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1.  Receptor Concentration and Diffusivity Control Multivalent Binding of Sv40 to Membrane Bilayers 
PLoS Computational Biology  2013;9(11):e1003310.
Abstract
Incoming Simian Virus 40 particles bind to their cellular receptor, the glycolipid GM1, in the plasma membrane and thereby induce membrane deformation beneath the virion leading to endocytosis and infection. Efficient membrane deformation depends on receptor lipid structure and the organization of binding sites on the internalizing particle. To determine the role of receptor diffusion, concentration and the number of receptors required for stable binding in this interaction, we analyze the binding of SV40 to GM1 in supported membrane bilayers by computational modeling based on experimental data. We measure the diffusion rates of SV40 virions in solution by fluorescence correlation spectroscopy and of the receptor in bilayers by single molecule tracking. Quartz-crystal microbalance with dissipation (QCM-D) is used to measure binding of SV40 virus-like particles to bilayers containing the viral receptor GM1. We develop a phenomenological stochastic dynamics model calibrated against this data, and use it to investigate the early events of virus attachment to lipid membranes. Our results indicate that SV40 requires at least 4 attached receptors to achieve stable binding. We moreover find that receptor diffusion is essential for the establishment of stable binding over the physiological range of receptor concentrations and that receptor concentration controls the mode of viral motion on the target membrane. Our results provide quantitative insight into the initial events of virus-host interaction at the nanoscopic level.
doi:10.1371/journal.pcbi.1003310
PMCID: PMC3828148  PMID: 24244125
2.  RNA encapsidation by SV40-derived nanoparticles follows a rapid two-state mechanism 
Remarkably, uniform virus-like particles self-assemble in a process that appears to follow a rapid kinetic mechanism. The mechanisms by which spherical viruses assemble from hundreds of capsid proteins around nucleic acid, however, are yet unresolved. Using Time-Resolved Small-Angle X-ray Scattering (TR-SAXS) we have been able to directly visualize SV40 VP1 pentamers encapsidating short RNA molecules (500 mers). This assembly process yields T = 1 icosahedral particles comprised of 12 pentamers and one RNA molecule. The reaction is nearly 1/3 complete within 35 milliseconds, following a two–state kinetic process with no detectable intermediates. Theoretical analysis of kinetics, using a master equation, shows that the assembly process nucleates at the RNA and continues by a cascade of elongation reactions in which one VP1 pentamer is added at a time, with a rate of approximately 109 M−1 s−1. The reaction is highly robust and faster than the predicted diffusion limit. The emerging molecular mechanism, which appears to be general to viruses that assemble around nucleic acids, implicates long-ranged electrostatic interactions. The model proposes that the growing nucleo-protein complex acts as an electrostatic antenna that attracts other capsid subunits for the encapsidation process.
doi:10.1021/ja2110703
PMCID: PMC3365646  PMID: 22329660
Time-resolved small-angle X-ray scattering; SV40; virus-like-particles; self-assembly; virus assembly kinetics; virus assembly mechanism
3.  Integrin-Mediated Signaling Induced by Simian Virus 40 Leads to Transient Uncoupling of Cortical Actin and the Plasma Membrane 
PLoS ONE  2013;8(2):e55799.
Simian Virus 40 (SV40) is a paradigm pathogen with multivalent binding sites for the sphingolipid GM1, via which it induces its endocytosis for infection. Here we report that SV40 also utilizes cell surface integrins to activate signaling networks required for infection, even in the absence of the previously implicated glycosphingolipids. We identify ILK, PDK1, the RhoGAP GRAF1 and RhoA as core nodes of the signaling network activated upon SV40 engagement of integrins. We show that integrin-mediated signaling through host SV40 engagement induces the de-phosphorylation of Ezrin leading to uncoupling of the plasma membrane and cortical actin. Our results provide functional evidence for a mechanism by which SV40 activates signal transduction in human epithelial cells via integrins in the context of clathrin-independent endocytosis.
doi:10.1371/journal.pone.0055799
PMCID: PMC3567119  PMID: 23409046
4.  Effect of capsid confinement on the chromatin organization of the SV40 minichromosome 
Nucleic Acids Research  2012;41(3):1569-1580.
