI of 4 and prepared for thin-section EM one, two, or four hours after infection. Cells infected with MVM exhibited dramatic alterations in nuclear shape. Scale bar, 2 µm. (Figure originally published by Cohen et al., 2006. Copyright Society for General Microbiology).  51  High magnification EM analysis showed ultra-structural changes in the NE of MVMinfected cells. At one hour and two hours post infection we observed numerous invaginations of the NE (Fig. 3-4), and viral particles often seemed to be associated with the NE inside these invaginations. We also observed distension of the ONM and INM (Fig. 3-4, top right), and disruptions of the NE that were similar to those observed in Xenopus oocytes (Fig. 3-4, bottom right). These disruptions increased in size and frequency with time after infection (Fig. 3-5a). Similar to in microinjected Xenopus oocytes, NE disruptions first appeared in the ONM at one hour post infection, and later affected both the inner and outer nuclear membranes. In addition, we again noticed changes in chromatin organization at the NE in MVM-infected cells. In mock-infected cells chromatin is densely packed at the nuclear face of the NE, except in proximity to NPCs. In MVM-infected cells, however, chromatin distribution at the NE was sparse and disrupted.  In order to quantify membrane damage, micrographs of regions of approximately 2.0 – 3.0 µm from 30 different cells were studied. For each region, the number of breaks on the ONM was counted, and the length of these breaks measured. The total length of the breaks was divided by the length of the NE on the micrographs (2.0 – 3.0 µm) to find the proportion of membrane damaged in each micrograph. Break-length and proportion of membrane damaged increased with time, as indicated in Figs. 3-5b and 3-5c. The disruptions of the ONM observed in mock-infected cells were small and rare, and likely represent junctions where the NE is continuous with the endoplasmic reticulum, which occasionally appear as breaks in thin section EM. Consistent with this explanation, no breaks were observed in the INM of mock-infected cells.  52  Figure 3-4: MVM infection causes alterations in nuclear morphology. LA9 cells were mock infected or infected with MVM at an MOI of 4 and prepared for thin-section EM one or two hours after infection. MVM-infected cells exhibited invaginations of the NE, distension of the ONM and INM, and disruption of the NE (indicated by brackets). The cytoplasm is indicated by c, nucleus by n. Scale bar, 200 nm. (Figure originally published by Cohen et al., 2006. Copyright Society for General Microbiology).  53  Mock  (a)  c  500  (c)  400 300 200 100 0  Mock  1 hour  2 hours  4 hours  Proportion of ONM damaged  (b)  Length of ONM Breaks (nm)  4 hours  2 hours  1 hour  n  0.3 0.2 0.1 0  Mock  1 hour  2 hours  4 hours  Figure 3-5: MVM induces disruption of the NE in infected fibroblast cells. (a) LA9 cells were mock infected or infected with MVM at an MOI of 4 and prepared for thinsection EM one hour, two hours, or four hours after infection. While mock-infected cells yielded intact nuclear membranes, MVM-infected cells showed disruptions (indicated by brackets) in the NE with dimensions that increased with time. c, cytoplasm, n, nucleus. Scale bar, 100 nm. (b) Bar graph of the length of the ONM breaks measured from EM cross-sections of cells from experiments performed as indicated in (a). (c) Bar graph of the proportion of ONM damage found in our micrographs. This was calculated as the length of the ONM breaks divided by the length of the NE on micrographs (2.0 – 3.0 µm) from EM cross-sections of cells from experiments performed as indicated in (a-d). Each bar graph (b-c) shows the mean value and standard error measured for 30 micrographs examined for each condition. (Figure originally published by Cohen et al., 2006. Copyright Society for General Microbiology). 54  3.2.4 Timing of nuclear envelope disruption is consistent with entry of MVM into the nucleus To examine the distribution of MVM in the cell at the time of NE disruption, mock- and MVM-infected cells were visualized by immunogold EM at two hours post infection. A mouse anti-MVM monoclonal antibody which recognizes a discontinuous epitope specific for the intact capsid (Lombardo et al., 2000, Lopez-Bueno et al., 2003) was used. Although gold was very rarely observed in mock-infected cells, 10-nm gold particles – indicating the location of intact MVM capsids – were observed in multiple locations throughout infected cells. Gold particles were often observed near the plasma membrane (Figs. 3-6a and 3-6b) and throughout the cytoplasm. Occasionally these gold particles were associated with more electron-dense structures (Fig. 3-6b), possibly representing endocytic compartments. In addition, a small proportion of gold particles were observed at the cytoplasmic side of the NE (Fig. 3-6c), in the perinuclear space between the ONM and INM (Fig. 3-6c), or in the nucleus (Fig. 3-6d). This suggests that the timing of MVM-induced NE disruption is consistent with the timing of entry of MVM into the nucleus. The implications of this result are more fully explored in Chapter 5. In addition, this data is consistent with reports that nuclear entry of parvoviruses is an inefficient process. Unfortunately, NE ultrastructure could not be carefully examined in immunogold-labeled samples, since the osmium tetroxide fixation step necessary to clearly visualize membranes was not included in the preparation of the samples because it destroys the antigenicity of proteins.  55  Figure 3-6: Immunogold localization of MVM capsids at two hours post infection. LA9 cells were infected with MVM at an MOI of 4, prepared for thin-section EM two hours after infection, and immunolabeled with anti-capsid antibody and 10 nm goldconjugated secondary antibody. Gold particles (indicated by arrows) were observed near the plasma membrane (a, b), at the NE (c), and in the nucleus (d). pm, plasma membrane, c, cytoplasm, n, nucleus. Scale bar, 200 nm.  56  3.2.5 MVM disrupts the nuclear lamina of infected fibroblast cells We next asked whether MVM infection has an effect on the host nuclear lamina, the protein meshwork that underlies the NE. We reasoned that disruption of the NE would be associated with disruption of the nuclear lamina as well. To examine this, we used double immunolabeling fluorescence microscopy. Lamin A/C was immunolabeled to visualize the nuclear lamina, and intact MVM capsids were labeled with an anti-MVM antibody. In addition to allowing us to visualize the nuclear lamina, which is difficult to observe by EM, this technique allowed us to better characterize the distribution of MVM throughout the cell at different time points after infection, since whole cells could be visualized rather than the 50-nm thin sections obtained for EM and described above.  The distribution of MVM and lamin A/C in the cell was examined at 0 minutes, 10 minutes, 30 minutes, one hour and two hours post infection (Fig 3-7). When cells were fixed and prepared for IF microscopy immediately after infection, very little MVM entered the cell. At 10 minutes post infection, MVM immunostaining displayed a punctate pattern at the cell periphery, likely representing the uptake of MVM into endosomes. By 30 minutes post infection, the MVM staining pattern was still punctate, but MVM-containing compartments were clustered at one pole of the nucleus. The asymmetrical perinuclear accumulation of MVM we observed is similar to that previously reported for MVM and other parvoviruses (Bartlett et al., 2000, Ros & Kempf, 2004, Vihinen-Ranta et al., 1998), and probably represents accumulation of MVM in late endosomes near the microtubule organizing centre. At one and two hours post infection, MVM capsids remained localized predominantly on one side of the nucleus, but the staining pattern became less punctate and more dispersed. This change in staining is likely due to the escape of capsids from endocytic compartments to the cytoplasm.  57  Figure 3-7: MVM infection causes disruption of the nuclear lamina. LA9 cells were infected with MVM at an MOI of 4 and prepared for indirect IF microscopy at 0 minutes, 10 minutes, 30 minutes, one hour or two hours post infection. For IF microscopy the cells were labeled with anti-lamin A/C (green) and anti-MVM (red) antibodies, and with DAPI to detect DNA (blue). At the later time points gaps appeared in the in the lamin A/C immunostaining of MVM-infected cells (indicated by arrows). Scale bar, 5 µm.  58  In addition, we also observed alterations in the nuclear lamin A/C immunostaining of MVM-infected cells. Fibroblast cells infected with MVM for 0-30 minutes displayed normal continuous nuclear rim staining of lamin A/C. However, consistent with our EM results, we found large abnormal gaps in the lamin A/C immunostaining of MVM-infected cells at one and two hours post infection. The gaps in the nuclear lamina of infected cells coincided with the location of the immunolabeling of MVM. While none of the mockinfected cells exhibited gaps in lamin A/C immunostaining, approximately 20% of MVMinfected cells showed gaps in lamin A/C that were detectable in our IF images at one and two hours post infection (1 hour: 20 ± 6%; 2 hours: 25 ± 6%).  Lastly, we examined the nuclear lamina by IF microscopy at later time points during infection. Unlike at two hours post infection, at 21 hours post infection no nuclear rim gaps could be seen in the lamin-A/C immunostaining of MVM infected cells (Fig. 3-8), suggesting that the nuclear lamina is repaired later during infection. The massive amount of MVM immunostaining observed at 21 hours post infection indicates that synthesis of progeny capsids has occurred by this time. These results indicate that the MVM-induced NE disruptions that we observe early during infection are a local, transient event.  3.3 Discussion The data in this chapter further characterize the effect of MVM on the host NE. Microinjection experiments revealed that the ability to induce disruption of the NE is not specific to MVM, but is a property shared by at least three autonomous parvoviruses, MVM, H1 and CPV (Fig. 3-2). Previous work by Hansen and colleagues showing that AAV can enter purified nuclei independently of the NPC (Hansen et al., 2001) suggests  59  Figure 3-8: MVM-induced disruption of the nuclear lamina is transient. LA9 cells were infected with MVM at an MOI of 4 and prepared for indirect IF microscopy at two or 21 hours post infection. The cells were labeled with anti-lamin A/C (green) and anti-MVM (red) antibodies, and with DAPI to detect DNA (blue). Gaps in the lamin A/C immunostaining of MVM-infected cells at two hours post infection are indicated by arrows. Scale bar, 5 µm.  60  that non-autonomous parvoviruses may share this feature as well. Importantly, we have now shown that disruption of the NE occurs during infection of mouse fibroblast cells with MVM (Fig. 3-5), indicating that this observation is not an artifact of our microinjection system. In addition, immunogold EM revealed that the timing of entry of MVM capsids into the nucleus is consistent with the timing of NE disruption (Fig. 3-6). Thus, MVM-induced NE disruption may play a role in facilitating nuclear entry of the viral genome. This data also supports the notion that the viral entity entering the nucleus is in fact an intact capsid, since antibodies recognizing a capsid structure not present on individual capsid proteins (Lombardo et al., 2000, Lopez-Bueno et al., 2003) were detected in the nucleus. IF microscopy suggests that in addition to disruption of the NE, the underlying nuclear lamina is also disrupted in MVM-infected cells (Fig. 3-7). In addition, based on the pattern of MVM immunostaining, it seems that the timing of NE disruption coincides with escape of capsids from endocytic compartments. This suggests that MVM mediates NE disruption through an interaction occurring in the cytoplasm or at the NE, rather than by inducing signaling from the plasma membrane or within an endosome. The fact that microinjected virus can also induce NE disruption is in agreement with this conclusion. Lastly, MVM-induced lamina disruption was no longer detectible at 21 hours post infection (Fig. 3-8), indicating that this disruption is a transient event which likely plays an important role in the viral replication cycle, rather than a nonspecific response to infection.  61  4. Mechanism of MVM-Induced Nuclear Envelope Disruption* 4.1 Introduction To follow up on the results described in Chapter 3, we next investigated the molecular mechanism of MVM-induced NE and nuclear lamina disruption. It has been shown that the MVM capsid has PLA2 activity, which is necessary for escape of virions from endosomes (Farr et al., 2005). Since the viral PLA2 is the only known enzymatic domain on the MVM capsid, we hypothesized that viral PLA2 activity may be required for MVMinduced NE disruption. If the viral PLA2 is not required, an alternate hypothesis is that MVM hijacks a cellular program for NEBD such as those used during mitosis or apoptosis. As described in section 1.5, the main enzymes involved in these two processes are kinases and caspases (Fahrenkrog, 2006, Guttinger et al., 2009). This chapter describes an investigation of the involvement of both viral PLA2 activity and host enzymes in MVM-induced NE disruption.  