pH stability and disassembly mechanism of wild-type simian virus 40.

Viruses are remarkable self-assembled nanobiomaterial-based machines, exposed to a wide range of pH values. Extreme pH values can induce dramatic structural changes, critical for the function of the virus nanoparticles, including assembly and genome uncoating. Tuning cargo-capsid interactions is essential for designing virus-based delivery systems. Here we show how pH controls the structure and activity of wild-type simian virus 40 (wtSV40) and the interplay between its cargo and capsid. Using cryo-TEM and solution X-ray scattering, we found that wtSV40 was stable between pH 5.5 and 9, and only slightly swelled with increasing pH. At pH 3, the particles aggregated, while capsid protein pentamers continued to coat the virus cargo but lost their positional correlations. Infectivity was only partly lost after the particles were returned to pH 7. At pH 10 or higher, the particles were unstable, lost their infectivity, and disassembled. Using time-resolved experiments we discovered that disassembly began by swelling of the particles, poking a hole in the capsid through which the genetic cargo escaped, followed by a slight shrinking of the capsids and complete disassembly. These findings provide insight into the fundamental intermolecular forces, essential for SV40 function, and for designing virus-based nanobiomaterials, including delivery systems and antiviral drugs.

Pathogen receptor discovery with a microfluidic human membrane protein array.

The discovery of how a pathogen invades a cell requires one to determine which host cell receptors are exploited. This determination is a challenging problem because the receptor is invariably a membrane protein, which represents an Achilles heel in proteomics. We have developed a universal platform for high-throughput expression and interaction studies of membrane proteins by creating a microfluidic-based comprehensive human membrane protein array (MPA). The MPA is, to our knowledge, the first of its kind and offers a powerful alternative to conventional proteomics by enabling the simultaneous study of 2,100 membrane proteins. We characterized direct interactions of a whole nonenveloped virus (simian virus 40), as well as those of the hepatitis delta enveloped virus large form antigen, with candidate host receptors expressed on the MPA. Selected newly discovered membrane protein-pathogen interactions were validated by conventional methods, demonstrating that the MPA is an important tool for cellular receptor discovery and for understanding pathogen tropism.

Effect of capsid confinement on the chromatin organization of the SV40 minichromosome.

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.

Receptor concentration and diffusivity control multivalent binding of Sv40 to membrane bilayers.

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.

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 (500mers). This assembly process yields T = 1 icosahedral particles comprised of 12 pentamers and one RNA molecule. The reaction is nearly one-third complete within 35 ms, 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 10(9) 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.

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.

Simian virus 40 infection triggers a balanced network that includes apoptotic, survival, and stress pathways.

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 (PLCgamma). 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 PLCgamma 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.

Empty SV40 capsids increase survival of septic rats by eliciting numerous host signaling networks that participate in a number of systemic functions.

Sepsis is an excessive, dysregulated immune response to infection that activates inflammatory and coagulation cascades, which may lead to tissue injury, multiple organ dysfunction syndrome and death. Millions of individuals die annually of sepsis. To date, the only treatment available is antibiotics, drainage of the infection source when possible, and organ support in intensive care units. Numerous previous attempts to develop therapeutic treatments, directed at discreet targets of the sepsis cascade, could not cope with the complex pathophysiology of sepsis and failed. Here we describe a novel treatment, based on empty capsids of SV40 (nanocapsids - NCs). Studies in a severe rat sepsis model showed that pre-treatment by NCs led to a dramatic increase in survival, from zero to 75%. Transcript analyses (RNAseq) demonstrated that the NC treatment is a paradigm shift. The NCs affect multiple facets of biological functions. The affected genes are modified with time, adjusting to the recovery processes. The NCs effect on normal control rats was negligible. The study shows that the NCs are capable of coping with diseases with intricate pathophysiology. Further studies are needed to determine whether when applied after sepsis onset, the NCs still improve outcome.

Effect of Calcium Ions and Disulfide Bonds on Swelling of Virus Particles.

