Simian Virus 40 Infection Research Articles

Simian virus 40 transformation, malignant mesothelioma and brain tumors

Simian virus 40 (SV40) is a DNA virus isolated in 1960 from contaminated polio vaccines, that induces mesotheliomas, lymphomas, brain and bone tumors, and sarcomas, including osteosarcomas, in hamsters. These same tumor types have been found to contain SV40 DNA and proteins in humans. Mesotheliomas and brain tumors are the two tumor types that have been most consistently associated with SV40, and the range of positivity has varied about from 6 to 60%, although a few reported 100% of positivity and a few reported 0%. It appears unlikely that SV40 infection alone is sufficient to cause human malignancy, as we did not observe an epidemic of cancers following the administration of SV40-contaminated vaccines. However, it seems possible that SV40 may act as a cofactor in the pathogenesis of some tumors. In vitro and animal experiments showing cocarcinogenicity between SV40 and asbestos support this hypothesis.

BiP and Multiple DNAJ Molecular Chaperones in the Endoplasmic Reticulum Are Required for Efficient Simian Virus 40 Infection

ABSTRACTSimian virus 40 (SV40) is a nonenveloped DNA virus that traffics through the endoplasmic reticulum (ER) en route to the nucleus, but the mechanisms of capsid disassembly and ER exit are poorly understood. We conducted an unbiased RNA interference screen to identify cellular genes required for SV40 infection. SV40 infection was specifically inhibited by up to 50-fold by knockdown of four different DNAJ molecular cochaperones or by inhibition of BiP, the Hsp70 partner of DNAJB11. These proteins were not required for the initiation of capsid disassembly, but knockdown markedly inhibited SV40 exit from the ER. In addition, BiP formed a complex with SV40 capsids in the ER in a DNAJB11-dependent fashion. These experiments identify five new cellular proteins required for SV40 infection and suggest that the binding of BiP to the capsid is required for ER exit. Further studies of these proteins will provide insight into the molecular mechanisms of polyomavirus infection and ER function.

Evidence of simian virus 40 infection in multiple organ transplant recipients with renal dysfunction

Electron microscopy (EM), real-time polymerase chain reaction (PCR) and conventional PCR were used to identify viruses associated with infection in 2 transplantation patients. An autologous haematopoietic stem cell, liver and renal transplant recipient was found to be positive for simian virus 40 (SV40). Dual BK virus and SV40 infection was found in a heart and renal transplantation patient. SV40 infection can occur in immunocompromised patients.

Multiple DNA Damage Signaling and Repair Pathways Deregulated by Simian Virus 40 Large T Antigen

We demonstrated previously that expression of simian virus 40 (SV40) large T antigen (LT), without a viral origin, is sufficient to induce the hallmarks of a cellular DNA damage response (DDR), such as focal accumulation of gamma-H2AX and 53BP1, via Bub1 binding. Here we expand our characterization of LT effects on the DDR. Using comet assays, we demonstrate that LT induces overt DNA damage. The Fanconi anemia pathway, associated with replication stress, becomes activated, since FancD2 accumulates in foci, and monoubiquitinated FancD2 is detected on chromatin. LT also induces a distinct set of foci of the homologous recombination repair protein Rad51 that are colocalized with Nbs1 and PML. The FancD2 and Rad51 foci require neither Bub1 nor retinoblastoma protein binding. Strikingly, wild-type LT is localized on chromatin at, or near, the Rad51/PML foci, but the LT mutant in Bub1 binding is not localized there. SV40 infection was previously shown to trigger ATM activation, which facilitates viral replication. We demonstrate that productive infection also triggers ATR-dependent Chk1 activation and that Rad51 and FancD2 colocalize with LT in viral replication centers. Using small interfering RNA (siRNA)-mediated knockdown, we demonstrate that Rad51 and, to a lesser extent, FancD2 are required for efficient viral replication in vivo, suggesting that homologous recombination is important for high-level extrachromosomal replication. Taken together, the interplay of LT with the DDR is more complex than anticipated, with individual domains of LT being connected to different subcomponents of the DDR and repair machinery.