In addition, we have previously shown that the ability of MVM to disrupt membranes does not apply to all organelles (Cohen & Panté, 2005). A unique feature of the ONM is the presence of Klarsicht/Anc-1/Syne-1 homology (KASH) domain proteins, called nesprins in mammalian cells (reviewed by Burke & Roux, 2009). These large proteins are localized to the ONM through interaction with the INM proteins Sun1 and Sun2 (Crisp et al., 2006, Padmakumar et al., 2005). Nesprins and Sad1p/Unc-84 homology *  A version of this chapter has been submitted for publication: • Cohen, S., Marr, A.K., Garcin, P., and Panté, N. Nuclear envelope disruption involving host caspases plays a role in the parvovirus replication cycle.  62  (SUN) domain proteins are thought to form a bridge linking the nucleoskeleton to the cytoskeleton; this structure is referred to as the linker of the nucleoskeleton and cytoskeleton (LINC) complex (Crisp et al., 2006). We hypothesized that nesprins may play a role in the specificity of MVM-induced membrane disruption for the NE. Thus, the role of ONM proteins in MVM-mediated NE disruption was also tested.  4.2 Results 4.2.1 MVM-induced nuclear envelope disruption does not require viral phospholipase A2 activity We began by characterizing the PLA2 activity of MVM using an enzymatic activity assay (Fig. 4-1). While untreated MVM capsids exhibited PLA2 activity of 0.029 ± 0.003 µmol/min/ml, the PLA2 activity of capsids treated with the inhibitor manoalide was reduced to 0.004 ± 0.002 µmol/min/ml, representing an approximately 7-fold reduction. However, these untreated MVM capsids correspond to a conformation where the VP1 unique portion, which contains the PLA2 domain, is buried within the capsid (Cotmore et al., 1999). It has previously been shown that exposing MVM capsids to high pH results in a dramatic boost in PLA2 activity (Farr et al., 2005). Indeed, when capsids were exposed to 0.6 M NaOH, PLA2 activity increased to 0.089 ± 0.015 µmol/min/ml, or 3-fold more than untreated. Treatment of MVM capsids exposed to high pH with manoalide brought PLA2 activity back down to 0.011 ± 0.003 µmol/min/ml (Fig. 4-1).  We next tested whether viral PLA2 activity was necessary for MVM-induced NE disruption using microinjection in Xenopus oocytes followed by EM. It was necessary to use microinjection rather than infection because under conditions of PLA2 inhibition MVM capsids are unable to escape from endosomes (Farr et al., 2005), preventing  63  Figure 4-1: Characterization of the PLA2 activity of MVM. Colorimetric assay for PLA2 activity of untreated and manoalide-treated MVM. Bar graph shows the mean values and standard deviation measured from three independent experiments. **, p<0.01 (unpaired Student’s t-test).  64  access to the NE. Therefore, oocytes were injected with MVM containing an H42R point mutation within the PLA2 active site of the capsid protein VP1 (H24R-MVM). This mutation has been extensively characterized and shown to completely abrogate the PLA2 activity of the MVM capsid (Farr et al., 2005). As controls, oocytes were mock injected with buffer or injected with wild-type MVM. Less MVM was injected than in previously published experiments (Cohen & Panté, 2005) in order to more closely approximate the MOI used in our infection experiments (MOI of 4). While the mockinjected oocytes yielded intact nuclear membranes, the MVM-injected oocytes showed frequent ONM disruptions of about 50 – 100 nm at two hours post injection (Fig. 4-2a). The H42R-MVM-injected oocytes also displayed frequent NE disruptions, suggesting that the viral PLA2 activity is not necessary for MVM-induced NE disruption to occur. To confirm these results, we also performed experiments inhibiting the PLA2 activity of wild type MVM with manoalide. MVM treated with manoalide was injected into Xenopus oocytes. Consistent with the results from H42R-MVM, the manoalide-treated MVM also disrupted the NE of the injected oocytes (Fig. 4-2a).  To better quantify nuclear membrane damage, three Xenopus oocytes were examined for each condition. For each oocyte micrographs of 10 NE regions of approximately 1.5 µm were studied. The number of disruptions in the ONM was counted, the length of the disruptions measured, and the total length of the disruptions was divided by the length of the ONM in order to find the proportion of the ONM damaged (Fig. 4-2b). In mockinjected oocytes the proportion of membrane damaged was 1.0 ± 0.4% - in fact the gaps observed in the ONM of mock-injected cells probably do not correspond to membrane disruptions, but are likely junctions where the ONM is continuous with the endoplasmic reticulum, which can appear as disruptions in thin section EM. In contrast, in MVM-  65  Figure 4-2: MVM-induced NE disruption does not involve viral PLA2 activity. (a) Views of NE cross-sections with adjacent cytoplasm (c) and nucleus (n) from Xenopus oocytes that have been mock injected or injected with wild-type MVM, H42R-MVM, or manoalide-treated MVM. Two representative NE views are shown for each condition. After injection oocytes were incubated for two hours at room temperature and processed for embedding and thin section EM. Brackets indicate disruptions in the NE caused by MVM. Scale bar, 100 nm. (b) Bar graph of the proportion of NE damage calculated as the length of the ONM breaks divided by the total length of the ONM from electron micrographs of experiments performed as indicated in (a). Shown are the mean values and standard error measured for 30 micrographs obtained from three different oocytes examined for each condition. **, p<0.01, as compared to MVM (unpaired Student’s ttest).  66  injected oocytes the proportion of ONM damaged was 4.7 ± 0.6%, while in H42R-MVMinjected oocytes it was 4.6 ± 0.5%, and in oocytes injected with manoalide-treated MVM it was 4.2 ± 0.6%. This supports the conclusion that the viral PLA2 is not responsible for MVM-induced NE disruption.  4.2.2 Establishment of a digitonin-permeabilized cell assay to identify inhibitors of MVM-induced nuclear envelope disruption Since the viral PLA2 was found not to be required for MVM-induced NE disruption, we hypothesized that MVM might be hijacking a cellular program for NEBD normally used during mitosis or apoptosis. To test the involvement of various host enzymes in MVMinduced NE disruption, we modified a previously established in vitro nuclear disassembly system used to study mitotic NEBD (Muhlhausser & Kutay, 2007). In this assay, HeLa cells expressing GFP-LAP2β as an NE marker were semipermeabilized with digitonin, which permeabilizes the plasma membrane but not the NE. Semipermeabilized cells were then incubated with MVM at an MOI of 4, and MVM-induced NE permeabilization was visualized by monitoring nuclear influx of a large (155-kDa) TRITC-labeled dextran (Fig. 4-3a). In the absence of MVM (mock-incubated cells), the NE remained impermeable to the dextran over a 30 minute time period, and the nuclei appeared black (Fig. 4-3a, Mock). However, in the presence of MVM the TRITC-dextran entered the nucleus (Fig. 4-3a, MVM). Thus, similar to MVM-infected cells, MVM disrupts the NE in the semipermeabilized cell system.  4.2.3 Caspases are involved in MVM-induced nuclear envelope disruption We next performed the semipermeabilized cell assay for MVM-induced NE permeabilization in the presence of an array of inhibitors of host cell kinases and proteases implicated in mitotic or apoptotic NEBD (data not shown). Of the inhibitors  67  Figure 4-3: MVM-induced NE disruption in semipermeabilized cells depends on caspases. (a) HeLa cells stably expressing GFP-LAP2β (green) were permeabilized with digitonin and then mock incubated or incubated with MVM (MOI of 4) in the presence of 155 kDa TRITC dextran (red). In the absence of MVM the NE remained impermeable to the dextran and the nuclei appear black. However, in the presence of MVM the NE was disrupted and the TRITC-dextran entered the nucleus. (b) GFP-LAP2β HeLa cells were assayed for MVM-induced NE disruption as indicated in (A) in the presence of MVM (MOI of 4) and the pancaspase inhibitor zVAD-fmk (200 µM), the caspase-3 inhibitor zDEVD-fmk (50 µM), or the caspase-6 inhibitor Z-VEID-fmk (50 µM). While zVAD-fmk and zDEVD-fmk completely inhibited dextran leakage into the nucleus, zVEID-fmk did not. Scale bar, 5 µm. 68  tested, addition of the pancaspase inhibitor zVAD-fmk, a cell-permeable broad spectrum inhibitor of caspases (Ekert et al., 1999), most efficiently inhibited MVM-induced NE permeabilization. Caspases are proteases known to be responsible for cleaving NE proteins such as nucleoporins and nuclear lamins during apoptotic NEBD (Fahrenkrog, 2006, Robertson et al., 2000). As illustrated in Fig. 4-3b, like the nuclei of mockincubated semipermeabilized cells, the nuclei of semipermeabilized cells incubated with MVM in the presence of 200 µM zVAD-fmk remained impermeable to the dextran over a 30 minute time period.  We subsequently attempted to narrow down which caspase might be involved. Nuclear lamins are known to be cleaved by caspase-3 and caspase-6 during apoptosis (Orth et al., 1996, Slee et al., 2001, Takahashi et al., 1996). Thus, we decided to test the caspase-3 inhibitor zDEVD-fmk and the caspase-6 inhibitor zVEID-fmk in our semipermeabilized cell assay (Ekert et al., 1999, Thornberry et al., 1997). As illustrated in Fig. 4-3b, while the caspase-3 inhibitor zDEVD-fmk (50 µM) completely inhibited the influx of fluorescent dextran into the nucleus in the presence of MVM, the caspase-6 inhibitor zVEID-fmk (50 µM) did not. The lower concentration was used to increase inhibitor specificity. Therefore, it seems that caspase-3 is especially important for MVMinduced NE permeabilization.  We wanted to confirm that the increase in NE permeability that we observed in our semipermeabilized cell assay correlated with the NE disruptions we previously observed by EM, and was not due to some other effect of the virus. Therefore, we mock injected or injected MVM into Xenopus oocytes in the absence or presence of the caspase inhibitors tested above. As in previous microinjection experiments, the mock-injected oocytes yielded intact nuclear membranes, while the MVM-injected oocytes showed 69  Figure 4-4: Caspase inhibitors prevent MVM-induced NE disruption in microinjected Xenopus oocytes. (a) Views of NE cross-sections with adjacent cytoplasm (c) and nucleus (n) from Xenopus oocytes that have been mock injected, injected with MVM, or co-injected with MVM and one of three caspase inhibitors: the pancaspase inhibitor zVAD-fmk, the caspase-3 inhibitor zDEVD-fmk, or the caspase-6 inhibitor zVEID-fmk. Two representative NE views are shown for each condition. After injection oocytes were incubated for two hours at room temperature and processed for embedding and thin section EM. Brackets indicate disruptions in the NE caused by MVM. While zVAD-fmk and zDEVD-fmk completely inhibited disruption of the NE, zVEID-fmk did not. Scale bar, 100 nm. (b) Bar graph of the proportion of NE damage calculated as the length of the ONM breaks divided by the total length of the ONM from electron micrographs of experiments performed as indicated above. Shown are the mean values and standard error measured for 30 micrographs obtained from three different oocytes examined for each condition. **, p<0.01, as compared to MVM (unpaired Student’s t-test).  70  frequent ONM disruptions at two hours post injection (Fig. 4-4a). Co-injection of MVM with either the pancaspase inhibitor zVAD-fmk or the caspase-3 inhibitor zDEVD-fmk completely prevented these NE disruptions, while co-injection of MVM with the caspase6 inhibitor zVEID-fmk did not (Fig. 4-4a). Again, nuclear membrane damage was quantified as described above (Fig. 4-4b). In mock-injected oocytes the proportion of ONM damaged was 0.6 ± 0.2%. In contrast, in MVM-injected oocytes the proportion of ONM damaged was 4.1 ± 0.6%. Co-injection of MVM with zVAD-fmk, zDEVD-fmk, or zVEID-fmk reduced the proportion of ONM damaged to 0.7 ± 0.3%, 0.8 ± 0.3%, and 2.0 ± 0.4%, respectively. Clearly, as in the semipermeabilized cell assay, the pancaspase and caspase-3 inhibitors were more effective at inhibiting MVM-induced NE disruption than was the caspase-6 inhibitor. In addition we conclude that the changes in NE permeability that we observed in the semipermeabilized cell assay correlate with the nuclear membrane disruptions visualized by EM, since both are observed under the same conditions.  Having found that caspases were implicated in MVM-induced NE disruption in semipermeabilized cells and microinjected Xenopus oocytes, we set out to examine the effect of caspase inhibitors on nuclear lamin immunostaining in MVM-infected mouse fibroblast cells using IF microscopy. As in Fig. 3-6, while mock-infected cells displayed continuous nuclear-rim immunostaining of lamin A/C, abnormal gaps were observed in the lamin-A/C immunostaining of MVM-infected cells at two hours post infection (Fig. 45). These gaps coincided with the location of the virus in the cell. In addition, a “cloud” of disassembled lamin proteins was observed around these nuclear-rim gaps. However, when cells were infected with MVM in the presence of the pancaspase inhibitor zVADfmk, gaps in the lamin-A/C immunostaining were no longer observed (Fig. 4-5).  