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ABSTRACT: 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.
Nucleus (Austin, Texas) 07/2011; 2(4):320-30.
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ABSTRACT: 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.
Journal of virological methods 12/2009; 164(1-2):145-7. · 2.13 Impact Factor
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Nilly Shimony,
Moise Bendayan,
Gregory Elkin, Orly Ben-nun-Shaul,
Mahmoud Abd-El-Latif,
Pnina Scherzer,
Ofer Arbel,
Ehud Ziv,
Lina Krasny,
Galina Pizov,
Ariella Oppenheim,
Yosef S Haviv
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ABSTRACT: Viral vector uptake into the pancreas is rare. The few viral vectors reported to transduce in vivo pancreatic islets after systemic injection required additional physical measures, such as direct pancreatic injection or hepatic vessel clamping. Because pancreatic islet uptake of the human polyomavirus family member BK virus was previously reported in hamsters after systemic administration, we hypothesized that SV40, a polyomavirus member remarkably similar to BK virus, may also infect the pancreas.
We injected intravenously a low dose of SV40, unaided by any other physical or chemical means, and evaluated viral uptake by pancreatic islets and pancreatic exocrine tissue via polymerase chain reaction, Western blot, electron microscopy, immunofluorescent microscopy, and protein A-gold immunocytochemistry.
Pancreatic uptake of SV40 was comparable to other major organs (ie, liver and spleen). SV40 viral particles were detected in both pancreatic islets and acini. In pancreatic islets, all islet cell types were infected by SV40, albeit the infection rate of glucagon-producing alpha cells surpassed beta- and delta-islet cells. Low-dose SV40 administration was not sufficient to induce heterologous gene expression in the pancreas.
Our study shows that pancreatic islet and acinar cell uptake of SV40 is feasible with a single, low-dose intravenous injection. However, this dose did not result in gene delivery into the murine pancreas.
Pancreas 06/2008; 36(4):411-6. · 2.39 Impact Factor
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ABSTRACT: 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 approximately 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.
PLoS ONE 02/2007; 2(8):e765. · 4.09 Impact Factor
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ABSTRACT: Chronic HBV infection, a world-wide epidemic, can lead to chronic hepatitis and eventually to cirrhosis and hepatocellular carcinoma. The liver poses obstacles for many available gene-transfer vectors. SV40-based vectors can transduce human hepatic and hematopoietic cells. We studied the effect of HBV on the transduction - efficiency of human hepatic cells by SV40 - based vectors.
A SV40-vector carrying the luciferase gene, and wild-type SV40, were used to assess transduction efficiency of human HBV-positive and HBV-negative hepatic cells. Transduction efficiency was measured as luciferase activity or by T-antigen staining. To evaluate whether differences in transduction efficiency are due to cell recognition and/or nuclear transport, MHC-I receptors were measured by FACS analysis and SV40-DNA was extracted from the nuclei of transduced cells and quantified.
Two HBV-positive cell-lines, HepG2.2.2.15 and FLC4-A10II, were transduced significantly more efficiently than their parental HBV-negative cell-lines. Transient transfection of HuH-7 cells with the HBV genome also increased transduction efficiency. The level of MHC-I, the cellular receptor for SV40, was comparable in all the cell-lines studied. However, soon after infection with SV40, the nuclei of HepG2.2.2.15 contained >6-fold more SV40-DNA than HepG2.
HBV increases transduction by SV40-vectors. This is due to enhanced vector entry and/or transport into the nucleus. SV40-vectors appear to have a potential for gene therapy for the treatment of HBV infections.
Journal of Hepatology 04/2004; 40(3):520-6. · 9.26 Impact Factor
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ABSTRACT: Simian virus 40 (SV40) vectors are efficient vehicles for gene delivery to hematopoietic and hepatic cells. To ensure their replication incompetence and because of safety considerations, it is critical that the vectors do not contain T-antigen sequences. Available packaging cell lines for T-antigen replacement vectors, COS and CMT4, contain considerable sequence identity with the vectors, leading to homologous recombination and reacquisition of the T-antigen gene. We constructed a packaging cell line, COT18, with minimal sequence identity to the vector. Vector stocks produced by passaging on COT18 had high transducing activity and undetectable levels of T-antigen-positive, replication-competent contaminants. This cell line provides a means for the preparation of safe SV40 vector stocks.
Virology 01/2003; 304(2):155-9. · 3.35 Impact Factor
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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 (VP1(5)) avidly interact with VP2/3, utilizing the same VP2/3 domain as described for polyomavirus. We conclude that VP1(5)-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 VP1(5)-VP2/3-DNA-Sp1 complex.
Journal of Virology 07/2002; 76(12):5915-24. · 5.40 Impact Factor
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ABSTRACT: 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.
Human Gene Therapy 02/2002; 13(2):299-310. · 4.22 Impact Factor
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ABSTRACT: Chromatin structure and protein-protein interactions play an important role in eukaryotic gene function. Nucleosomal rearrangement at the simian virus 40 (SV40) regulatory region occurs at the late stages of the viral life cycle preceding viral assembly. The SV40 capsid proteins are required for this nucleosomal rearrangement suggesting that they participate in turning-off the viral promoters. In aiming to elucidate the role of the capsid proteins in gene regulation, we studied the interaction between VP3, an internal capsid protein, and the cellular transcription factor Sp1, a major regulator of both the early and late viral promoters. Our results showed that VP3 repressed transcription from the viral early promoter in vitro. We found significant cooperativity between Sp1 and VP3 in specific DNA-binding to the Sp1 binding site. In addition, protein-protein interactions between VP3 and Sp1 in the absence of DNA were observed. These findings have led us to conclude that the novel host-viral Sp1-VP3 complex down regulates viral transcription and further suggest that Sp1 participates in recruiting VP3 to the SV40 minichromosome in SV40 assembly.
Journal of Molecular Biology.
