Orly Ben-nun-Shaul

Cellular Transcription Factor Sp1 Recruits Simian Virus 40 Capsid Proteins to the Viral Packaging Signal, ses

ABSTRACT 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.

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.

Simian virus 40 induces lamin A/C fluctuations and nuclear envelope deformation during cell entry

The canonical gate of viruses and viral genomes into the nucleus in non-dividing cells is the nuclear pore, embedded within the nuclear envelope. However, we found that for SV40, the nuclear envelope poses a major hurdle to infection: FISH analysis revealed that the majority of viral DNA remains trapped in the ER; silencing of Lamin A/C rendered the cells more susceptible to infection; and proliferating cells are more susceptible to infection than quiescent cells. Surprisingly, we observed that following SV40 infection the nuclear envelope, including lamins A/C, B1, B2 and the nuclear pore complex, was dramatically deformed, as seen by immunohistochemistry. The infection induced fluctuations in the level of lamin A/C, dephosphorylation of an unknown epitope and leakage to the cytoplasm just prior to and during nuclear entry. Deformations were transient, and the spherical structure of the nuclear envelope was restored subsequent to nuclear entry. Nuclear envelope deformations and lamin A/C dephosphorylation depended on caspase-6 cleavage of lamin A/C. Notably, we have previously reported that inhibition of caspase-6 abolishes SV40 infection. Taken together the results suggest that alterations of the nuclear lamina, induced by the infecting virus, are involved in the nuclear entry of the SV40 genome. We propose that SV40 utilize this unique, previously unknown mechanism for direct trafficking of its genome from the ER to the nucleus. As SV40 serves as a paradigm for the pathogenic human BK, JC and Merkel cell polyomavirus, this study suggests nuclear entry as a novel drug target for these infections.

High cooperativity of the SV40 major capsid protein VP1 in virus assembly.

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.

Rapid method for SV40 titration

SV40 titer is determined traditionally by the conventional plaque assay. Plaques appear after several rounds of infection and the assay takes around two weeks, which may delay research. A simpler assay was developed, based on detection of T-antigen in the infected cells by flow cytometry. Cells grown in 6-well plates are infected with serial dilutions of the viral stock, harvested 48h post-infection, stained and analyzed for T-antigen using a flow cytometer. The viral titer is calculated based on the percentage of T-antigen positive cells. The procedure is accomplished in 2 days. Unexpectedly we found that titers on different permissive African Green Monkey kidney cell lines were consistently different, suggesting variable susceptibility to SV40 infection. The method described, optimized for SV40 titration, may be adapted readily to other viruses.

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.

The SV40 Capsid Is Stabilized by a Conserved Pentapeptide Hinge of the Major Capsid Protein VP1

The simian virus 40 (SV40) outer shell is composed of 72 pentamers of VP1. The core of the VP1 monomer is a beta-barrel with jelly-roll topology and extending N- and C-terminal arms. A pentapeptide hinge, KNPYP, tethers the C-arm to the VP1 beta-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 six 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 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 reduced only to a small extent. However, 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 a 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.

SV40 assembly in vivo and in vitro

The Simian virus 40 (SV40) capsid is a T = 7d icosahedral lattice ∼45 nm in diameter surrounding the ∼5 kb circular minichromosome. The outer shell is composed of 360 monomers of the major capsid protein VP1, tightly bound in 72 pentamers. VP1 is a jellyroll β-barrel, with extending N- and C-terminal arms. The N-terminal arms bind DNA and face the interior of the capsid. The flexible C-arms tie together the 72 pentamers in three distinct kinds of interactions, thus facilitating the formation of a T = 7 icosahedron from identical pentameric building blocks. Assembly in vivo was shown to occur by addition of capsomers around the DNA. We apply a combination of biochemical and genetic approaches to study SV40 assembly. Our in vivo and in vitro studies suggest the following model: one or two capsomers bind at a high affinity to ses, the viral DNA encapsidation signal, forming the nucleation centre for assembly. Next, multiple capsomers attach concomitantly, at lower affinity, around the minichromosome. This increases their local concentration facilitating rapid, cooperative assembly reaction. Formation of the icosahedron proceeds either by gradual addition of single pentamers to the growing shell or by concerted assembly of pentamer clusters.

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.