71  Figure 4-5: Caspase inhibitors prevent nuclear lamina disruption in MVM-infected cells. LA9 cells were mock infected or infected with MVM (MOI of 4) for two hours in the presence or absence of 200 µM of the pancaspase inhibitor zVAD-fmk, the caspase-3 inhibitor zDEVD-fmk, or the caspase-6 inhibitor zVEID-fmk. Cells were immunolabeled with antibodies against lamin A/C (green) and MVM (red). DNA was detected with DAPI (blue). While mock-infected cells showed continuous nuclear rim-staining of the lamin A/C, gaps were seen in the lamin-A/C immunostaining of MVM-infected cells (indicated by arrows). zVAD-fmk and zDEVD-fmk completely inhibited the MVM-induced nuclear lamina disruption, but zVEID-fmk did not. Scale bar, 5 µm.  72  Moreover, like zVAD-fmk, the caspase-3 inhibitor zDEVD-fmk also prevented the appearance of gaps in the nuclear lamin immunostaining of MVM-infected cells, while the caspase-6 inhibitor zVEID-fmk did not (Fig. 4-5). These results indicate that caspase-3 is involved in MVM-mediated NE disruption during infection of live cells. In addition, we observed differences in the localization of MVM in the presence or absence of caspase inhibitors (Fig. 4-5). In infected cells untreated with inhibitor, or treated with the caspase-6 inhibitor zVEID-fmk, the virus displayed a fairly dispersed localization at two hours post infection. However, in infected cells treated with pancaspase or caspase3 inhibitor, virus capsids appeared to accumulate at the NE, suggesting that inhibition of MVM-induced NE disruption prevented nuclear entry of the virus, and perhaps also virus disassembly.  4.2.4 Caspases are involved in MVM-induced lamin cleavage in infected cells Since caspases are capable of cleaving lamins, and because we observed disruption of the nuclear lamina in MVM-infected cells, we next asked whether nuclear lamins were cleaved in MVM-infected cells. The mammalian nuclear lamina is composed of A/C-type lamins, which are products of alternative splicing, as well as lamin B1 and lamin B2, encoded by two additional genes (Dechat et al., 2008). We examined lysates from cells that were mock infected or infected with MVM for two hours for the presence of lamin cleavage products. Western blot with an anti-lamin A/C antibody yielded two bands of the predicted molecular weights, but no cleavage products were observed (Fig. 4-6a).  In contrast, Western blot with an anti-B-lamin antibody revealed the presence of a 16 kDa cleavage product in lysates from MVM-infected but not mock-infected cells (Fig. 46b, lanes mock and MVM, band 5). When cells were infected with MVM in the presence  73  Figure 4-6: B-type lamins, but not A/C-type lamins, are cleaved in a caspasedependent manner in MVM-infected cells. (a) Western blot for A/C-lamins in LA9 cells that were mock infected or infected with MVM (MOI of 4) for two hours in the presence or absence of 100 µM of the pancaspase inhibitor zVAD-fmk, the caspase-3 inhibitor zDEVD-fmk, or the caspase-6 inhibitor zVEID-fmk. Molecular weights are indicated on the left. (b) Western blot for B-lamins in LA9 cells prepared as in (a). Molecular weights are indicated on the left. Numbers on the right indicate the corresponding predicted cleavage product in (d) for each band. (c) Quantification of the band intensity of the 16 kDa cleavage product shown in (b). The bands were quantified using Image Pro Plus, and normalized so that mock was 0 and MVM was 100%. Bar graph shows the mean value and standard error from three films. **, p<0.01, as compared to MVM (unpaired Student’s t-test). (d) Schematic showing putative caspase-3 (DEVD) and caspase-6 (VEVD) cleavage sites in the B-type lamins, as well as the sizes of predicted cleavage products. Numbers in brackets indicate the corresponding band in (b).  74  of the pancaspase inhibitor zVAD-fmk or the caspase-3 inhibitor zDEVD-fmk, the amount of this 16 kDa cleavage product was dramatically reduced (Fig. 4-6b, lanes MVM+zVAD and MVM+zDEVD). In cells infected in the presence of the caspase-6 inhibitor zDEVD-fmk the abundance of the 16 kDa cleavage product was also reduced, but not as much as in the zVAD or zDEVD lanes (Fig. 4-6b, lane MVM+zVEID). Quantification of the band intensity from three separate experiments revealed that the 16 kDa band in the zVAD lane was reduced to 1 ± 2% of the band intensity for the MVM lane, while the band in the zDEVD lane was reduced to 4 ± 5%, and the band in the zVEID lane was reduced to 23 ± 13% (Fig. 4-6c). These results indicate that the 16 kDa band observed in MVM-infected cells is a caspase-3 cleavage product of either lamin B1 or lamin B2. Examination of the amino acid sequences of lamin B1 and lamin B2 (available in the GenBank database, http://www.ncbi.nlm.nih.gov/genbank/) suggested the presence of a caspase-3 consensus site (DEVD) at the appropriate location in lamin B2, but not lamin B1 (Fig. 4-6d). Thus, we propose that the 16 kDa band observed in MVM-infected cells is a caspase-3 cleavage product of lamin B2. The partial inhibition of this cleavage in MVM-infected cells treated with the caspase-6 inhibitor zVEID-fmk is likely due to non-specific effects of the inhibitor.  Cleavage at the lamin B2 DEVD site is expected to result in a 51 kDa cleavage product in addition to the observed 16 kDa fragment (Fig. 4-6d). Indeed, such a band was observed in MVM-infected, but not mock-infected cells (Fig. 4-6b, band 2). However, this band was less distinct than the 16 kDa band, and therefore difficult to quantify. Perhaps the 51 kDa lamin B2 fragment is less stable than the 16 kDa fragment, or not recognized as well by the polyclonal anti-lamin antibody which was used to detect it.  75  In addition to the 16 kDa and 51 kDa bands observed in some of the lanes, three additional bands with molecular weights of 67 kDa, 41 kDa, and 26 kDa were present in every lane (Fig. 4-6b, bands 1, 3 and 4). The 67 kDa band corresponds to full-length lamin B1 and lamin B2. Examination of the amino acid sequences of lamin B1 and lamin B2 revealed the presence of another putative caspase cleavage site in both lamins: a VEVD site resulting in cleavage products with predicted molecular weights of 26 kDa and 41 kDa (Fig. 4-6d). VEVD is very similar to the caspase-6 consensus site VEID. Thus, these two bands may correspond to caspase-6 cleavage products of the B-lamins. However, unlike the 16 kDa caspase-3 cleavage product, the amount of the caspase-6 cleavage products did not seem to be dramatically different between mock- and MVMinfected cells (Fig. 4-6b).  Our results suggest that during infection of cells with MVM, lamin B2 is cleaved by caspase-3. This then results in a change in the organization of A/C-type lamins, even though they themselves are not cleaved. The altered organization of A/C lamins is detectable by IF microscopy, as seen in Fig. 4-5.  4.2.5 Caspase-3 is not activated above basal levels in MVM-infected cells, but is relocalized Based on our results implicating caspase-3 in MVM-induced NE disruption, we next examined whether caspase-3 was activated in MVM-infected cells. A colorimetric caspase-3 activity assay revealed that there was no difference in the amount of active caspase-3 in lysates from mock-infected and MVM-infected cells at two hours post infection (Fig. 4-7). Lysates from mock-infected cells had caspase-3 activity of 150 ± 30 pmol/min/mg, while lysates from MVM-infected cells had caspase-3 activity of 130 ± 50 pmol/min/mg. This was in contrast to a positive control of lysates from cells induced to  76  Figure 4-7: Caspase-3 is not activated above basal levels in MVM-infected cells (enzymatic assay). Colorimetric assay for caspase-3 activity in lysates from mockinfected, MVM-infected (MOI of 4, two hours infection), and STS-treated cells. Bar graph shows the mean values and standard error measured for three independent experiments. **, p<0.01, as compared to mock (unpaired Student’s t-test).  77  undergo apoptosis by treatment with staurosporine (STS), which had robust caspase-3 activity of 3080 ± 190 pmol/min/mg. In an alternate approach, caspase activity of mockand MVM- infected cells was examined using fluorochrome inhibitor of caspases (FLICA) labeling, followed by flow cytometry. Unlike the enzymatic assay described above, which uses cell lysate and thus averages the caspase activity of many cells, flow cytometry allows the caspase activity of individual cells to be measured. However, this approach also revealed no difference in the amount of active caspase-3 between mockinfected and MVM-infected cells at two hours post infection (Fig. 4-8). In a representative experiment, in the mock-infected sample 61.0% of the events counted were caspasenegative cells of a normal size; 12.7% were caspase-positive cells; and 12.3% were debris. Similarly, in the MVM-infected sample 63.7% were caspase-negative cells; 10.4% were caspase-positive cells; and 12.9% were debris. This was in contrast to the STS-treated sample, in which only 30.0% of the events counted were caspase-negative cells; 15.2% were caspase-positive cells; and 33.7% were debris.  Although caspase-3 was not activated above basal levels in MVM-infected cells, both mock- and MVM-infected cells did exhibit some caspase-3 activity (Fig. 4-7 and 4-8). Caspase-3 exists as a proenzyme which must be proteolytically cleaved to become activated. Though caspase-3 is primarily cleaved and activated during apoptosis, low levels of active caspase-3 may exist at a basal state. When we visualized cleaved (activated) caspase-3 in mock- and MVM-infected cells by IF microscopy at two hours post infection, we noticed a change in the localization of cleaved caspase-3 in MVMinfected cells. While cleaved caspase-3 was completely excluded from the nucleus in mock-infected cells, in MVM-infected cells some of the cleaved caspase-3 appeared to enter the nucleus, in close proximity to the location of the virus in the cell (Fig. 4-9). This  78  Figure 4-8: Caspase-3 is not activated above basal levels in MVM-infected cells (FLICA). LA9 cells were (a) mock infected, (b) infected with MVM (MOI of 4, two hours infection), or (c) treated with STS. Cells were then labeled with FAM-VAD-fmk and analyzed using flow cytometry. The frequency histogram of the number of events (Y axis) versus fluorescein intensity (X axis) shows three peaks: cell debris occurs in the yellow region; caspase-negative cells occur in the red region; and caspase-positive cells occur in the green region.  79  Figure 4-9: Basally activated caspase-3 is relocalized in MVM-infected cells. LA9 cells were mock infected or infected with MVM at an MOI of 4 and prepared for indirect IF two hours after infection. Cells were immunolabeled with antibodies against cleaved caspase-3 (green) and MVM (red). DNA was detected with DAPI (blue). Cleaved caspase-3 was excluded from the nucleus in mock-infected cells, but entered the nucleus in MVM-infected cells. Nuclei have been demarcated in white. Scale bar, 5 µm.  80  suggests that previously cleaved, activated caspase-3 already present at low levels in the cytoplasm gains access to the nucleus because of MVM-induced nuclear membrane disruption; once inside the nucleus, caspase-3 likely plays a role in lamin cleavage and progression of NE disruption.  4.2.6 Outer nuclear membrane proteins are involved in MVM-induced nuclear envelope disruption To test whether ONM proteins are involved in mediating the specificity of MVM-induced membrane disruption for the NE, we used a strategy involving a dominant-negative form of Sun1. Expression of the soluble lumenal domain of Sun1 targeted to the perinuclear space and ER lumen (signal sequence-hemaglutinin-Sun1 lumenal-KDEL, SS–HA– Sun1L–KDEL) has been shown to cause mislocalization of nesprin 2 giant from the ONM to the ER, and distension of the outer and inner nuclear membranes (Crisp et al., 2006). Expression of this construct likely causes a similar mislocalization of the other KASH domain proteins as well.  Uninduced MC3T3 cells or MC3T3 cells induced to express SS–HA–Sun1L–KDEL were mock infected or infected with MVM for two hours, and prepared for immunolabeling fluorescence microscopy of lamin A/C and intact MVM capsids. As in LA9 cells, mockinfected MC3T3 cells displayed continuous nuclear-rim immunostaining of lamin A/C while MVM-infected MC3T3 cells displayed gaps in the lamin A/C immunostaining (Fig. 4-10). However, these gaps were completely abrogated in cells expressing SS–HA– Sun1L–KDEL (Fig. 4-10). This indicates that ONM proteins play a role in MVM-induced disruption of the nuclear lamina.  81  Figure 4-10: MVM does not induce NE disruption in cells with mislocalized ONM proteins. Un-induced MC3T3 cells or MC3T3 cells induced to express SS–HA–Sun1L– KDEL were mock infected or infected with MVM at an MOI of 4 and prepared for indirect IF microscopy at two hours post infection. The cells were labeled with anti-lamin A/C (green) and anti-MVM (red) antibodies. While mock-infected cells showed continuous nuclear rim-staining of the lamin A/C, gaps were seen in the lamin-A/C immunostaining of MVM-infected cells (indicated by arrows). Expression of SS–HA–Sun1L–KDEL completely inhibited the MVM-induced nuclear lamina disruption. Scale bar, 5 µm.  82  4.3 Discussion This chapter describes an investigation of the viral and cellular factors required for MVMinduced NE disruption. Microinjection of MVM under conditions of PLA2 inhibition revealed that viral PLA2 activity is not required for MVM-induced NE disruption (Fig. 42). This is not entirely surprising, since the parvoviral PLA2 is much more active under conditions of high calcium (Zadori et al., 2001). Thus, one would expect the PLA2 of MVM to function predominantly in an endocytic compartment; release of capsids into the cytoplasm, where the calcium concentration is extremely low, would result in dramatically reduced PLA2 activity.  