Using small-angle X-ray scattering, we determined the three-dimensional packing architecture of the minichromosome confined within the SV40 virus. In solution, the minichromosome, composed of closed circular dsDNA complexed in nucleosomes, was shown to be structurally similar to cellular chromatin. In contrast, we find a unique organization of the nanometrically encapsidated chromatin, whereby minichromosomal density is somewhat higher at the center of the capsid and decreases towards the walls. This organization is in excellent agreement with a coarse-grained computer model, accounting for tethered nucleosomal interactions under viral capsid confinement. With analogy to confined liquid crystals, but contrary to the solenoid structure of cellular chromatin, our simulations indicate that the nucleosomes within the capsid lack orientational order. Nucleosomes in the layer adjacent to the capsid wall, however, align with the boundary, thereby inducing a ‘molten droplet’ state of the chromatin. These findings indicate that nucleosomal interactions suffice to predict the genome organization in polyomavirus capsids and underscore the adaptable nature of the eukaryotic chromatin architecture to nanoscale confinement.
doi:10.1093/nar/gks1270
PMCID: PMC3561987  PMID: 23258701
5.  Virus strategies for passing the nuclear envelope barrier 
Nucleus  2012;3(6):526-539.
Viruses that replicate in the nucleus need to pass the nuclear envelope barrier during infection. Research in recent years indicates that the nuclear envelope is a major hurdle for many viruses. This review describes strategies to overcome this obstacle developed by seven virus families: herpesviridae, adenoviridae, orthomyxoviridae, lentiviruses (which are part of retroviridae), Hepadnaviridae, parvoviridae and polyomaviridae. Most viruses use the canonical nuclear pore complex (NPC) in order to get their genome into the nucleus. Viral capsids that are larger than the nuclear pore disassemble before or during passing through the NPC, thus allowing genome nuclear entry. Surprisingly, increasing evidence suggest that parvoviruses and polyomaviruses may bypass the nuclear pore by trafficking directly through the nuclear membrane. Additional studies are required for better understanding these processes. Since nuclear entry emerges as the limiting step in infection for many viruses, it may serve as an ideal target for antiviral drug development.
doi:10.4161/nucl.21979
PMCID: PMC3515536  PMID: 22929056
disassembly; nuclear entry; nuclear transport; virus entry; viruses
6.  Plasmalemmal vesicle associated protein (PV1) modulates SV40 virus infectivity in CV-1 cells 
Plasmalemmal vesicle associated protein (Plvap/PV1) is a structural protein required for the formation of the stomatal diaphragms of caveolae. Caveolae are plasma membrane invaginations that were implicated in SV40 virus entry in primate cells. Here we show that de novo Plvap/PV1 expression in CV-1 green monkey epithelial cells significantly reduces the ability of SV40 virus to establish productive infection, when cells are incubated with low concentrations of the virus. However, in presence of high viral titers PV1 has no effect on SV40 virus infectivity. Mechanistically, PV1 expression does not reduce the cell surface expression of known SV40 receptors such as GM1 ganglioside and MHC class I proteins. Furthermore, PV1 does not reduce the binding of virus-like particles made by SV40 VP1 protein to the CV-1 cell surface and does not impact their internalization when cells are incubated with either high or low VLP concentrations. These results suggest that PV1 protein is able to block SV40 infectivity at low but not at high viral concentration either by interfering with the infective internalization pathway at the cell surface or at a post internalization step.
doi:10.1016/j.bbrc.2011.07.063
PMCID: PMC3171970  PMID: 21827737
7.  Simian virus 40 induces lamin A/C fluctuations and nuclear envelope deformation during cell entry 
Nucleus  2011;2(4):320-330.