Binding of p110 Retinoblastoma Protein Inhibits Nuclear Import of Simian Virus SV40 Large Tumor Antigen

Nuclear import of the simian virus 40 large tumor antigen (T-ag) is dependent on its nuclear localization signal (NLS) within amino acids 126-132 that is recognized by the importin alpha/beta1 heterodimer, as well as a protein kinase CK2 site at serine 112 upstream of the NLS, which enhances the interaction approximately 50-fold. Here we show for the first time that T-ag nuclear import is negatively regulated by N-terminal sequences (amino acids 102-110), which represent the binding site (BS) for the retinoblastoma (Rb) tumor suppressor protein (p110(Rb)). Quantitative confocal laser scanning microscopic analysis of the transport properties of T-ag constructs with or without Rb binding site mutations in living transfected cells or in a reconstituted nuclear transport system indicates that the presence of the RbBS significantly reduces nuclear accumulation of T-ag. A number of approaches, including the analysis of T-ag nuclear import in an isogenic cell pair with and without functional p110(Rb) implicate p110(Rb) binding as being responsible for the reduced nuclear accumulation, with the Ser(106) phosphorylation site within the RbBS appearing to enhance the inhibitory effect. Immunoprecipitation experiments confirmed association of T-ag and p110(Rb) and dependence thereof on negative charge at Ser(106). The involvement of p110(Rb) in modulating T-ag nuclear transport has implications for the regulation of nuclear import of other proteins from viruses of medical significance that interact with p110(Rb), and how this may relate to transformation.

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.

Polyomavirus Infection and Its Impact on Renal Function and Long-term Outcomes After Lung Transplantation

Polyomavirus infection causes nephropathy after kidney transplantation but has not been thoroughly investigated in nonrenal organ transplantation. Ninety lung transplant recipients were enrolled, and they provided urine samples for over 4.5 years. Samples were analyzed for BK virus (BKV), JC virus (JCV), and simian virus 40 (SV40) by conventional and quantitative real-time polymerase chain reaction. Fifty-nine (66%) patients had polyomavirus detected at least once, including 38 patients (42%) for BKV, 25 patients (28%) for JCV, and six patients (7%) for SV40. Frequency of virus shedding in serial urine samples by patients positive at least once varied significantly among viruses: JCV, 64%; BKV, 48%; and SV40, 14%. Urinary viral loads for BKV (10 copies/mL) and JCV (10 copies/mL) were higher than for SV40 (10 copies/mL; P=0.001 and 0.0003, respectively). Polyomavirus infection was associated with a pretransplant diagnosis of chronic obstructive pulmonary disease (odds ratio 6.0; P=0.016) but was less common in patients with a history of acute rejection (odds ratio 0.28; P=0.016). SV40 infection was associated with sirolimus-based immunosuppression (P=0.037). Reduced survival was noted for patients with BKV infection (P=0.03). Patients with polyomavirus infection did not have worse renal function than those without infection, but in patients with BKV infection, creatinine clearances were lower at times when viral shedding was detected (P=0.038). BKV and JCV were commonly detected in the urine of lung transplant recipients; SV40 was found at low frequency. No definite impact of polyomavirus infection on renal function was documented. BKV infection was associated with poorer survival.

The molecular and cellular basis of infection—Perspectives from the first advanced summer school in Africa

The molecular and cellular basis of infection—Perspectives from the first advanced summer school in Africa

Productive Simian Virus 40 Infection of Neurons in Immunosuppressed Rhesus Monkeys