Multivalent ions affect the structure and organization of virus nanoparticles. Wild-type simian virus 40 (wt SV40) is a nonenveloped virus belonging to the polyomavirus family, whose external diameter is 48.4 nm. Calcium ions and disulfide bonds are involved in the stabilization of its capsid and are playing a role in its assembly and disassembly pathways. Using solution small-angle X-ray scattering (SAXS), we found that the volume of wt SV40 swelled by about 17% when both of its calcium ions were chelated by ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid and its disulfide bonds were reduced by dithiothreitol. By applying osmotic stress, the swelling could be reversed. DNA-containing virus-like particles behaved in a similar way. The results provide insight into the structural role of calcium ions and disulfide bonds in holding the capsid proteins in compact conformation.

Simian virus 40 vectors for pulmonary gene therapy.

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.

Crystallization, Reentrant Melting, and Resolubilization of Virus Nanoparticles.

Crystallization is a fundamental and ubiquitous process that is well understood in the case of atoms or small molecules, but its outcome is still hard to predict in the case of nanoparticles or macromolecular complexes. Controlling the organization of virus nanoparticles into a variety of 3D supramolecular architectures is often done by multivalent ions and is of great interest for biomedical applications such as drug or gene delivery and biosensing, as well as for bionanomaterials and catalysis. In this paper, we show that slow dialysis, over several hours, of wild-type Simian Virus 40 (wt SV40) nanoparticle solution against salt solutions containing MgCl2, with or without added NaCl, results in wt SV40 nanoparticles arranged in a body cubic center crystal structure with Im3m space group, as a thermodynamic product, in coexistence with soluble wt SV40 nanoparticles. The nanoparticle crystals formed above a critical MgCl2 concentrations. Reentrant melting and resolubilization of the virus nanoparticles took place when the MgCl2 concentrations passed a second threshold. Using synchrotron solution X-ray scattering we determined the structures and the mass fraction of the soluble and crystal phases as a function of MgCl2 and NaCl concentrations. A thermodynamic model, which balances the chemical potentials of the Mg2+ ions in each of the possible states, explains our observations. The model reveals the mechanism of both the crystallization and the reentrant melting and resolubilization and shows that counterion entropy is the main driving force for both processes.

p53 elevation in human cells halt SV40 infection by inhibiting T-ag expression.

SV40 large T-antigen (T-ag) has been known for decades to inactivate the tumor suppressor p53 by sequestration and additional mechanisms. Our present study revealed that the struggle between p53 and T-ag begins very early in the infection cycle. We found that p53 is activated early after SV40 infection and defends the host against the infection. Using live cell imaging and single cell analyses we found that p53 dynamics are variable among individual cells, with only a subset of cells activating p53 immediately after SV40 infection. This cell-to-cell variabilty had clear consequences on the outcome of the infection. None of the cells with elevated p53 at the beginning of the infection proceeded to express T-ag, suggesting a p53-dependent decision between abortive and productive infection. In addition, we show that artificial elevation of p53 levels prior to the infection reduces infection efficiency, supporting a role for p53 in defending against SV40. We further found that the p53-mediated host defense mechanism against SV40 is not facilitated by apoptosis nor via interferon-stimulated genes. Instead p53 binds to the viral DNA at the T-ag promoter region, prevents its transcriptional activation by Sp1, and halts the progress of the infection. These findings shed new light on the long studied struggle between SV40 T-ag and p53, as developed during virus-host coevolution. Our studies indicate that the fate of SV40 infection is determined as soon as the viral DNA enters the nucleus, before the onset of viral gene expression.

Downregulation of the stress-induced ligand ULBP1 following SV40 infection confers viral evasion from NK cell cytotoxicity.