Rather than using a viral enzyme, it seems that MVM utilizes a cellular mechanism for NEBD to induce NE disruption. A semi-permeabilized cell assay for MVM-induced NE disruption was used to test for the involvement of various host kinases and proteases. Of the inhibitors tested, a pan-caspase inhibitor was the most effective at preventing MVMmediated permeabilization of the NE (Fig. 4-3). Further investigation revealed that caspase-3 in particular was involved; microinjection and lamin immunostaining experiments supported this conclusion (Figs. 4-4 and 4-5). Consistent with a role for caspase-3 in MVM-induced NE disruption, we observed cleavage of lamin B2 but not of A/C-type lamins in cells infected with MVM at two hours post infection (Fig. 4-6). It has been shown that during apoptosis lamin A is cleaved by caspase-6, while a B lamin is cleaved by caspase-3 (Slee et al., 2001). Immunostaining of the nuclear lamina suggests that the cleavage of lamin B2 by caspase-3 has an impact on the organization of the A/C lamins, even though they are not cleaved (Fig. 4-5).  83  Two assays for caspase activity, an enzymatic assay (Fig. 4-7) and one based on flow cytometry of cells labeled for active caspase (Fig. 4-8), indicated that caspase-3 was not activated above basal levels in MVM-infected cells. However, basally active caspase-3 appeared to relocalize to the nucleus in MVM infected cells (Fig. 4-9). Thus, we propose a model in which MVM induces disruption of the nuclear membranes by an unknown mechanism, not involving viral PLA2 activity. This membrane disruption allows previously cleaved and activated caspases in the cytoplasm to gain access to the nucleus, where the proteases cleave lamin B2, resulting in disruption of the nuclear lamina structure and progression of NE disruptions. The initial trigger of NE disruption remains to be determined.  Intriguingly, KASH domain ONM proteins also seem to be involved in MVM-induced NE disruption, since mislocalization of these proteins by expression of a dominant-negative form of Sun1 completely inhibited lamina disruption in MVM-infected cells (Fig. 4-10). It is currently difficult to interpret these results. In the future, an additional control involving overexpression of wild-type Sun1 should be included. In addition, several KASH domain proteins have been reported to link the NE to microtubules via interaction with the motor proteins dynein or kinesin (reviewed by Burke & Roux, 2009). Thus, it is possible that mislocalizing KASH proteins impacts the organization of the microtubule network, and impairs trafficking of MVM towards the nucleus. However, it is also possible that ONM proteins play a currently unappreciated role in cellular NEBD, and that MVM hijacks this function to induce NE disruption during infection. This idea is further discussed in section 7.2.2.  84  5. Role of Nuclear Envelope Disruption in the MVM Replication Cycle* 5.1 Introduction Having identified some of the host proteins required for MVM-induced NE disruption, we next set out to investigate the role of this disruption in the parvovirus replication cycle. We first asked whether the NE disruption observed early during infection with MVM was due to the induction of apoptosis. However, after finding that apoptosis was not induced in infected cells until much later during infection, pharmacological inhibition was used to test the hypothesis that NE disruption plays a specific role in the parvovirus replication cycle. The effect of inhibiting NE disruption on gene expression and nuclear entry of MVM was tested. In addition, the compartmentalization of host proteins during infection with MVM was examined.  5.2 Results 5.2.1 Nuclear envelope disruption is not due to induction of apoptosis The involvement of caspases in MVM-induced lamin cleavage and NE disruption (Chapter 4) suggested that at least part of the cellular apoptosis machinery was being used. Previous work has shown that apoptosis occurs in parvovirus-infected cells at 72 hours after infection, once viral replication has occurred (Ikeda et al., 1998, Morey et al., 1993, Poole et al., 2004, Rayet et al., 1998). Whether apoptosis begins earlier during infection with parvovirus has not been investigated. We therefore examined LA9 cells  *  A version of this chapter has been submitted for publication: • Cohen, S., Marr, A.K., Garcin, P., and Panté, N. Nuclear envelope disruption involving host caspases plays a role in the parvovirus replication cycle.  85  infected with MVM (MOI of 4) for signs of apoptosis at earlier time points after infection. Mouse fibroblast cells were infected and stained for double-stranded DNA breaks using a TUNEL assay at 24, 48 or 72 hours post infection. We did not observe TUNEL staining in infected cells until 48 hours after infection, at which time a small proportion of the cells began to stain positive for double-stranded DNA breaks (Fig. 5-1). By 72 hours after infection, approximately half of the cells stained positive for double-stranded DNA breaks (Fig. 5-1), consistent with previous results from other groups (Ikeda et al., 1998, Morey et al., 1993, Poole et al., 2004, Rayet et al., 1998). These results suggest that MVMinduced NE disruption early during infection is not due to the induction of apoptosis. This led us to hypothesize that NE disruption induced early during infection rather plays a specific role in the replication cycle of parvoviruses.  5.2.2 Nuclear envelope disruption is important for the replication cycle of MVM at a stage prior to gene expression To investigate whether NE disruption is in fact important for the replication cycle of MVM we examined the effect of the caspase-3 inhibitor zDEVD-fmk, which inhibits MVMinduced NE disruption (Figs. 4-3, 4-5 and 4-6), on expression of the viral protein NS1. NS1 is not present on the MVM capsid in large amounts (Cotmore & Tattersall, 1989). Therefore, the presence of NS1 in MVM-infected cells indicates that the viral genome has successfully entered the nucleus of the host cell, and expression of viral proteins has begun.  We began by characterizing the time course of NS1 expression during infection by IF microscopy. While NS1 was never detected in mock-infected cells, NS1 was first detected in MVM-infected cells at 6 hours post infection. At this time, NS1 showed a punctate nuclear pattern (Fig. 5-2). At 8-10 hours post infection some of these NS1 foci  86  Figure 5-1: MVM does not cause apoptosis until 48 hours after infection. LA9 cells were infected with MVM (MOI of 4) for 24, 48, or 72 hours. Double-stranded DNA breaks were detected using TUNEL, and DNA was detected with DAPI. Scale bar, 50 µm.  87  Figure 5-2: Time course of NS1 expression in MVM-infected cells. LA9 cells were infected with MVM (MOI of 4) for 6, 8, 10 or 12 hours. Cells were immunolabeled with an antibody against NS1. Scale bar, 5 µm.  88  had become enlarged, and by 12 hours post infection NS1 filled the whole nucleus, although brighter foci were still present (Fig. 5-2). Because asynchronously dividing cells were used, a mixture of these phenotypes was observed at each time point. The time indicated in Fig. 5-2 represents the earliest time at which the corresponding phenotype was observed. This sequence of events is very similar to that reported for CPV (Ihalainen et al., 2007); the distribution of NS1 at early time points after infection has not been reported for other parvoviruses. However, the NS1 foci we observe likely correspond to subnuclear compartments termed the autonomous parvovirus-associated replication (APAR) bodies. APAR bodies contain parvoviral DNA, NS1, cyclin A and cellular DNA polymerases, and are the sites of parvoviral DNA replication (Bashir et al., 2001).  When cells were infected with MVM (MOI of 4) for 15 hours followed by visualization of NS1 by IF microscopy, threshold analysis detected NS1 in approximately 15% of the cells (Fig. 5-3). Because caspase inhibitors are unstable and needed to be replaced frequently, this time point was chosen to investigate the effect of inhibition of NE disruption on viral gene expression. When cells were infected in the presence of 200 µM of the caspase-3 inhibitor zDEVD-fmk, the proportion of cells detectibly expressing NS1 decreased by half (Fig. 5-3). This indicates that caspase-mediated NE disruption is important for the MVM replication cycle at a stage prior to virus gene expression.  5.2.3 MVM-induced nuclear envelope disruption mediates nuclear entry of the capsid Several observations led to the hypothesis that MVM-induced NE disruption plays a role in entry of MVM into the nucleus. We previously observed virions in close proximity to disruptions of the ONM, and in the intermembrane space between the INM and ONM,  89  Figure 5-3: Caspase-mediated NE disruption is important for the parvovirus replication cycle. (a) LA9 cells were infected with MVM (MOI of 4) for 15 hours in the presence or absence of 200 µM of the caspase-3 inhibitor zDEVD-fmk. Cells were immunolabeled with an antibody against NS1, and DNA was detected with DAPI. The proportion of cells expressing NS1 was dramatically reduced in the presence of zDEVDfmk. Scale bar, 20 µm. (b) Quantification of the proportion of cells expressing NS1 in experiments performed as described in (a). Bar graph shows the mean values and standard error measured from four independent experiments; approximately 2000 cells were counted for each condition for each experiment. **, p<0.01, as compared to MVM (unpaired Student’s t-test).  90  after injection of MVM into Xenopus oocytes (Cohen & Panté, 2005). In addition, the timing of MVM-induced NE disruption is consistent with the timing of nuclear entry of the capsids (Figs. 3-5 and 3-6), and IF microscopy revealed MVM capsids accumulated at the NE of infected cells when NE disruption was inhibited with the caspase inhibitors zVAD-fmk or zDEVD-fmk (Fig. 4-5). Lastly, inhibition of NE disruption by zDEVD-fmk in MVM-infected cells resulted in a dramatic reduction in NS1 expression (Fig. 5-3), suggesting that NE disruption plays a role prior to virus gene expression.  To test the hypothesis that NE disruption mediates nuclear entry of MVM, cells were infected with MVM (MOI of 4) for two hours in the presence or absence of 100 µM of the caspase-3 inhibitor zDEVD-fmk. Cells were then prepared for immunogold EM and labeled for intact MVM capsids as in section 3.2.4. The localization of MVM capsids within infected cells was examined. While gold was frequently observed in the nucleus of cells infected with MVM in the absence of zDEVD-fmk (Fig. 5-4a), nuclear gold particles were rare in cells infected in the presence of zDEVD-fmk. Instead, consistent with our IF results, gold particles were observed to accumulate at the cytoplasmic side of the NE (Fig. 5-4b). To quantify the number of MVM capsids reaching the nucleus, 60 cells from two independent experiments were carefully examined for each condition, and the number of gold particles inside the nucleus was counted. The histogram in Fig. 5-4c shows that the distribution of the number of gold particles/nucleus is markedly skewed in MVM-infected cells treated with zDEVD-fmk, as compared with the number of gold particles/nucleus in cells infected with MVM in the absence of the drug. For example, in the absence of zDEVD-fmk, on average 14/30 cells did not have any gold particles in the nucleus, while in the presence of zDEVD-fmk this increased to 22/30. Similarly, while on average 10/30 cells infected with MVM in the absence of zDEVD-fmk had 2 or more gold particles in the nucleus, this was reduced to 3/30 cells infected in the presence of 91  Figure 5-4: Inhibition of caspase-mediated NE disruption prevents nuclear entry of MVM. LA9 cells were infected with MVM (MOI of 4) for two hours in the absence or presence of 100 µM of the caspase-3 inhibitor zDEVD-fmk, then fixed and prepared for immunogold EM. Cells were immunolabeled with an antibody against MVM capsids and an appropriate secondary antibody conjugated to 10-nm gold (indicated by arrows). (a) In cells infected with MVM in the absence of zDEVD-fmk, gold was frequently observed in the nucleus. (b) In contrast, in cells infected with MVM in the presence of zDEVD-fmk, gold was rarely observed in the nucleus; instead, an accumulation of gold particles was observed at the cytoplasmic side of the NE. The cytoplasm is indicated by c, nucleus by n. Because membranes are not easily visualized when samples are prepared for immunogold EM, the nuclear membranes have been outlined. Arrowhead indicates an NPC. Scale bar, 100 nm. (c) Histogram showing the mean number of gold particles/nucleus and standard error for 60 cells from two independent experiments performed as described above.  92  zDEVD-fmk. This strongly suggests that NE disruption involving caspase-3 facilitates nuclear entry of MVM.  5.2.4 MVM-induced nuclear envelope disruption alters the compartmentalization of cellular proteins The change in localization of caspase-3 that we observed in MVM-infected cells (Fig. 49) led to the hypothesis that MVM-induced NE disruption may also affect the compartmentalization of other host proteins. To examine this, cells were transiently transfected with a construct encoding 5 green fluorescent protein molecules in tandem (5-GFP, construct courtesy of Dr. G. Lukacs), and the localization of the 5-GFP in mockand MVM-infected cells at two hours post infection was examined. We chose 5-GFP because this is the smallest number of tandem GFP molecules that is completely excluded from the nucleus under normal conditions (Wang & Brattain, 2007). Similar to cleaved caspase-3, 5-GFP was completely excluded from the nucleus in mock-infected cells; however, in MVM-infected cells some of the 5FP leaked into the nucleus, again in close proximity to the location of the virus in the cell (Fig. 5-5). Thus, infection with MVM seems to alter the compartmentalization of multiple host proteins early on during infection. Interestingly, when cells transiently-transfected with 5-GFP were infected with MVM for 24 hours, the cytoplasmic compartmentalization of 5-GFP was restored (Fig. 56). This is consistent with our observation that the nuclear lamina is disrupted in MVMinfected cells early during infection (Fig. 3-7), but is repaired later on (Fig. 3-8).  