In non-dividing cells, the nuclear pore complex provides the major route for viruses and viral genomes to enter the nucleus. However, SV40 infection of non-dividing cells is very inefficient suggesting that the nuclear envelope prevents most viral genomes from entering the nucleus. Surprisingly, we observed that following infection of quiescent CV-1 cells with SV40, the nuclear envelope was dramatically deformed, as seen by immunohistochemistry detection of lamins A/C, B1, B2 and the nuclear pore complexes. Accompanying deformation of the nuclear envelope, we also observed fluctuations in the levels of lamin A/C, dephosphorylation of an unknown epitope on lamin A/C and accumulation of lamin A in the cytoplasm. The nuclear envelope deformations occured just prior to and during nuclear entry of the viral genome and were transient and the spherical structure of the nuclear envelope was restored subsequent to nuclear entry. Nuclear envelope deformation and lamin A/C dephosphorylation required caspase-6 cleavage of a small fraction of lamin A/C. Taken together the results suggest that virus-induced alterations of the nuclear lamina, are involved in the nuclear entry of the SV40 genome in non-dividing cells. We propose that SV40 utilize this unique, previously unknown mechanism for direct trafficking of its genome from the ER to the nucleus.
doi:10.4161/nucl.2.4.16371
PMCID: PMC3260569  PMID: 21941111
nuclear envelope; lamina; lamin A/C; SV40; nuclear entry; caspase-6
8.  Simian Virus 40 Infection Triggers a Balanced Network That Includes Apoptotic, Survival, and Stress Pathways▿ †  
Journal of Virology  2010;84(7):3431-3442.
The infection process by simian virus 40 (SV40) and entry of its genome into nondividing cells are only partly understood. Infection begins by binding to GM1 receptors at the cell surface, cellular entry via caveolar invaginations, and trafficking to the endoplasmic reticulum, where the virus disassembles. To gain a deeper insight into the contribution of host functions to this process, we studied cellular signaling elicited by the infecting virus. Signaling proteins were detected by Western blotting and immunofluorescence staining. The study was assisted by a preliminary proteomic screen. The contribution of signaling proteins to the infection process was evaluated using specific inhibitors. We found that CV-1 cells respond to SV40 infection by activating poly(ADP-ribose) polymerase 1 (PARP-1)-mediated apoptotic signaling, which is arrested by the Akt-1 survival pathway and stress response. A single key regulator orchestrating the three pathways is phospholipase C-gamma (PLCγ). The counteracting apoptotic and survival pathways are robustly balanced as the infected cells neither undergo apoptosis nor proliferate. Surprisingly, we have found that the apoptotic pathway, including activation of PARP-1 and caspases, is absolutely required for the infection to proceed. Thus, SV40 hijacks the host defense to promote its infection. Activities of PLCγ and Akt-1 are also required, and their inhibition abrogates the infection. Notably, this signaling network is activated hours before T antigen is expressed. Experiments with recombinant empty capsids, devoid of DNA, indicated that the major capsid protein VP1 alone triggers this early signaling network. The emerging robust signaling network reflects a delicate evolutionary balance between attack and defense in the host-virus relationship.
doi:10.1128/JVI.01735-09
PMCID: PMC2838102  PMID: 20089643
9.  The SV40 capsid is stabilized by a conserved pentapeptide hinge of the major capsid protein VP1 
Journal of molecular biology  2008;386(5):1382-1391.