There currently is no animal model of JC virus-associated progressive multifocal leukoencephalopathy (PML). Reactivation of simian virus 40 (SV40) in immunosuppressed rhesus monkeys, however, rarely causes a PML-like illness. We sought to isolate a neurotropic clone of SV40 and determine its pathogenic potential in monkeys. The clone SV40CNS1 was amplified by polymerase chain reaction from the brain DNA of a simian/human immunodeficiency virus-infected monkey that had developed PML and meningoencephalitis. Compared with the SV40 prototype 776, SV40CNS1 had a small number of single-amino-acid mutations and caused a productive infection in monkey fibroblasts. It was inoculated into 2 SV40-negative, simian/human immunodeficiency virus-immunosuppressed monkeys. Both animals developed meningoencephalitis with productive SV40 infection of cerebral cortical neurons and glia in the superficial layers of the cortex and at the gray-white junction. Focal SV40-infected cells were also found in the cerebellar molecular and granule cell layers and white matter. Both animals also developed disseminated SV40 infection with nephritis and pneumonitis. Thus, SV40CNS1 is infectious and pathogenic in immunosuppressed monkeys, but it induces encephalitis with fulminant productive infection in cortical neurons and systemic disease, rather than PML. These findings shed new light on SV40 neurotropism and expand the host cell range of this virus.

SV40 large T antigen‐specific human T cell memory responses

The continued presence of simian virus 40 (SV40), a monkey polyomavirus, in man is confirmed by the regular detection of SV40-specific antibodies in 5-10% of children who are unlikely to have received contaminated polio-vaccines. The aim of our experiments was to find cellular immunological evidence of SV40 infection in humans by testing memory T cell responses to SV40 large T antigen (Tag). As there is some indication that the virus may be present in malignant pleural mesothelioma (MPM) cells, we analyzed T cell responses in MPM patients and in healthy donors. The frequencies of responding T cells to overlapping Tag peptides were tested by cytokine flow cytometry. CD8+ T cells from 4 of 32 MPM patients responded (above twofold of control) to SV40 Tag peptides, while no positive responses were detected in 12 healthy donors. Within SV40 Tag we identified three 15 amino acid-long immunogenic sequences and one 9 amino acid-long T cell epitope (p138) (138FPSELLSFL146), the latter including a HLA-B7-restriction motif. T cell responses to p138 were SV40-specific as T cells stimulated with p138 did not cross-react with the corresponding sequences of Tag of human polyomaviruses BKV and JCV. Similarly, the relevant BKV and JCV Tag peptides did not generate T cell responses against SV40 TAg p138. Peptide-stimulated T cells also killed SV40 Tag-transfected target cells. This article demonstrates the presence, and provides a detailed analysis, of SV40-specific T cell memory in man.

Mesothelioma Epidemiology, Carcinogenesis, and Pathogenesis

The incidence of mesothelioma has gone from almost none to the current 2500-3000 cases per year in the USA. This estimate is an extrapolation based on information available from the Surveillance, Epidemiology and End Results (SEER) Program that collects information on approximately 12% of the US population. Mesothelioma is a cancer that is linked to exposure to carcinogenic mineral fibers. Asbestos and erionite have a proven causative role; the possible role of other mineral fibers in causing mesothelioma is being investigated. Asbestos is considered the main cause of mesothelioma in the US and in the Western world. The capacity of asbestos to induce mesothelioma has been linked to its ability to cause the release of TNF-alpha (that promotes mesothelial cells survival), other cytokines and growth factors, and of mutagenic oxygen radicals from exposed mesothelial cells and nearby macrophages. Some investigators proposed that as a consequence of the regulations to prevent exposure and to forbid and/or limit the use of asbestos, the incidence of mesothelioma in the US (and in some European countries) should have started to decline before or around the year 2000, and sharply decline thereafter. Unfortunately, there are no data available yet to support this optimistic hypothesis. Simian virus 40 (SV40) infection and radiation exposure are additional causes, although their contribution to the overall incidence of mesothelioma is unknown. Recent data from several laboratories indicate that asbestos exposure and SV40 infection are co-carcinogens in causing mesothelioma in rodents and in causing malignant transformation of human mesothelial cells in tissue culture. An exciting new development comes from the discovery that genetic susceptibility to mineral fiber carcinogenesis plays a critical role in the incidence of this cancer in certain families. It is hoped that the identification of this putative mesothelioma gene will lead to novel mechanistically driven preventive and therapeutic approaches.