5.3 Discussion The results described in this chapter indicate that MVM-induced NE disruption is not a general result of the induction of apoptosis. Instead, this NE disruption seems to play a specific role in the virus replication cycle. When NE disruption was pharmacologically  93  Figure 5-5: MVM-induced NE disruption alters the compartmentalization of cellular proteins at two hours post infection. LA9 cells transiently expressing 5-GFP (green) were mock infected or infected with MVM at an MOI of 4 and prepared for indirect IF microscopy two hours after infection. Cells were immunolabeled with antibodies against MVM (red) and DNA was detected with DAPI (blue). The 5-GFP was excluded from the nucleus in mock-infected cells, but entered the nucleus in MVM-infected cells. Nuclei have been demarcated in white. Scale bar, 5 µm.  94  Figure 5-6: The compartmentalization of host proteins is restored at 24 hours post infection. LA9 cells transiently expressing 5-GFP (green) were mock infected or infected with MVM at an MOI of 4 and prepared for indirect IF microscopy 24 hours after infection. Cells were immunolabeled with antibodies against MVM (red) and DNA was detected with DAPI (blue). At 24 hours post infection, 5-GFP was excluded from the nucleus in both mock- and MVM-infected cells. Nuclei have been demarcated in white. Scale bar, 5 µm.  95  inhibited with the caspase-3 inhibitor zDEVD-fmk, NS1 expression was dramatically reduced in MVM-infected cells (Fig. 5-3). This indicates that NE disruption is required for a stage in the virus replication cycle prior to early gene expression. Immunogold EM revealed that inhibition of NE disruption resulted in far fewer MVM capsids reaching the nucleus (Fig. 5-4), suggesting that NE disruptions actually mediate entry of MVM into the nucleus. This is a novel nuclear entry mechanism, unlike any previously described for other viruses. In addition, the compartmentalization of host proteins was altered in MVMinfected cells early during infection. Both cleaved caspase-3 (Fig. 4-9) and exogenously expressed 5-GFP (Fig. 5-5) were exclusively cytoplasmic in mock-infected cells, but leaked into the nucleus at two hours post infection in MVM-infected cells. Interestingly, these proteins did not disperse throughout the nucleus, but remained restricted to a small area close to where the virus was located in the cell. This effect is very similar to that observed when cells infected with human cytomegalovirus (HCMV) were loaded with large fluorescent dextran and then visualized at 72 hours post infection (Buchkovich et al., 2010). Like MVM, HCMV also induces disruption of the nuclear lamina; however, HCMV accomplishes this by recruiting PKC, which then phosphorylates the nuclear lamins (Muranyi et al., 2002). Unlike during MVM infection, lamina disruption occurs late during infection with HCMV, and plays a role in egress of progeny virions from the nucleus (Sanchez & Spector, 2002). That these two unrelated viruses both disrupt the nuclear lamina indicates that alteration to the NE and associated structures in virusinfected cells may be a common theme. In addition, the restricted localization of cytoplasmic markers to a small region of the nucleus suggests that there are structures in the nucleus which prevent rapid movement of these molecules. Perhaps incomplete disruption of the nuclear lamina causes proteins or dextrans to become trapped between the NE and the partially dissolved lamin meshwork.  96  The change in compartmentalization of cellular proteins during infection with MVM may also play a role in the virus replication cycle. It is possible that relocalization of certain cytoplasmic proteins to the nucleus is beneficial for viral replication. However, the observation that normal compartmentalization is restored at 24 hours post infection (Fig. 5-6) suggests that if this is the case, these cytoplasmic proteins are no longer required later during infection, at the time when assembly of progeny virions is occurring.  97  6. Proteomic Approaches for Studying MVM Infection 6.1 Introduction Many viruses induce post-translational modification of a multitude of cellular proteins during infection of host cells. These modifications include cleavage, phosphorylation, and ubiquitination (reviewed by Isaacson & Ploegh, 2009, Munter et al., 2006). In addition, host proteins may be upregulated or downregulated in response to infection.  A comprehensive study of post-translational modifications in cells infected with parvovirus has not been carried out. Thus, we used 2D-DIGE in an attempt to identify host proteins that are modified in MVM-infected cells. Based on the results described in Chapter 3, we expected to observe differences in the amount or modification state of nuclear lamins and perhaps nucleoporins or other proteins associated with the NE. We also hoped to observe differences in other proteins pertaining to virus trafficking or replication, for example components of the cytoskeleton, proteins involved in cell signaling, or transcription factors.  Also, in addition to the candidate approach to identifying host proteins involved in MVMinduced NE disruption described in Chapter 4, we sought to use an unbiased approach. Thus, IP of MVM capsids followed by mass spectrometry was used to identify putative binding partners of MVM. Using this approach, we hoped to identify NE or signaling proteins involved in induction of NE disruption through direct interaction with the MVM capsid. Other anticipated binding partners not involved in NE disruption would include a cell surface receptor or co-receptor, cytoskeleton-associated motor proteins, and signaling proteins such as kinases or phosphatases, as well as transcription factors.  98  6.2 Results 6.2.1 Post-translational modifications in cells infected with MVM Post-translational modifications to host proteins during infection with MVM were investigated using 2D-DIGE. Mouse fibroblast cells were mock infected or infected with MVM (MOI of 2) for two or 24 hours. The cells were then lysed, and the proteins fluorescently labeled. Samples were run on the same gel, and analyzed by 2D-DIGE.  At two hours post infection, there were very few differences between the protein profiles of mock- and MVM-infected cells (Fig. 6-1). However, the most dramatic difference between the samples was the appearance of three red spots (Fig. 6-1, circled) corresponding to proteins present in cells infected with MVM for two hours, but not in mock-infected cells. Excision of these three spots from the gel followed by MALDI-ToF mass spectrometry revealed that these three proteins were cleavage products of the intermediate filament protein vimentin (Table 6-1). While full-length mouse vimentin has a molecular weight of approximately 53.7 kDa, the cleavage products were 50 kDa, 48 kDa, and 45 kDa. These cleavage products had an MVM-infected to mock-infected volume ratio of 1.53, 1.63, and 2.17, respectively.  At 24 hours post infection, more differences between the protein profiles of mock- and MVM-infected cells were apparent (Fig. 6-2). Of the spots selected for analysis by mass spectrometry, 9 proteins which increased or decreased by more than 1.4-fold in MVMinfected cells (at either two or 24 hours post infection) were successfully identified (Table 6-1). Interestingly, at 24 hours post infection the vimentin cleavage products observed at two hours post infection were no longer present at higher levels than in mock-infected  99  Figure 6-1: Post-translational modifications to host proteins in MVM-infected cells at two hours post infection. 2D-DIGE analysis of mock- and MVM-infected cells at two hours post infection. Proteins from mock-infected cells are shown in green, while proteins from MVM-infected cells are shown in red; where the two samples are similar, the gel appears yellow. The most dramatic difference between the samples is the appearance of three red spots (circled) present in MVM- but not mock-infected cells. Spot numbers correspond to protein names in Table 6-1, as identified by MALDI-ToF mass spectrometry following excision from the gel.  100  Table 6-1: Post-translational modifications in MVM infected cells Spot a 1 2 3 4 5 6 7 8 9  Protein Name Vimentin, 45 kDa Vimentin, 48 kDa Vimentin, 50 kDa Farnesyl diphosphate synthetase Acidic ribosomal phosphoprotein P0 Dihydropyrimidinase-like 2 Acetyl co-enzyme A dehydrogenase, medium chain Phosphoglycerate kinase 1 Eukaryotic elongation factor 2  Volume Ratio b,c 2 hours 24 hours -1.13 2.17 1.01 1.63 -1.03 1.53 1.05 1.85 1.21 1.71 1.00 1.46 1.04 1.42 -1.24 -1.42  -1.52 -1.72  a  Location of spot on gel. Spot numbers correspond to the numbers in Figs. 6-1 and 6-2. Volume ratio represents the fold change in protein abundance in lysates derived from MVM-infected as compared to mock-infected cells. A positive volume ratio indicates that the protein was more abundant in MVM-infected cells, while a negative volume ratio indicates that it was less abundant in infected cells. c Changes in protein abundance of more than 1.4-fold are indicated in bold. b  101  Figure 6-2: Post-translational modifications to host proteins in MVM-infected cells at 24 hours post infection. 2D-DIGE analysis of mock- and MVM-infected cells at 24 hours post infection. Proteins from mock-infected cells are shown in green, while proteins from MVM-infected cells are shown in red; where the two samples are similar, the gel appears yellow. Spot numbers correspond to protein names in Table 6-1, as identified by MALDI-ToF mass spectrometry following excision from the gel.  102  cells. However, the amount of farnesyl phosphate synthetase, acidic ribosomal protein P0, dihydropyrimidinase-like 2, and the acetyl co-enzyme A dehydrogenase medium chain were all increased in MVM-infected cells at 24 hours. In contrast, phosphoglycerate kinase 1 and eukaryotic elongation factor 2 were both decreased in MVM-infected cells at 24 hours.  6.2.2 Putative binding partners of the MVM capsid In order to identify putative binding partners of the MVM capsid, sepharose beads conjugated to an anti-MVM capsid antibody were mock incubated or incubated with purified MVM, washed, and then incubated with lysate prepared from mouse fibroblast LA9 cells. When the immunoprecipitated proteins were run on an SDS-polyacrylamide gel and stained with Coomassie blue, three MVM capsid proteins were clearly detectible in the sample with MVM (+MVM) but not in the sample without MVM (-MVM) (Fig. 6-3). Therefore, we proceeded to analyze samples by mass spectrometry.  Two rounds of IP followed by mass spectrometry were performed. In both experiments, quantitative mass spectrometry detected MVM capsid proteins at a ratio of >10-fold in the +MVM sample as compared with the -MVM sample, indicating successful IP of MVM. Several host proteins were enriched in the +MVM sample in both experiments as well (Table 6-2). However, in the second experiment conditions were further optimized, and a larger number of host proteins were enriched in the +MVM sample. Although these results need to be confirmed in another independent experiment, proteins enriched by more than 1.5-fold in the +MVM sample in one out of two experiments will be discussed in this thesis (Table 6-3). Several interesting themes emerge. Most of the putative binding partners of the MVM capsid which we identified fall into one of 6 categories. These include proteins involved in vesicular traffic and/or NE integrity, cell surface  103  Figure 6-3: IP of MVM and putative cellular binding partners. Lanes, from left to right: protein standards, purified MVM, IP (-MVM), IP (+MVM). Stars indicate the MVM capsid proteins present in lanes 2 and 4.  104  Table 6-2: Putative binding partners of the MVM capsid: proteins with a MVM/mock ratio of >1 in two rounds of mass spectrometry Protein Name Galectin-3-binding protein Heat shock protein 73 (Hsp73) Proteasome subunit α3 Elongation factor 1γ Myosin-9  MVM/Mock (Average) 1.92 1.42 1.35 1.30 1.25  105  Table 6-3: Putative binding partners of the MVM capsid: proteins with a MVM/mock ratio of >1.5 in one round of mass spectrometry Protein Name Vesicular traffic and NE integrity Sac1 phosphatidylinositide phosphatase Coatomer subunit α Clathrin heavy chain 1 Dis3 mitotic control homolog Cell surface Galectin-3 Cytoskeleton Isoform 1 of cytoplasmic FMR1-interacting protein 1 T-complex protein 1 subunit αA Protein turnover Proteasome non-ATPase regulatory subunit 3 Translation Ribosomal protein S7 Ribosomal protein L30 Ribosomal protein L6 Eukaryotic initiation factor 4A1 Eukaryotic initiation factor 3B Ribosomal protein S8 Ribosomal protein L13a Ribosomal protein S3 Ribosomal protein S16 Ribosomal protein L7 Metabolism Carbamoyl-phosphate synthetase 2 Aspartyl-tRNA synthetase β-glucuronidase Asparagine synthetase Fatty acid synthase Phosphofructokinase Other Rik hypothetical protein LOC330286  MVM/Mock 5.28 2.85 1.62 1.57 1.55 2.45 2.07 1.82 2.48 2.27 2.26 2.10 1.85 1.69 1.64 1.61 1.57 1.56 2.98 2.81 2.29 2.06 1.95 1.76 6.54  106  proteins, proteins associated with the cytoskeleton, and proteins involved in protein turnover, translation, and metabolism.  