Summary
The SV40 outer shell is composed of 72 pentamers of VP1. The core of the VP1 monomer is a β-barrel with jelly-roll topology, with extending N and C-terminal arms. A pentapeptide hinge, KNPYP, tethers the C-arm to the VP1 β-barrel core. The five C-arms that extend from each pentamer insert into the neighbouring pentamers, tying them together through different types of interactions. In the mature virion, this element adopts either of 6 conformations, according to their location in the capsid. We found that the hinge is conserved among 16 members of the polyomaviridae, attesting to its importance in capsid assembly and/or structure. We have used site-directed mutagenesis in order to gain an understanding into the structural requirements of this element: Y299 was changed to A, F and T and P300 to A and G. The mutants showed reduction in viability to varying degrees. Unexpectedly, assembly was only reduced to a small extent. Interestingly the data showed that the mutants were highly unstable. The largest effect was observed for mutations of P300, indicating a role of the proline in the virion structure. P300G was more unstable than P300A, indicating requirement for rigidity of the pentapeptide hinge. Y299T and Y299A were more defective in viability than Y299F, highlighting the importance of an aromatic ring at this position. Structural inspection showed that this aromatic ring contacts C-arms of neighbouring pentamers. Computational modelling predicted loss of stability of the Y mutants in concordance with the experimental results. This study provides insights into the structural details of the pentapeptide hinge that are responsible for capsid stability.
doi:10.1016/j.jmb.2008.11.034
PMCID: PMC2718546  PMID: 19094992
SV40; major capsid protein VP1; site-directed mutagenesis; computational binding prediction; protein structure-function
10.  DNA-Free Recombinant SV40 Capsids Protect Mice from Acute Renal Failure by Inducing Stress Response, Survival Pathway and Apoptotic Arrest 
PLoS ONE  2008;3(8):e2998.
Viruses induce signaling and host defense during infection. Employing these natural trigger mechanisms to combat organ or tissue failure is hampered by harmful effects of most viruses. Here we demonstrate that SV40 empty capsids (Virus Like Particles-VLPs), with no DNA, induce host Hsp/c70 and Akt-1 survival pathways, key players in cellular survival mechanisms. We postulated that this signaling might protect against organ damage in vivo. Acute kidney injury (AKI) was chosen as target. AKI is critical, prevalent disorder in humans, caused by nephrotoxic agents, sepsis or ischemia, via apoptosis/necrosis of renal tubular cells, with high morbidity and mortality. Systemic administration of VLPs activated Akt-1 and upregulated Hsp/c70 in vivo. Experiments in mercury-induced AKI mouse model demonstrated that apoptosis, oxidative stress and toxic renal failure were significantly attenuated by pretreatment with capsids prior to the mercury insult. Survival rate increased from 12% to >60%, with wide dose response. This study demonstrates that SV40 VLPs, devoid of DNA, may potentially be used as prophylactic agent for AKI. We anticipate that these finding may be projected to a wide range of organ failure, using empty capsids of SV40 as well as other viruses.
doi:10.1371/journal.pone.0002998
PMCID: PMC2515219  PMID: 18714386
11.  Simian virus 40 vectors for pulmonary gene therapy 
Respiratory Research  2007;8(1):74.
Background
Sepsis remains the leading cause of death in critically ill patients. One of the primary organs affected by sepsis is the lung, presenting as the Acute Respiratory Distress Syndrome (ARDS). Organ damage in sepsis involves an alteration in gene expression, making gene transfer a potential therapeutic modality. This work examines the feasibility of applying simian virus 40 (SV40) vectors for pulmonary gene therapy.
Methods
Sepsis-induced ARDS was established by cecal ligation double puncture (2CLP). SV40 vectors carrying the luciferase reporter gene (SV/luc) were administered intratracheally immediately after sepsis induction. Sham operated (SO) as well as 2CLP rats given intratracheal PBS or adenovirus expressing luciferase served as controls. Luc transduction was evaluated by in vivo light detection, immunoassay and luciferase mRNA detection by RT-PCR in tissue harvested from septic rats. Vector abundance and distribution into alveolar cells was evaluated using immunostaining for the SV40 VP1 capsid protein as well as by double staining for VP1 and for the surfactant protein C (proSP-C). Immunostaining for T-lymphocytes was used to evaluate the cellular immune response induced by the vector.
Results
Luc expression measured by in vivo light detection correlated with immunoassay from lung tissue harvested from the same rats. Moreover, our results showed vector presence in type II alveolar cells. The vector did not induce significant cellular immune response.