Evidence of simian virus 40 exposure in a colony of captive baboons

Simian virus 40 (SV40) is a polyomavirus for which non-human primates are the permissive host. The baboon ( Papio spp.) is an old world monkey that is used in a variety of research investigations; however, natural infection of SV40 among baboons has not been thoroughly examined or reported. Initially, we were interested in determining the prevalence of SV40 infection among a captive colony of baboons based on the presence of antibodies to SV40 large T-antigen (Tag). An overall seroprevalence rate of > 50% was found after screening sera from 142 baboons in the colony based on ELISA. Endpoint titer values for serum antibody binding to SV40 Tag reached as high as 1280 for 5 out of 142 baboons. Peptide binding assays revealed that a range of SV40 Tag epitopes are immunogenic in the baboon, and that individual animals differ in their humoral immune responses to SV40 Tag based on epitope recognition. Specificity to SV40 Tag and not some other primate polyomavirus encoded large Tag was further examined by serologic reactivity to peptide epitopes unique to SV40 Tag. Additional serology was performed to assess SV40 Tag reactivity by Western blot and whether antibodies were capable of neutralizing SV40 infectivity in vitro. Although antibodies with high levels of SV40 neutralization were observed in a number of the baboons, there was a lack of correlation between viral neutralization and antibodies to SV40 Tag. Further examination using molecular-based diagnosis and SV40 Tag specific real-time quantitative PCR determined that some of the baboons appeared to be exposed to SV40. DNA sequence analysis of the PCR products confirmed that SV40 Tag specific sequences were detected in baboons.

SV40 reporter viruses

Three simian virus 40 (SV40) reporter viruses were constructed in this study. One expresses the green fluorescent protein (GFP) as a fusion protein with the first exon of large-T (LT) antigen and is useful for live-cell imaging. A second reporter virus has a FLAG epitope tag at the C-terminus of large-T antigen (vC-LT FLAG), and a third has the FLAG tag at the N-terminus of LT (vN-LT FLAG). The vC-LT FLAG construct grows to titers near those of wild-type (WT) virus and functions well as a reporter virus for SV40 infection. The vN-LT FLAG construct, while viable, has a defect in the production and spread of infectious particles. All three viruses are useful in detecting superinfecting virus in cells in which nuclear LT is already present, such as persistently infected human mesothelial cells.

Ataxia Telangiectasia-Mutated Damage-Signaling Kinase- and Proteasome-Dependent Destruction of Mre11-Rad50-Nbs1 Subunits in Simian Virus 40-Infected Primate Cells

Although the mechanism of simian virus 40 (SV40) DNA replication has been extensively investigated with cell extracts, viral DNA replication in productively infected cells utilizes additional viral and host functions whose interplay remains poorly understood. We show here that in SV40-infected primate cells, the activated ataxia telangiectasia-mutated (ATM) damage-signaling kinase, gamma-H2AX, and Mre11-Rad50-Nbs1 (MRN) assemble with T antigen and other viral DNA replication proteins in large nuclear foci. During infection, steady-state levels of MRN subunits decline, although the corresponding mRNA levels remain unchanged. A proteasome inhibitor stabilizes the MRN complex, suggesting that MRN may undergo proteasome-dependent degradation. Analysis of mutant T antigens with disrupted binding to the ubiquitin ligase CUL7 revealed that MRN subunits are stable in cells infected with mutant virus or transfected with mutant viral DNA, implicating CUL7 association with T antigen in MRN proteolysis. The mutant genomes produce fewer virus progeny than the wild type, suggesting that T antigen-CUL7-directed proteolysis facilitates virus propagation. Use of a specific ATM kinase inhibitor showed that ATM kinase signaling is a prerequisite for proteasome-dependent degradation of MRN subunits as well as for the localization of T antigen and damage-signaling proteins to viral replication foci and optimal viral DNA replication. Taken together, the results indicate that SV40 infection manipulates host DNA damage-signaling to reprogram the cell for viral replication, perhaps through mechanisms related to host recovery from DNA damage.