6.3 Discussion Proteomic approaches to investigating MVM infection have resulted in some promising insights. Analysis of the protein profile of MVM-infected cells by 2D-DIGE did not detect changes to any NE proteins, but revealed that the intermediate filament protein vimentin is cleaved at two hours post infection (Fig. 6-1). Caspases are known to cleave vimentin (Byun et al., 2001, Muller et al., 2001, Prasad et al., 1998). Indeed, the cleavage profile of vimentin we observed - 50 kDa, 48 kDa, and 45 kDa fragments – corresponds particularly well with the cleavage profile when vimentin is cleaved by caspase-3 (Byun et al., 2001). Thus, it is possible that the cleavage of vimentin and the B-lamin cleavage described in Chapter 4 are related. Further discussion of the significance of these observations follows in section 7.4.  The changes in the protein profile of MVM-infected cells at 24 hours post infection (Fig. 6-2) are more difficult to interpret. At this point viral proteins have been expressed in the infected cell, and the viral genome has been replicated. Changes to host protein levels may be a result of either viral processes or the host response to infection. Interestingly, the acidic ribosomal phosphoprotein P0 was more abundant in MVM-infected cells at 24 hours post infection, while eukaryotic elongation factor 2 was less abundant. These results, combined with the large number of putative binding partners of MVM which are involved in translation, suggest that MVM may modulate host translation. Many viruses, including adenoviruses, herpesviruses, picornaviruses and poxviruses, are known to interfere with host translation (reviewed by Roberts et al., 2009).  107  Lastly, IP of MVM capsids followed by mass spectrometry identified a large number of putative binding partners (Tables 6-2 and 6-3). Proteins identified in two separate experiments include galectin-3-binding protein (G3BP), heat shock protein 73 (Hsp73), proteasome subunit α3, elongation factor 1γ, and myosin-9. The proteasome has been implicated in MVM-infection. Proteasome inhibitors reduce viral replication in MVMinfected cells, and it has been suggested that the proteasome may play a role in proteolytic processing of the MVM capsid (Ros et al., 2002, Ros & Kempf, 2004). This reassures us that at least some of the proteins identified by IP/mass spectrometry play a relevant role in the MVM replication cycle. In addition, our results suggest that the MVM capsid interacts physically with the proteasome subunit α3 (Table 6-2) and possibly the proteasome non-ATPase regulatory subunit 3 (Table 6-3).  Putative MVM-binding proteins identified in one out of two experiments await confirmation. Intriguingly, three of the proteins identified have been implicated in NE integrity (Table 6-3), and could be involved in the NE disruption induced by MVM. These are the Sac1 phosphatidylinositide phosphatase (with a MVM/mock ratio of 5.28), the coatomer subunit α (α-coatomer; MVM/mock ratio of 2.85), and the Dis3 mitotic control homologue (MVM/mock ratio of 1.57). Sac1 is a lipid phosphatase which localizes to the ER and Golgi, and interacts with members of the COP1 complex (Rohde et al., 2003), while α-coatomer is part of the COP1 complex. The COP1 complex coats vesicles involved in intra-Golgi transport and transport from the Golgi to the ER (reviewed by Bonifacino & Glick, 2004). This complex has been implicated in mitotic NEBD, likely by participating in vesiculation of the NE (Liu et al., 2003). In addition, Dis3 mitotic control homologue binds directly to the GTPase Ran (Noguchi et al., 1996), which plays many roles in the coordination of nuclear transport and mitosis, including being involved in NE reformation at the end of mitosis (reviewed by Clarke & Zhang, 2008). The other proteins 108  identified include cell surface proteins, proteins associated with the cytoskeleton, and proteins involved in protein turnover, translation, and metabolism (Table 6-3). A complete discussion of the potential significance of these interactions as well as future directions follows in section 7.5.  In addition to being confirmed in a third round of IP/mass spectrometry, any putative binding partners of MVM will need to be validated by other methods. In our experiments, we have incubated MVM capsids directly with cell lysate, which abolishes the compartmentalization of the cell. Therefore, it will be important to determine whether these interactions actually take place in the context of the infected cell. This could be validated by IP of MVM from infected cells followed by Western blot to detect putative binding partners, as well as IF microscopy to look for co-localization between MVM and various binding partners in infected cells.  The design of our IP/mass spectrometry experiment may also have resulted in the failure to detect certain binding partners. During infection, the parvovirus capsid undergoes several conformational transitions (reviewed by Harbison et al., 2008). By incubating MVM capsids with lysate rather than immunoprecipitating MVM from infected cells we may have missed conditional binding partners which bind only after certain conformational transitions have taken place in the MVM capsid. Thus, the IP/mass spectrometry experiment could be repeated either by immunoprecipitating MVM from infected cells (which requires a huge amount of material), or by using MVM capsids that have been exposed to low pH in order to mimic endocytic processing. This type of experiment may result in the identification of additional binding partners that interact with the MVM capsid following escape from endosomes.  109  7. Conclusion As parvoviruses are only 26 nm in diameter, it has been assumed that they enter the nucleus through the NPC. I have shown for the first time that the parvovirus MVM can use an alternate mechanism, which involves disruption of the NE and entry through the resulting gaps. This is a novel nuclear entry mechanism, unlike those previously described for any virus or protein. I propose that disruption of the NE and entry through the resulting gaps comprises a fifth viral nuclear entry strategy, in addition to the four strategies that have been described for other viruses (Fig. 7-1).  In this thesis I have addressed the objectives laid out in Chapter 1. These included characterizing the effect of the parvovirus MVM on the host NE (Chapter 3); investigating the molecular mechanism by which MVM induces NE disruption (Chapter 4); determining the role of NE disruption in the replication cycle of MVM (Chapter 5); examining post-translational modifications to host proteins in MVM-infected cells (Chapter 6); and identifying putative host binding partners of the MVM capsid (Chapter 6). Each of these objectives will be discussed below.  7.1 Effect of MVM on the host nuclear envelope In Chapter 3, I characterized the effect of MVM on the host NE. Microinjection experiments revealed that the ability to induce disruptions of the NE is not specific to MVM, but is a property shared by at least three autonomous parvoviruses, MVM, H1 and CPV. Importantly, I showed by EM that disruption of the NE occurs during infection of mouse fibroblast cells with MVM, indicating that this observation is not an artifact of microinjection. Immunogold EM revealed that the timing of entry of MVM capsids into the nucleus is consistent with the timing of NE disruption (discussed further in 7.3.1), while  110  Figure 7-1: How viruses access the nucleus – revised. (1) The MLV PIC gains access to the nucleus during mitosis, when the NE is temporarily disassembled. (2) Influenza A virus undergoes extensive disassembly in the cytoplasm. The cytoplasmic released vRNPs contain NLSs and are thereby able to cross the NPC using the host transport machinery. (3) HSV-1 capsids use importins to attach to the cytoplasmic side of the NPC. Interaction with the NPC then triggers the release of the viral genome, which then enters the nucleus through the NPC. (4) Capsids of the baculovirus AcMNPV cross the NPC intact. Genome release presumably occurs inside the nucleus. (5) Unlike all the other strategies, which involve the NPC, parvoviruses transiently disrupt the NE and nuclear lamina, and enter the nucleus through the resulting gaps. (Modified from a figure originally published by Cohen et al., 2011. Copyright Elsevier).  111  IF microscopy of the nuclear lamina suggested that NE disruption is a transient event which likely plays a specific role in the viral replication cycle, rather than a non-specific response to infection. In addition, I found that MVM causes dramatic alterations in the nuclear morphology of infected cells, including the induction of amorphous invaginations of the NE, as well as alterations in chromatin structure. These effects are similar to those seen when the HIV-1 protease is microinjected into cells (Shoeman et al., 1990), and may be related to the cleavage of vimentin we observed by 2D-DIGE in Chapter 6 (see section 7.4 for further discussion).  7.2 Mechanism of MVM-induced nuclear envelope disruption In Chapter 4, I investigated the molecular mechanism of MVM-induced NE disruption. While microinjection of MVM into Xenopus oocytes under conditions of PLA2 inhibition indicated that viral phospholipase activity is dispensable for the induction of NE disruption by MVM, I found that cellular proteins involved in NEBD are required. Caspase-3 in particular seems to be involved, while ONM proteins may also play a role.  7.2.1 The role of caspases in MVM-induced nuclear envelope disruption Four separate assays, including a semi-permeabilized cell assay for NE permeability, EM visualization of the NE of microinjected Xenopus oocytes, lamin immunostaining and Western blot of lamins in MVM-infected cells, all support a role for cellular caspase activity in MVM-mediated disruption of the NE (Chapter 4). Western blot results suggest that lamin B2 was cleaved at a caspase-3 consensus site in MVM-infected cells. Thus, I have focused primarily on the role of caspase-3 in MVM-induced NE disruption. Although caspase-mediated disruption of the nuclear lamina was observed in MVMinfected cells early during infection (Chapter 4), apoptosis leading to double stranded DNA breaks did not occur until 48 hours post infection (Chapter 5). While caspases are  112  usually described as apoptotic proteases, a variety of non-apoptotic functions have also been discovered for these enzymes (Feinstein-Rotkopf & Arama, 2009). For example, caspase-3 is upregulated and activated just prior to mitosis, suggesting that it may play a role in the G2/M transition (Hsu et al., 2006). Here we propose a non-apoptotic role for caspase-3 in the parvovirus replication cycle.  Caspase-3 was not activated above basal levels in cells infected with MVM; however, previously cleaved caspase-3 present at low levels under basal conditions showed relocalization to the nucleus in MVM-infected cells (Chapter 4). Based on these observations I propose the model shown in Fig. 7.2. In this model, MVM induces disruption of the nuclear membranes by an as yet unknown mechanism, not involving viral PLA2 activity; this mechanism may involve ONM proteins or the COP1 complex (see 7.2.2 and 7.5.1 below); nuclear membrane disruption allows previously cleaved and activated caspases in the cytoplasm to gain access to the nucleus, where the proteases cleave lamin B1, resulting in disruption of the nuclear lamina structure and progression of NE disruptions. The fact that lamin B1 was cleaved in MVM-infected cells while A/Ctype lamins were not is consistent with a recently proposed model of the nuclear lamina in which the regular structure directly underlying the NE is composed of B-type lamins, while A/C-type lamins form sparser bundles which lie beneath (Goldberg et al., 2008). Thus, caspases gaining access to the nuclear lamina from the cytoplasmic side would primarily encounter B-type lamins rather than A/C-type lamins. I propose that caspasemediated NE disruption plays a role in delivery of the MVM genome into the nucleus of the host cell (see 7.3.1). MVM-induced NE disruption also alters the compartmentalization of cellular proteins other than cleaved caspase-3 (Chapter 5). Therefore, it is possible that relocalization of certain cytoplasmic proteins to the nucleus is beneficial for viral replication (see 7.3.4). 113  Figure 7-2: Model of MVM-induced NE disruption. MVM induces disruption of the nuclear membranes by a mechanism that does not involve viral PLA2 activity, but may involve ONM proteins or the COP1 complex (1). Previously cleaved, activated caspases in the cytoplasm gain access to the nucleus (2), where they cleave B-type lamins (3). This cleavage is necessary for sustained disruption of the NE, which in turn mediates nuclear entry of the MVM capsid and possibly other cellular proteins required by the virus for replication (4).  114  7.2.2 The role of outer nuclear membrane proteins in MVM-induced nuclear envelope disruption In addition to caspase-3, preliminary results suggest that KASH domain ONM proteins may play a role in MVM-induced NE disruption. Mislocalization of these proteins through expression of dominant-negative Sun1 completely inhibited nuclear lamina disruption in MVM-infected cells (Chapter 4). As mentioned, it is difficult to interpret these results because KASH domain proteins have been reported to link the NE to microtubules via interaction with motor proteins (reviewed by Burke & Roux, 2009). Thus, it is possible that mislocalizing KASH proteins impacts the organization of the microtubule network, and impairs trafficking of MVM towards the nucleus. This could be tested by using IF microscopy to visualize the localization of MVM capsids in conjunction with various organelle markers in infected cells expressing dominant-negative Sun1. For example, it is possible that mislocalizing KASH proteins to the ER results in targeting of MVM to the ER rather than to the NE. The distribution of microtubules in cells expressing dominantnegative Sun1 could also be examined using IF microscopy. In addition, drugs which prevent microtubule assembly could be tested to see whether their effects on MVM infection are similar to those observed in cells expressing dominant-negative Sun1.  