Conclusion
In the present study we have demonstrated efficient uptake and expression of an SV40 vector in the lungs of animals with sepsis-induced ARDS. These vectors appear to be capable of in vivo transduction of alveolar type II cells and may thus become a future therapeutic tool.
doi:10.1186/1465-9921-8-74
PMCID: PMC2238754  PMID: 17967178
12.  High Cooperativity of the SV40 Major Capsid Protein VP1 in Virus Assembly 
PLoS ONE  2007;2(8):e765.
SV40 is a small, non enveloped DNA virus with an icosahedral capsid of 45 nm. The outer shell is composed of pentamers of the major capsid protein, VP1, linked via their flexible carboxy-terminal arms. Its morphogenesis occurs by assembly of capsomers around the viral minichromosome. However the steps leading to the formation of mature virus are poorly understood. Intermediates of the assembly reaction could not be isolated from cells infected with wt SV40. Here we have used recombinant VP1 produced in insect cells for in vitro assembly studies around supercoiled heterologous plasmid DNA carrying a reporter gene. This strategy yields infective nanoparticles, affording a simple quantitative transduction assay. We show that VP1 assembles under physiological conditions into uniform nanoparticles of the same shape, size and CsCl density as the wild type virus. The stoichiometry is one DNA molecule per capsid. VP1 deleted in the C-arm, which is unable to assemble but can bind DNA, was inactive indicating genuine assembly rather than non-specific DNA-binding. The reaction requires host enzymatic activities, consistent with the participation of chaperones, as recently shown. Our results demonstrate dramatic cooperativity of VP1, with a Hill coefficient of ∼6. These findings suggest that assembly may be a concerted reaction. We propose that concerted assembly is facilitated by simultaneous binding of multiple capsomers to a single DNA molecule, as we have recently reported, thus increasing their local concentration. Emerging principles of SV40 assembly may help understanding assembly of other complex systems. In addition, the SV40-based nanoparticles described here are potential gene therapy vectors that combine efficient gene delivery with safety and flexibility.
doi:10.1371/journal.pone.0000765
PMCID: PMC1942081  PMID: 17712413
13.  The Abundant Nuclear Enzyme PARP Participates in the Life Cycle of Simian Virus 40 and Is Stimulated by Minor Capsid Protein VP3 
Journal of Virology  2003;77(7):4273-4282.
The abundant nuclear enzyme poly(ADP-ribose) polymerase (PARP) functions in DNA damage surveillance and repair and at the decision between apoptosis and necrosis. Here we show that PARP binds to simian virus 40 (SV40) capsid proteins VP1 and VP3. Furthermore, its enzymatic activity is stimulated by VP3 but not by VP1. Experiments with purified mutant proteins demonstrated that the PARP binding domain in VP3 is localized to the 35 carboxy-terminal amino acids, while a larger peptide of 49 amino acids was required for full stimulation of its activity. The addition of 3-aminobenzamide (3-AB), a known competitive inhibitor of PARP, demonstrated that PARP participates in the SV40 life cycle. The titer of SV40 propagated on CV-1 cells was reduced by 3-AB in a dose-dependent manner. Additional experiments showed that 3-AB did not affect viral DNA replication or capsid protein production. PARP did not modify the viral capsid proteins in in vitro poly(ADP-ribosylation) assays, implying that it does not affect SV40 infectivity. On the other hand, it greatly reduced the magnitude of the host cytopathic effects, a hallmark of SV40 infection. Additional experiments suggested that the stimulation of PARP activity by VP3 leads the infected cell to a necrotic pathway, characterized by the loss of membrane integrity, thus facilitating the release of mature SV40 virions from the cells. Our studies identified a novel function of the minor capsid protein VP3 in the recruitment of PARP for the SV40 lytic process.
doi:10.1128/JVI.77.7.4273-4282.2003
PMCID: PMC150672  PMID: 12634384
14.  Cellular Transcription Factor Sp1 Recruits Simian Virus 40 Capsid Proteins to the Viral Packaging Signal, ses 
Journal of Virology  2002;76(12):5915-5924.