Polyomaviruses are a diverse family of viruses which are prevalent in the human population. However, the interactions of these viruses with the immune system are not well characterized. We have previously shown that two human polyomaviruses, JC and BK, use an identical microRNA to evade immune attack by Natural Killer (NK) cells. We showed that this viral microRNA suppresses ULBP3 expression, a stress induced ligand for the killer receptor NKG2D. Here we show that Simian Virus 40 (SV40) also evades NK cell attack through the down regulation of another stress-induced ligand of NKG2D, ULBP1. These findings indicate that NK cells play an essential role in fighting polyomavirus infections and further emphasize the importance of various members of the ULBP family in controlling polyomavirus infection.

Liver-targeted gene therapy by SV40-based vectors using the hydrodynamic injection method.

Efficient reconstitution of defective genes in hepatocytes could be used to treat various liver and systemic diseases through gene therapy. To explore the potential of SV40-based vectors in liver gene therapy, we constructed SV/luc, an SV40 T-antigen replacement transduction vector, that was propagated on COS and COT cells, which supply the SV40 T-antigen in trans. For liver targeting, BALB/C mice were injected via the tail vein with SV/luc stocks containing 3 x 10(6) to 10(8) transducing units in a volume of 1-2 ml. Luciferase activity was monitored with a light-detection cooled charged-coupled device (CCCD) camera, which enables continuous in vivo measurement of luc expression. The SV40 vector proved to be efficient in gene delivery to the liver, leading to long-term (> or =107 days) transgene expression in hepatocytes. Optimal results were obtained with 3 x 10(6) to 3 x 10(7) transducing units. The hydrodynamic vector delivery method caused transient liver inflammatory changes, with full recovery within days. Low levels of SV40-neutralizing antibodies were detected in the sera of treated mice; however, there was no indication of vector or transgene-specific cellular immune responses. Vectors packaged in vitro, using recombinant capsid proteins and plasmid DNA, were also effective in liver transduction. These results suggest that SV40 vectors may be useful for liver gene therapy.

Y chromosome evidence for a founder effect in Ashkenazi Jews.

Recent genetic studies, based on Y chromosome polymorphic markers, showed that Ashkenazi Jews are more closely related to other Jewish and Middle Eastern groups than to their host populations in Europe. However, Ashkenazim have an elevated frequency of R-M17, the dominant Y chromosome haplogroup in Eastern Europeans, suggesting possible gene flow. In the present study of 495 Y chromosomes of Ashkenazim, 57 (11.5%) were found to belong to R-M17. Detailed analyses of haplotype structure, diversity and geographic distribution suggest a founder effect for this haplogroup, introduced at an early stage into the evolving Ashkenazi community in Europe. R-M17 chromosomes in Ashkenazim may represent vestiges of the mysterious Khazars.

High cloning capacity of in vitro packaged SV40 vectors with no SV40 virus sequences.

In vitro packaging of plasmid DNA using recombinant SV40 capsid proteins is a potentially useful procedure that overcomes some restrictions of the other SV40 systems such as the requirement for SV40 sequences and the limitation in size of DNA that can be packaged. The in vitro packaging system uses the four SV40 proteins (VP1, VP2, VP3, and agno) or VP1 only. The ability to confer drug resistance by three ABC transporter genes (MDR 1, MRP 1, or MXR) was determined using the surrogate fluorescent substrates rhodamine-123 or calcein AM and their specific inhibitors, or by using specific antibodies to the transporters to detect cell surface expression by fluorescence-activated cell sorter analysis (FACS). A green fluorescent protein plasmid (EGFP-C1) was also used to monitor gene transfer. The packaged plasmids ranged in size from 4.2 to 17.6 kb, and only slightly affected particle size as determined by electron microscopy. When 9.5 kb and larger plasmids were packaged using all SV40 proteins, MDR1 expression was decreased compared to VP1 alone. The size of the 15.2 kb DNA after packaging was the same as the original DNA. Packaging with SV40 capsid proteins in vitro does not require any SV40 sequences. Using either the MDR1 or the GFP gene we could demonstrate enhanced expression when cells were pretreated with phorbol 12-myristate 13-acetate (PMA) at low concentrations. Interferon-gamma did not alter expression. We conclude that in vitro packaging is more flexible then previously realized, permitting packaging of at least 17 kb plasmid DNA without the requirement for any viral sequences. This system combines efficient gene delivery of the SV40 viral vector with the presumed safety of nonviral vectors.

