Investigation of simian virus 40 (SV40) and human JC, BK, MC, KI, and WU polyomaviruses in glioma.

The gliomagenesis remains not fully established and their etiological factors still remain obscure. Polyomaviruses were detected and involved in several human tumors. Their potential implication in gliomas has been not yet surveyed in Africa and Arab World. Herein, we investigated the prevalence of six polyomaviruses (SV40, JCPyV, BKPyV, MCPyV, KIPyV, and WUPyV) in 112 gliomas from Tunisian patients. The DNA sequences of polyomaviruses were examined by PCR assays. Viral infection was confirmed by DNA in situ hybridization (ISH) and/or immunohistochemistry (IHC). The relationships between polyomavirus infection and tumor features were evaluated. Specific SV40 Tag, viral regulatory, and VP1 regions were identified in 12 GBM (10.7%). DNA ISH targeting the whole SV40 genome and SV40 Tag IHC confirmed the PCR findings. Five gliomas yielded JCPyV positivity by PCR and DNA ISH (2.7%). However, no BKPyV, KIPyV, and WUPyV DNA sequences were identified in all samples. MCPyV DNA was identified in 30 gliomas (26.8%). For GBM samples, MCPyV was significantly related to patient age (p = 0.037), tumor recurrence (p = 0.024), and SV40 (p = 0.045) infection. No further significant association was identified with the remaining tumor features (p > 0.05) and patient survival (Log Rank, p > 0.05). Our study indicates the presence of SV40, JCPyV, and MCPyV DNA in Tunisian gliomas. Further investigations are required to more elucidate the potential involvement of polyomaviruses in these destructive malignancies.

Towards dynamic monitoring of cell cultures using high throughput sequencing.

We used a combination of DOP-PCR with high throughput sequencing (HTS) to study infected cell cultures over time to assess the feasibility of using this technique to provide a read-out other than cytopathic effect in cell culture infectivity assays. Because DOP-PCR primers feature a short constant sequence at their 3' terminus, the procedure yields a reproducible representational library of products from a given PCR template, including viral nucleic acids. Using SV40- and MVM-infected cultures harvested at different times, we show that the number of viral matches among DOP-PCR products parallels the quantity of virus as shown by real-time PCR, and further show that HTS analysis of specific DOP-PCR products that increase in quantity over time could be used to identify the infecting virus with a sensitivity similar to that of typical cell-culture assays that rely on cytopathic effect.

Polyomavirus Persistence.

Mammalian polyomaviruses are characterized by establishing persistent infections in healthy hosts and generally causing clinical disease only in hosts whose immune systems are compromised. Despite the fact that these viruses were discovered decades ago, our knowledge of the mechanisms that govern viral persistence and reactivation is limited. Whereas mouse polyomavirus has been studied in a fair amount of detail, our understanding of the human viruses in particular is mostly inferred from experiments aimed at addressing other questions. In this review, we summarize the state of our current knowledge, draw conclusions when possible, and suggest areas that are in need of further study.

Transcription enhancers as major determinants of SV40 polyomavirus growth efficiency and host cell tropism.

The non-coding control region (NCCR) of polyomaviruses includes the promoters for early and late genes, a transcription enhancer and the origin of DNA replication. Particularly virulent variants of the human pathogens BKPyV and JCPyV, as well as of simian virus 40 (SV40), occur in vitro and in vivo. These strains often harbour rearrangements in their NCCR, typically deletions of some DNA segment(s) and/or duplications of others. Using an SV40-based model system we provide evidence that duplications of enhancer elements, whether from SV40 itself or from the related BKPyV and JCPyV, increase early gene transcription and replicative capacity. SV40 harbouring subsegments of the strong cytomegalovirus (HCMV) enhancer replicated better than the common 'wild-type' SV40 in the human cell lines HEK293 and U2OS. In conclusion, replacing the SV40 enhancer with heterologous enhancers can profoundly influence SV40's infective capacity, underscoring the potential of small DNA viruses to overcome cell type and species barriers.

An SV40 mutant defective in VP4 expression exhibits a temperature-sensitive growth defect.

On reexamination of temperature-sensitive D-type (tsD) mutants of simian virus 40 (SV40), we found that the tsD222 mutant is identical to the VP2 M228I mutant, which is defective in VP4 expression, at the nucleotide level. Although a previous study reported that lack of VP4 caused defects in viral dissemination in BSC-1 cells, this mutant showed a temperature-sensitive growth defect in CV-1 cells. tsD101:VP3 Q113K and tsD202:VP3 P108S exhibited a growth phenotype similar to that of tsD222, and they retained the VP4 open reading frame (ORF). These three mutants did not complement each other, suggesting that their defects were functionally indistinguishable. Transduction of the SV40 vector expressing wild-type VP4 in tsD222-infected cells did not ameliorate the growth defect at the non-permissive temperature. The results indicate that tsD mutation in minor capsid proteins has a more profound impact on viral propagation, and that lack of VP4 ORF seems to have little influence on viral growth.

Unique susceptibility of a human lung carcinoid tumor cell line to infection with BK virus.