Simian virus 40 (SV40) capsid assembly occurs in the nucleus. All three capsid proteins bind DNA nonspecifically, raising the dilemma of how they attain specificity to the SV40 minichromosome in the presence of a large excess of genomic DNA. The SV40 packaging signal, ses, which is required for assembly, is composed of multiple DNA elements that bind transcription factor Sp1. Our previous studies showed that Sp1 participates in SV40 assembly and that it cooperates in DNA binding with VP2/3. We hypothesized that Sp1 recruits the capsid proteins to the viral minichromosome, conferring upon them specific DNA recognition. Here, we have tested the hypothesis. Computer analysis showed that the combination of six tandem GC boxes at ses is not found at cellular promoters and therefore is unique to SV40. Cooperativity in DNA binding between Sp1 and VP2/3 was not abolished at even a 1,000-fold excess of cellular DNA, providing strong support for the recruitment hypothesis. Sp1 also binds VP1 and cooperates with VP1 in DNA binding. VP1 pentamers (VP15) avidly interact with VP2/3, utilizing the same VP2/3 domain as described for polyomavirus. We conclude that VP15-VP2/3 building blocks are recruited by Sp1 to ses, where they form the nucleation center for capsid assembly. By this mechanism the virus ensures that capsid formation is initiated at a single site around its minichromosome. Sp1 enhances the formation of SV40 pseudovirions in vitro, providing additional support for the model. Analyses of Sp1 and VP3 deletion mutants showed that Sp1 and VP2/3 bind one another and cooperate in DNA binding through their DNA-binding domains, with additional contacts outside these domains. VP1 contacts Sp1 at residues outside the Sp1 DNA-binding domain. These and additional data allowed us to propose a molecular model for the VP15-VP2/3-DNA-Sp1 complex.
doi:10.1128/JVI.76.12.5915-5924.2002
PMCID: PMC136189  PMID: 12021324
15.  Caveolar Endocytosis of Simian Virus 40 Is Followed by Brefeldin A-Sensitive Transport to the Endoplasmic Reticulum, Where the Virus Disassembles 
Journal of Virology  2002;76(10):5156-5166.
Simian virus 40 (SV40) enters cells by atypical endocytosis mediated by caveolae that transports the virus to the endoplasmic reticulum (ER) instead of to the endosomal-lysosomal compartment, which is the usual destination for viruses and other cargo that enter by endocytosis. We show here that SV4O is transported to the ER via an intermediate compartment that contains β-COP, which is best known as a component of the COPI coatamer complexes that are required for the retrograde retrieval pathway from the Golgi to the ER. Additionally, transport of SV40 to the ER, as well as infection, is sensitive to brefeldin A. This drug acts by specifically inhibiting the ARF1 GTPase, which is known to regulate assembly of COPI coat complexes on Golgi cisternae. Moreover, some β-COP colocalizes with intracellular caveolin-1, which was previously shown to be present on a new organelle (termed the caveosome) that is an intermediate in the transport of SV40 to the ER (L. Pelkmans, J. Kartenbeck, and A. Helenius, Nat. Cell Biol. 3:473-483, 2001). We also show that the internal SV40 capsid proteins VP2 and VP3 become accessible to immunostaining starting at about 5 h. Most of that immunostaining overlays the ER, with some appearing outside of the ER. In contrast, immunostaining with anti-SV40 antisera remains confined to the ER.
doi:10.1128/JVI.76.10.5156-5166.2002
PMCID: PMC136127  PMID: 11967331
16.  Initiation Points for DNA Replication in Nontransformed and Simian Virus 40-Transformed BALB/c 3T3 Cells 
Journal of Virology  1978;25(1):450-452.
The number of initiation points for DNA synthesis per unit length of DNA in rapidly growing cells is greater for simian virus 40-transformed than for nontransformed BALB/c 3T3 cells.
PMCID: PMC353948  PMID: 202755

Results 1-16 (16)