Nuclear factor of activated T-cells (NFAT) plays a role in SV40 infection

Recent evidence highlighted a role for the transcription factor, nuclear factor of activated T-cells (NFAT), in the transcription of the human polyomavirus JCV. Here we show that NFAT is also important in the transcriptional control of the related polyomavirus, Simian Virus 40 (SV40). Inhibition of NFAT activity reduced SV40 infection of Vero, 293A, and HeLa cells, and this block occurred at the stage of viral transcription. Both NFAT3 and NFAT4 bound to the SV40 promoter through κB sites located within the 72 bp repeated enhancer region. In Vero cells, NFAT was involved in late transcription, but in HeLa and 293A cells both early and late viral transcription required NFAT activity. SV40 large T-Ag was found to increase NFAT activity and provided a positive feedback loop to transactivate the SV40 promoter.

Influence of the Viral Regulatory Region on Tumor Induction by Simian Virus 40 in Hamsters

Most of the simian virus 40 (SV40) genome is conserved among isolates, but the noncoding regulatory region and the genomic region encoding the large T-antigen C terminus (T-ag-C) may exhibit considerable variation. We demonstrate here that SV40 isolates differ in their oncogenic potentials in Syrian golden hamsters. Experimental animals were inoculated intraperitoneally with 10(7) PFU of parental or recombinant SV40 viruses and were observed for 12 months to identify genetic determinants of oncogenicity. The viral regulatory region was found to exert a statistically significant influence on tumor incidence, whereas the T-ag-C played a minor role. Viruses with a single enhancer (1E) were more oncogenic than those with a two-enhancer (2E) structure. Rearrangements in the 1E viral regulatory region were detected in 4 of 60 (6.7%) tumors. Viral loads in tumors varied, with a median of 5.4 SV40 genome copies per cell. Infectious SV40 was rescued from 15 of 37 (40%) cell lines established from tumors. Most hamsters with tumors and many without tumors produced antibodies to T antigen. All viruses displayed similar transforming frequencies in vitro, suggesting that differences in oncogenic potential in vivo were due to host responses to viral infection. This study shows that SV40 strains differ in their biological properties, suggests that SV40 replicates to some level in hamsters, and indicates that the outcome of an SV40 infection may depend on the viral strain present.

N -Glycolyl GM1 Ganglioside as a Receptor for Simian Virus 40

Carbohydrate microarrays have emerged as powerful tools in analyses of microbe-host interactions. Using a microarray with 190 sequence-defined oligosaccharides in the form of natural glycolipids and neoglycolipids representative of diverse mammalian glycans, we examined interactions of simian virus 40 (SV40) with potential carbohydrate receptors. While the results confirmed the high specificity of SV40 for the ganglioside GM1, they also revealed that N-glycolyl GM1 ganglioside [GM1(Gc)], which is characteristic of simian species and many other nonhuman mammals, is a better ligand than the N-acetyl analog [GM1(Ac)] found in mammals, including humans. After supplementing glycolipid-deficient GM95 cells with GM1(Ac) and GM1(Gc) gangliosides and the corresponding neoglycolipids with phosphatidylethanolamine lipid groups, it was found that GM1(Gc) analogs conferred better virus binding and infectivity. Moreover, we visualized the interaction of NeuGc with VP1 protein of SV40 by molecular modeling and identified a conformation for GM1(Gc) ganglioside in complex with the virus VP1 pentamer that is compatible with its presentation as a membrane receptor. Our results open the way not only to detailed studies of SV40 infection in relation to receptor expression in host cells but also to the monitoring of changes that may occur with time in receptor usage by the virus.