In vitro-packaged SV40 pseudovirions as highly efficient vectors for gene transfer.

A procedure for in vitro packaging of plasmid DNA in recombinant SV40 capsid proteins was developed by Sandalon et al. (1997). Here, we report the highly efficient transduction into different human, murine and monkey cell lines using a scaled-up protocol for producing SV40 pseudovirions, packaged in vitro, carrying the human multidrug-resistance gene MDR1 encoding P-glycoprotein (P-gp) or the green fluorescent protein reporter gene (GFP) under control of SV40 and cytomegalovirus (CMV) promoters. The percentage of expressing cells was proportional to the number of transducing particles, with close to 100% of cells transduced at optimal ratios of transducing particles to cells. The ability to confer multidrug resistance was evaluated by measuring dye efflux and cell-surface expression in infected cells. The relative level of expression of P-gp driven by the different promoters varied among different cell lines. In human lymphoblastoid cells, which express high levels of major histocompatibility complex (MHC) class I (a surface receptor for SV40), constructs that carry an intron yield the highest expression. Our experiments further demonstrate that MDR1 and GFP expression driven by these promoters is transient; however, transduced cells remain MDR1-positive if selected in colchicine. Thus, the SV40 vectors are well suited to situations in which only short-term expression is required or expression is selected, such as for bone marrow protection during chemotherapy.

Scaffold properties are a key determinant of the size and shape of self-assembled virus-derived particles.

Controlling the geometry of self-assembly will enable a greater diversity of nanoparticles than now available. Viral capsid proteins, one starting point for investigating self-assembly, have evolved to form regular particles. The polyomavirus SV40 assembles from pentameric subunits and can encapsidate anionic cargos. On short ssRNA (≤814 nt), SV40 pentamers form 22 nm diameter capsids. On RNA too long to fit a T = 1 particle, pentamers forms strings of 22 nm particles and heterogeneous particles of 29-40 nm diameter. However, on dsDNA SV40 forms 50 nm particles composed of 72 pentamers. A 7.2-Å resolution cryo-EM image reconstruction of 22 nm particles shows that they are built of 12 pentamers arranged with T = 1 icosahedral symmetry. At 3-fold vertices, pentamers each contribute to a three-helix triangle. This geometry of interaction is not seen in crystal structures of T = 7 viruses and provides a structural basis for the smaller capsids. We propose that the heterogeneous particles are actually mosaics formed by combining different geometries of interaction from T = 1 capsids and virions. Assembly can be trapped in novel conformations because SV40 interpentamer contacts are relatively strong. The implication is that by virtue of their large catalog of interactions, SV40 pentamers have the ability to self-assemble on and conform to a broad range of shapes.

Integrin-mediated signaling induced by simian virus 40 leads to transient uncoupling of cortical actin and the plasma membrane.

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.

Pathogens use structural mimicry of native host ligands as a mechanism for host receptor engagement.

A pathogen's ability to engage host receptors is a critical determinant of its host range and interspecies transmissibility, key issues for understanding emerging diseases. However, the identification of host receptors, which are also attractive drug targets, remains a major challenge. Our structural bioinformatics studies reveal that both bacterial and viral pathogens have evolved to structurally mimic native host ligands (ligand mimicry), thus enabling engagement of their cognate host receptors. In contrast to the structural homology, amino acid sequence similarity between pathogen molecules and the mimicked host ligands was low. We illustrate the utility of this concept to identify pathogen receptors by delineating receptor tyrosine kinase Axl as a candidate receptor for the polyomavirus SV40. The SV40-Axl interaction was validated, and its participation in the infection process was verified. Our results suggest that ligand mimicry is widespread, and we present a quick tool to screen for pathogen-host receptor interactions.