Alternatively, it is also possible that ONM proteins play a currently unappreciated role in cellular NEBD, and that MVM hijacks this function to induce NE disruption during infection. This would also explain the inhibition of MVM-induced NE disruption in cells with mislocalized ONM proteins. It has been shown that mitotic NEBD involves dyneinmediated tearing of the NE and transport of the resulting pieces of membrane along microtubules (Beaudouin et al., 2002, Salina et al., 2002). How dynein interacts with the NE during this process has not been determined. In C. elegans, the KASH domain  115  protein Zyg-12 binds dynein, and is important for nuclear positioning and coupling of the nucleus to the centrosome (Malone et al., 2003, Zhou et al., 2009). Thus, it seems likely that localization of dynein to the NE during mitotic NEBD is mediated by an as yet uncharacterized KASH domain protein. MVM-induced NE disruption may prove to be a useful model for investigating the role of ONM proteins in cellular NEBD.  To test whether ONM proteins play a role in the induction of NE disruption by MVM, cells expressing dominant-negative Sun1 could be used in the digitonin-permeabilized cell assay for MVM-induced NE disruption described in Chapter 4. In this assay, the cytoskeleton is removed, and MVM is added directly to the nuclei of permeabilized cells. Thus, if mislocalization of ONM proteins affects the ability of MVM to disrupt the NE in digitonin-permeabilized cells, it could not be because of defects in trafficking to the nucleus. If expression of dominant-negative Sun1 does prevent MVM-induced NE disruption in this system, then ONM proteins could be knocked down individually using RNA interference to determine which ONM protein is involved. It would also be interesting to use IF microscopy to see whether dynein relocalizes to the NE in MVMinfected cells, as it does during mitotic NEBD.  7.3 The role of MVM-induced nuclear envelope disruption in the viral replication cycle In Chapter 5, I investigated the role of MVM-induced NE disruption in the viral replication cycle. When NE disruption was pharmacologically inhibited with the caspase-3 inhibitor zDEVD-fmk, NS1 expression was dramatically reduced in MVM-infected cells. This indicates that NE disruption is required for a stage in the virus replication cycle prior to early gene expression. I propose that one function of MVM-induced NE disruption is to  116  mediate nuclear entry of the capsid (section 7.3.1). However, other functions such as recompartmentalization of host proteins may be important as well (7.3.4).  7.3.1 MVM-induced nuclear envelope disruption can mediate nuclear entry of the MVM capsid Several lines of evidence point to NE disruption as the mechanism of parvoviral entry to the nucleus. When MVM is microinjected into Xenopus oocytes and visualized by EM, virions are observed in close proximity to disruptions of the ONM, and in the intermembrane space between the INM and ONM (Cohen & Panté, 2005). In addition, in Xenopus oocytes pre-injected with the lectin wheat germ agglutinin (WGA) to block transport through the NPC, microinjection of MVM causes NE disruptions which support nuclear entry of proteins in an NPC-independent manner (Cohen & Panté, 2005). Consistent with this data, blocking NPCs with WGA does not prevent uptake of AAV into purified nuclei (Hansen et al., 2001). Lastly, both IF microscopy and immunogold EM showed that MVM capsids accumulated at the cytoplasmic side of the NE in infected cells when NE disruption was inhibited with the caspase inhibitor zDEVD-fmk (Chapters 4 and 5).  The parvoviral nuclear entry mechanism through disruptions of the NE is unlike that of any other virus known to date. The majority of viruses use nuclear localization signals (NLSs) in viral proteins to bind soluble cellular transport receptors, which then mediate nuclear import of the viral genome through the NPC (Greber & Fornerod, 2005, Helenius, 2007, Whittaker, 2003, Whittaker et al., 2000). Like other viruses, it has been shown that parvoviruses have functional NLSs in their capsid proteins (Lombardo et al., 2002, Vihinen-Ranta et al., 1997). It has been demonstrated that these NLSs are necessary for nuclear import of capsid protein trimers prior to virion assembly in the  117  nucleus (Riolobos et al., 2006). One of these NLSs, within VP2, is buried inside the capsid of assembled virions and could not be involved in nuclear entry of the incoming capsid during initial infection (Agbandje-McKenna et al., 1998, Lombardo et al., 2000). The others, within VP1, are initially buried within the capsid as well; these NLSs are located within a region of VP1 which becomes exposed during endocytic trafficking of the capsid (Cotmore et al., 1999, Mani et al., 2006), and could potentially participate in transport of the capsid through the NPC. However, it has not been demonstrated that these NLSs become sufficiently exposed to be able to interact with cellular import receptors in order to mediate nuclear import of the capsid. If the VP1 NLSs do become sufficiently exposed to interact with importins, then it is possible that these NLSs play a role in targeting the MVM capsid to the NE prior to nuclear entry via NE disruptions. In addition, it has recently been shown that an NLS-like sequence in the VP1 of AAV2 plays a role in targeting capsids to the nucleolus (Johnson et al., 2010). Thus, it is possible that the VP1 NLSs function primarily in intranuclear trafficking, rather than in trafficking of parvovirus capsids to the nucleus.  7.3.2 Can the nuclear pore complex also mediate nuclear entry of parvoviruses? Although the evidence described above suggests that NE disruption is an important nuclear entry route for parvoviruses, it is not currently possible to rule out an additional route through the NPC. To test this explicitly, it will be necessary to perform experiments blocking the NPCs and evaluating nuclear entry of MVM. Use of digitonin-permeabilized cells is a well established system for studying nuclear import (Adam et al., 1990). Digitonin permeabilizes the plasma membrane of cells by removing cholesterol, but leaves the NE intact. Thus, digitonin-permeabilized cells retain import-competent nuclei, but are depleted of cytosolic factors. These factors can be added back to identify which  118  are necessary for the import of a particular substrate. Inhibitors can also be easily introduced in this system. Import of a fluorescent substrate into the nuclei of digitoninpermeabilized cells can then be monitored using fluorescence microscopy (Adam et al., 1990). In order to determine whether MVM can enter the nucleus through the NPC, digitonin-permeabilized cells could be used to assay for nuclear import of fluorescentlylabeled MVM capsids under different conditions. This assay could be performed in the absence or presence of cytosol, caspase inhibitors to prevent NE disruption, or the lectin WGA, which blocks NPCs. Nuclear entry of fluorescently-labeled MVM could then be assayed and quantified under these different conditions in order to investigate the relative contribution of NE disruption-mediated versus NPC-mediated nuclear entry of MVM.  Digitonin-permeabilized cells are a convenient system for studying nuclear entry. However, MVM does not have the opportunity to undergo endocytic processing when it is added to digitonin-permeabilized cells, which may affect the exposure of putative NLSs. Thus, it will be important to perform experiments in infected cells as well. This could be done by transfecting cells with a dominant-negative mutant of importin β which binds to the NPC and blocks NLS-dependent protein import (Kutay et al., 1997). These cells could then be infected with MVM, and nuclear entry of MVM capsids evaluated using immunogold EM. The disadvantage of this experiment is that dominant-negative importin β affects many cellular processes, since it also blocks nuclear export of mRNA (Kutay et al., 1997). Nevertheless, similar experiments have been used to show that the baculovirus AcMNPV is transported into the nucleus via NPCs (Ohkawa et al., 2010).  119  7.3.3 Nuclear entry of MVM compared with other viruses So far, MVM is the only virus for which it has been demonstrated (in the current study) that the capsid can enter the nucleus through disruptions of the NE. However, in addition to parvoviruses, there are other viruses that may also use a similar strategy. Simian virus 40 (SV40) is a small (40 nm) non-enveloped DNA virus in the polyomavirus family. SV40 enters the cell by an unusual mechanism: the virus is taken up by caveolar endocytosis, and then traffics via caveosomes to the ER (Pelkmans et al., 2001). In the ER, the host chaperone protein ERp29 triggers a conformational change in the capsid (Magnuson et al., 2005). This results in the exposure of the capsid protein VP2, which then integrates into and perforates the ER membrane, releasing the capsid or subviral particle (Rainey-Barger et al., 2007). It is unclear whether SV40 escapes from the ER to the cytoplasm and then enters the nucleus through the NPC, or whether it enters the nucleus directly from the ER by penetrating the INM. The latter possibility would presumably require disruption of the nuclear lamina. Recent findings show that caspase6 is activated early on during infection of cells with SV40 (Butin-Israeli et al., 2010). Together with caspase-3, caspase-6 is one of the proteases involved in apoptotic cleavage of nuclear lamins (Slee et al., 2001). Thus, caspase-6 activation may be induced by SV40 in order to cause lamina disruption, which could then facilitate nuclear entry of the SV40 capsid via the INM.  In addition, it has been shown that overexpression of the HIV-1 protein Vpr induces ruptures of the NE (de Noronha et al., 2001). It has been suggested that these ruptures may mediate entry of the HIV PIC into the nucleus (Segura-Totten & Wilson, 2001). However, it is unclear whether Vpr-induced NE rupture actually occurs during infection of cells with HIV-1. Interestingly, MVM and HIV share another property as well: vimentin is  120  cleaved in HIV-infected cells (see section 7.4). That viruses as different as MVM, a single-stranded DNA virus, and HIV-1, a retrovirus, might share the abilities to disrupt the NE and cleave vimentin indicate that these processes likely represent solutions to problems faced by many viruses.  7.3.4 Other possible functions of nuclear envelope disruption in the MVM replication cycle It is currently puzzling what the advantages might be for a virus to use a nuclear entry strategy that involves disruption of the NE. I have shown that MVM-induced NE disruption results in localized changes in the compartmentalization of cellular proteins, for example exogenous 5-GFP (Chapter 5). It is possible that such changes in compartmentalization are beneficial for MVM, e.g. cytoplasmic proteins used by the virus for a replication step are able to leak into the nucleus. It is also possible that disruption of the ONM, which is continuous with the ER, results in release of calcium and that subsequent signaling plays a role in infection. Calcium signaling has recently been shown to play an important role in infection of cells with West Nile Virus (Scherbik & Brinton, 2010). The hypothesis that calcium-mediated signaling plays a role in parvoviral infection could be tested using live-cell imaging of MVM-infected cells loaded with calcium-sensitive dye. If this is the case, then infection with MVM should result in an increase in the concentration of cytoplasmic calcium.  7.4 Post-translational modifications in MVM-infected cells Proteomic analysis of MVM-infected versus mock-infected cells by 2D-DIGE led to one very striking observation: vimentin is cleaved early during infection with MVM (Chapter 6). The cleavage profile suggests that, like lamin B2 (Chapter 4), vimentin may be cleaved by caspase-3 under these circumstances (Chapter 6). Therefore, this protease  121  seems to play an important role in MVM infection. It is not clear what the function of vimentin cleavage is in the replication cycle of MVM. Intermediate filaments have been shown to play a role in localizing caspases within the cell (Dinsdale et al., 2004). Preliminary results indicate that in addition to being cleaved, the distribution of vimentin is altered in MVM-infected cells; at two hours post infection, vimentin is observed to accumulate on one side of the nucleus, near to where the virus is located in the cell (N. Fay and N. Panté, unpublished results). Thus, cleavage of vimentin and collapse of the vimentin network may relocalize caspase-3 near to the NE, where it could then play a role in the cleavage of nuclear lamins and NE disruption. In addition, vimentin has been shown to affect the localization of lysosomes; lysosomal acidification is also impaired in vimentin null cells (Styers et al., 2004). Therefore, cleavage of vimentin could also play a role in preventing the degradation of MVM capsids within lysosomes.  