Importance of Calcium-Binding Site 2 in Simian Virus 40 Infection

The exposure of molecular signals for simian virus 40 (SV40) cell entry and nuclear entry has been postulated to involve calcium coordination at two sites on the capsid made of Vp1. The role of calcium-binding site 2 in SV40 infection was examined by analyzing four single mutants of site 2, the Glu160Lys, Glu160Arg, Glu157Lys (E157K), and Glu157Arg mutants, and an E157K-E330K combination mutant. The last three mutants were nonviable. All mutants replicated viral DNA normally, and all except the last two produced particles containing all three capsid proteins and viral DNA. The defect of the site 1-site 2 E157K-E330K double mutant implies that at least one of the sites is required for particle assembly in vivo. The nonviable E157K particles, about 10% larger in diameter than the wild type, were able to enter cells but did not lead to T-antigen expression. Cell-internalized E157K DNA effectively coimmunoprecipitated with anti-Vp1 antibody, but little of the DNA did so with anti-Vp3 antibody, and none was detected in anti-importin immunoprecipitate. Yet, a substantial amount of Vp3 was present in anti-Vp1 immune complexes, suggesting that internalized E157K particles are ineffective at exposing Vp3. Our data show that E157K mutant infection is blocked at a stage prior to the interaction of the Vp3 nuclear localization signal with importins, consistent with a role for calcium-binding site 2 in postentry steps leading to the nuclear import of the infecting SV40.

SV40 and human cancer: A review of recent data

An unknown proportion of formalin-inactivated poliovirus vaccine lots administered to millions of US residents between 1955 and 1963 was contaminated with small amounts of infectious simian virus 40 (SV40), a polyomavirus of the rhesus macaque. It has been reported that mesothelioma, brain tumors, osteosarcoma and non-Hodgkin lymphoma (NHL) contain SV40 DNA sequences and that SV40 infection introduced into humans by the vaccine probably contributed to the development of these cancers. The Immunization Safety Review Committee of the Institute of Medicine (IOM) reviewed this topic in 2002. The present review of recent studies showed that the earlier results describing the recovery of SV40 DNA sequences from a large proportion of the above tumors were not reproducible and that most studies were negative. Contamination with laboratory plasmids was identified as a possible source of false positive results in some previous studies. The low-level immunoreactivity of human sera to SV40 was very likely the result of cross-reactivity with antibodies to the SV40-related human polyomaviruses BKV and JCV, rather than of authentic SV40 infection. SV40 sero-reactivity in patients with the suspect tumors was no greater than that in controls. In epidemiologic studies, the increased incidence of some of the suspect tumors in the 1970s to 1980s was not related to the risk of exposure to SV40-contaminated vaccines. In summary, the most recent evidence does not support the notion that SV40 contributed to the development of human cancers.

Is There a Role for SV40 in Human Cancer?

The question of whether Simian Virus 40 (SV40) can cause human tumors has been one of the most highly controversial topics in cancer research during the last 50 years. The longstanding debate began with the discovery of SV40 as a contaminant in poliovirus vaccine stocks that were used to inoculate approximately 100 million children and adults in the United States between 1955 and 1963, and countless more throughout the world. Concerns regarding the potential health risk of SV40 exposure were reinforced by studies demonstrating SV40's potential to transform human cells and promote tumor growth in animal models. Many studies have attempted to assess the relationship between the potential exposure of humans to SV40 and cancer incidence. Reports of the detection of SV40 DNA in a variety of cancers have raised serious concerns as to whether the inadvertent inoculation with SV40 has led to the development of cancer in humans. However, inconsistent reports linking SV40 with various tumor types has led to conflicting views regarding the potential of SV40 as a human cancer virus. Several recent studies suggest that older detection methodologies were flawed, and the limitations of these methods could account for most, if not all, of the positive correlations of SV40 in human tumors to date. Although many people may have been exposed to SV40 by polio vaccination, there is inadequate evidence to support widespread SV40 infection in the population, increased tumor incidence in those individuals who received contaminated vaccine, or a direct role for SV40 in human cancer.