Interestingly, it has been shown that the HIV-1 protease can cleave vimentin (Honer et al., 1991, Shoeman et al., 1990). Even more intriguing, the resulting vimentin peptides dramatically affected nuclear architecture when they were microinjected into cells, resulting in invagination of the nucleus and perturbation of chromatin organization (Shoeman et al., 2001). These effects are extremely similar to those we observe in MVM-infected cells (Chapter 3). Thus, cleavage of vimentin may contribute to the changes in nuclear architecture seen in MVM-infected cells. However, the mechanism of vimentin cleavage is likely to be different from that of HIV-1, since parvoviruses do not seem to encode any proteases. In addition, the importance of these nuclear changes for the replication cycle of either HIV-1 or parvoviruses is currently unclear.  122  7.5 Putative binding partners of MVM: significance and future directions In Chapter 6, IP of MVM followed by mass spectrometry was used to identify putative host binding partners of the MVM capsid. This experiment yielded a large number of potential interaction partners, which fell into 6 categories: proteins involved in vesicular traffic and/or NE integrity, cell surface proteins, proteins associated with the cytoskeleton, as well as proteins involved in protein turnover, translation, and metabolism. The potential significance of some of these proteins in the MVM replication cycle is discussed below.  7.5.1 Proteins involved in vesicular trafficking and nuclear envelope integrity As expected, several proteins implicated in NE integrity were identified as putative binding partners of the MVM capsid. These included α-coatomer, Sac1 lipid phosphatase, and Dis3 mitotic control homologue. The coat protein α-coatomer is part of the COP1 complex, which coats vesicles involved in intra-Golgi transport and transport from the Golgi to the ER (reviewed by Bonifacino & Glick, 2004), and has been implicated in mitotic NEBD (Liu et al., 2003). It has been shown that another subunit of the complex, β-coatomer, relocalizes from the Golgi to the NE during mitosis, and antibodies against β-coatomer prevented NEBD in a Xenopus reconstituted nucleus system (Liu et al., 2003). This led to a model in which the COP1 complex is recruited to the NE during mitosis, where it participates in vesiculation of the NE and redistribution of integral nuclear membrane proteins to the ER (Liu et al., 2003). Thus, MVM may initiate NE disruption by recruiting the COP1 complex to the NE. Consistent with this idea we previously observed vesicles close to NE disruptions in Xenopus oocytes microinjected  123  with MVM (Cohen & Panté, 2005). Sac1 lipid phosphatase, which interacts with members of the COP1 complex (Rohde et al., 2003), was also identified as a putative binding partner of the MVM capsid. In fact, Sac1 was among the most highly enriched of the identified proteins. This suggests that binding of MVM to coatomer subunit α may be via an indirect interaction mediated by Sac1. If these interactions can be confirmed in infected cells, then it will be interesting to determine whether infection with MVM results in relocalization of the COP1 complex to the NE, and whether inhibition of the COP1 complex using antibodies or RNA interference can prevent MVM-induced NE disruption. Interaction of MVM with Dis3 mitotic control homologue could also affect NE integrity. Dis3 binds directly to the GTPase Ran and enhances the nucleotide exchange activity of RCC1 on Ran, promoting Ran to be in the GTP-bound state (Noguchi et al., 1996). Many studies have suggested a role for Ran in reformation of the NE at the end of mitosis (reviewed by Clarke & Zhang, 2008). Intriguingly, disruption of RCC1 in fission yeast causes NE fragmentation (Demeter et al., 1995). Similarly, sequestration of Dis3 by MVM could promote NE disruption. Alternatively, interaction of MVM with Dis3 in the nucleus might stimulate NE repair, contributing to the transient nature of MVM-induced NE disruption (Chapter 3). If this interaction can be confirmed, it will be interesting to investigate whether RNA interference of Dis3 results in an increase or decrease in the amount of NE disruption occurring in MVM-infected cells.  In addition to α-coatomer, the coat protein clathrin heavy chain 1 was also identified as a putative binding partner of MVM. Clathrin coats vesicles at the trans-Golgi network and plasma membrane (reviewed by Bonifacino & Glick, 2004). In addition, during mitosis clathrin plays a role in stabilizing the mitotic spindle (Royle et al., 2005). Interestingly, heat shock protein 73, which mediates the uncoating of clathrin-coated vesicles (Braell  124  et al., 1984), was also identified as a putative MVM-binding partner. However, the potential significance of an interaction between MVM and clathrin is currently unclear.  7.5.2 Cell surface proteins Both G3BP and galectin-3 itself were identified as putative binding partners of the MVM capsid. Galectin-3 is an extracellular matrix protein which crosslinks ligands through binding to various β-galactosidase containing glycans (reviewed by Ochieng et al., 2004). Interestingly, it has recently been shown that galectin-1 promotes the infectivity of HIV-1 by stabilizing the attachment of virions to the host cell surface (Mercier et al., 2008). Thus, galectin-3 and/or G3BP may similarly play a role in MVM infection by promoting attachment at the cell surface. Alternatively, these proteins may actually act as a receptor for MVM. However this would be highly unusual, since viral receptors are typically transmembrane rather than extracellular or secreted proteins (Smith & Helenius, 2004). It will be of interest to determine whether knock down or overexpression of either galectin-3 or G3BP affects the rate of internalization of MVM into host cells.  7.5.3 Cytoskeleton-associated proteins Isoform 1 of cytoplasmic FMR1-interacting protein 1 and T-complex protein 1 subunit αA were both identified as putative binding partners of the MVM capsid. Cytoplasmic FMR1interacting protein 1 is also called specifically Rac1-associated protein (Sra1). Sra1 is involved in localizing the Rho-GTPase Rac1 at sites of actin assembly (Steffen et al., 2004). T-complex protein 1 subunit αA is part of the chaperonin containing TCP-1 (CCT), which mediates proper folding of both actin and tubulin (reviewed by Brackley & Grantham, 2009). MVM has been shown to cause rearrangement and degradation of filamentous actin starting at 10 hours post infection (Nuesch et al., 2005). Interaction of  125  MVM capsids or capsid proteins with either Sra1 or CCT could play a role in causing these effects. Alternatively, CCT could function to mediate proper folding and assembly of MVM capsid proteins. CCT has been implicated as a chaperone for the influenza virus polymerase subunit PB2 (Fislova et al., 2010), indicating that a role for CCT as a chaperone for viral proteins is not far-fetched.  In addition, the actin-based molecular motor myosin-9 was identified as a putative MVMbinding partner. It is possible that interaction of MVM with myosin-9 could mediate the transport of capsids through the cytoplasm, either after entry into the cell, or upon exit of progeny virions from the nucleus. The former possibility is contradictory to studies which have suggested that microtubules and dynein rather than actin are important for trafficking of incoming capsids from the cell periphery to the nucleus (Suikkanen et al., 2003a, Suikkanen et al., 2003b, Vihinen-Ranta et al., 2000). Therefore, the latter possibility may be more likely.  7.5.4 Regulators of protein-turnover The proteasome α3 and non-ATPase regulatory 3 subunits were both identified as putative binding partners of the MVM capsid. The proteasome, which is responsible for degradation of ubiquitinated proteins, has been implicated in MVM-infection. Proteasome inhibitors reduce viral replication in MVM-infected cells, suggesting that the proteasome plays a role in proteolytic processing of the MVM capsid after escape from endosomes (Ros et al., 2002). Because less virus appeared to enter the nucleus by IF microscopy in the presence of proteasome inhibitors, it has been proposed that this proteolytic processing is necessary for subsequent trafficking steps (Ros & Kempf, 2004). Our results suggest that the MVM capsid interacts physically with the  126  proteasome, in particular with subunit α3 and possibly the non-ATPase regulatory subunit 3.  7.5.5 Proteins involved in translation Many proteins involved in translation were identified as putative interaction partners of the MVM capsid, including eukaryotic initiation factor (eIF) 4A1 and eIF3B, elongation factor 1γ and many ribosomal proteins. Ribosomal proteins are extremely abundant in the cell, and may have been immunoprecipitated non-specifically. However, it is also possible that MVM actively modulates host translation. Other viruses that modulate translation to favour the production of viral proteins include adenoviruses, herpesviruses, picornaviruses and poxviruses (reviewed by Roberts et al., 2009). There are two general strategies used by viruses to modulate translation: either cap-dependent translation is stimulated and translation of host mRNAs is somehow inhibited, giving the virus a selective advantage, or cap-dependent translation is inhibited and cap-independent translation involving internal ribosome entry is used by the virus (Roberts et al., 2009). Some herpesviruses and poxviruses use the former strategy, while picornaviruses use the latter (Roberts et al., 2009).  The incoming MVM capsid may interact with the translation machinery. It is also possible that newly synthesized capsid proteins or capsid protein oligomers interact with the translation machinery and play a previously unrecognized role in modulating host translation. The effect of parvovirus infection on host translation has not been well characterized. This could be investigated by pulse-labeling cells with [35S]methionine at various time points after infection to see whether translation of host proteins decreases as translation of viral proteins increases. Even if host translation is not affected by infection with MVM, it is possible that the virus recruits host translation machinery to  127  specialized sites of viral translation. For example, the poxvirus vaccinia virus has been shown to recruit eIF4E and eIF4G to its replication factories (Katsafanas & Moss, 2007).  In addition, some of the ribosomal proteins identified as putative binding partners of the MVM capsid have extraribosomal functions. For example, ribosomal protein S7 sequesters Hdm2, which is an E3 ubiquitin ligase, while ribosomal protein S3 is a DNA endonuclease (reviewed by Warner & McIntosh, 2009). Thus, MVM may interact with some of these ribosomal proteins not in their capacity as components of the ribosome, but to promote or inhibit their other functions.  7.5.6 Proteins involved in metabolism Lastly, a number of proteins involved in metabolism were enriched following IP of MVM. It is not currently clear how modulating any of these metabolic pathways could be advantageous for the virus. In addition to being enriched in the samples containing MVM, a number of (different) metabolic proteins were enriched in the samples that did not contain MVM. Thus, these proteins are likely to have been immunoprecipitated nonspecifically due to being very abundant in the cell, rather than because of a specific interaction with the MVM capsid. These putative binding partners await confirmation through a third round of IP/mass spectrometry.  7.6 Concluding remarks The intracellular trafficking pathways of non-enveloped viruses are in general poorly understood. In this thesis, I have investigated the nuclear entry of a model nonenveloped virus, the parvovirus MVM. I have identified an entirely novel nuclear entry mechanism, which involves transient disruption of the NE and entry of viral capsids  128  through the resulting disruptions. This MVM-mediated NE disruption is accomplished by the hijacking of host proteins involved in apoptotic and possibly mitotic NEBD.  My work opens the door to many other questions. It is still unclear whether nuclear entry of parvovirus via NE disruptions functions instead of or in addition to a pathway through the NPC. It remains to be determined whether NE disruption plays other roles in the parvovirus replication cycle in addition to mediating nuclear entry of the capsid. It will also be of interest to determine whether this nuclear entry mechanism is shared by other viruses. Results from other groups suggest that the non-enveloped virus SV40 may use a similar mechanism. Thus, it will be interesting to examine whether additional nuclearreplicating, non-enveloped viruses share this strategy. In addition, proteomic approaches to studying MVM infection have resulted in many new avenues of research to pursue. These include investigation of the roles of the COP1 complex in MVM-induced NE disruption, of galectin-3 in entry of MVM into cells, and of various cytoskeleton associated proteins in the MVM replication cycle. In addition, it will be interesting to investigate whether, like many other viruses, parvoviruses modulate host translation to favour the production of viral proteins. This question has hardly been addressed.  Viruses have been used as tools to investigate a variety of cellular processes. Similarly, parvoviruses may be useful in the study of mitotic and apoptotic NEBD. For example, I have implicated ONM proteins in MVM-induced NE disruption. 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