Carcinoid syndrome is caused by the unregulated secretion of bioactive amines from neuroendocrine tumors arising primarily in the gastrointestinal tract and lungs. The incidence of carcinoid syndrome is 1-2/100,000 and the syndrome is thought to be increasing. Carcinoid tumors are relatively slow growing but can become highly metastatic. Currently, there is no effective therapy to inhibit cell proliferation or metastasis of neuroendocrine tumor (NET) disease. Polyomaviruses are a family of viruses that are able to transform cells and promote tumor formation. In this study, the polyomaviruses SV40, JCV, and BKV were used to assess the ability of polyomaviruses to productively infect a range of human carcinoid cell lines. Infection was assessed by the immunofluorescence detection of T antigen and V antigen. Viruses and cell lines that exhibited productive infections were subsequently assayed by FACS analysis for cell binding and dual promoter luciferase assay for early and late promoter activity. Most carcinoid cell lines were not susceptible to infection by polyomaviruses. However, BKV efficiently infected the pulmonary carcinoid H727 cell line but did not infect a control, non-carcinoid lung cell line (A549). BKV was found to bind to both the susceptible H727 cells and to the non-susceptible A549 cells but viral genes were only efficiently expressed in the H727 cell line. The data demonstrate that BKV can infect human pulmonary carcinoid cells. Infection does not seem to be solely mediated by the virus' ability to bind to cells, as the virus will also bind to non-carcinoid control cells. Both early and late gene expression are supported by the pulmonary carcinoid cells.

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.

Inhibition of Simian virus 40 large T antigen helicase activity by fluoroquinolones.

BACKGROUND: Fluoroquinolones represent a potent group of antibiotics that inhibit bacterial DNA replication by targeting the essential bacterial enzymes gyrase and topoisomerase IV. Inhibition of gyrase activity by quinolones involves the interaction of these drugs with the helicase component of bacterial gyrase. DNA tumour viruses also encode helicases that are essential for their DNA replication in the host. METHODS: In this study we have evaluated the effect of fluoroquinolones on viral DNA replication using the DNA tumour virus Simian virus 40 (SV40) as our model. Four different fluoroquinolones, namely, levofloxacin, trovafloxacin, ciprofloxacin and ofloxacin, were tested for their ability to inhibit viral DNA replication. RESULTS: We show here that all four quinolones tested were effective in the inhibition of SV40 plaque formation and DNA replication in CV1-P cells. In addition, we found that each of these quinolones was inhibitory to the helicase activity of SV40 large tumour antigen. CONCLUSIONS: Fluoroquinolones and their derivates may therefore be useful in the treatment and/or prevention of infection by SV40-homologous human DNA viruses that encode helicase activity for their survival.

The carboxyl-terminal domain of large T antigen rescues SV40 host range activity in trans independent of acetylation.

The host range activity of SV40 has been described as the inability of mutant viruses with deletions in the C terminal region of large T Ag to replicate in certain types of African green monkey kidney cells. We constructed new mutant viruses expressing truncated T Ag proteins and found that these mutant viruses exhibited the host range phenotype. The host range phenotype was independent of acetylation of T Ag at lysine 697. Co-expression of the C terminal domain of T Ag (aa 627-708) in trans increased both T Ag and VP1 mRNA as well as protein levels for host range mutant viruses in the restrictive cell type. In addition, the T Ag 627-708 fragment promoted the productive lytic infection of host range mutant viruses in the nonpermissive cell type. The carboxyl-terminal region of T Ag contains a biological function essential for the SV40 viral life cycle.

Distinct patterns of MCM protein binding in nuclei of S phase and rereplicating SV40-infected monkey kidney cells.

BACKGROUND: Simian Virus 40 (SV40) infection of growth-arrested monkey kidney cells stimulates S phase entry and the continued synthesis of both viral and cellular DNA. Infected cells can attain total DNA contents as high as DNA Index, DI = 5.0-6.0 (10-12C), with host cell DNA representing 70-80% of the total. In this study, SV40-infected and uninfected control cells were compared to determine whether continued DNA replication beyond DI = 2.0 was associated with rebinding of the minichromosome maintenance (MCM) hexamer, the putative replicative helicase, to chromatin. METHOD: Laser scanning cytometry was used to measure the total expression per cell and the chromatin/matrix-association of two MCM subunits in relation to DNA content. RESULTS: MCM2 and MCM3 proteins that were associated with the chromatin/matrix fraction in G1 phase of both uninfected and SV40-infected cells were gradually released during progression through S phase. However, in SV40-infected cells that progressed beyond DI = 2.0, chromatin/matrix-associated MCM2 and MCM3 remained at the low levels observed at the end of S phase. Rereplication was not preceded by an obvious rebinding of MCM proteins to chromatin, as was observed in G1 phase. CONCLUSIONS: The rereplication of host cell DNA in the absence of the reassociation of MCM proteins with chromatin indicates that SV40 infection induces a novel mechanism of licensing cellular DNA replication.

Insights into hRPA32 C-terminal domain--mediated assembly of the simian virus 40 replisome.

Simian virus 40 (SV40) provides a model system for the study of eukaryotic DNA replication, in which the viral protein, large T antigen (Tag), marshals human proteins to replicate the viral minichromosome. SV40 replication requires interaction of Tag with the host single-stranded DNA-binding protein, replication protein A (hRPA). The C-terminal domain of the hRPA32 subunit (RPA32C) facilitates initiation of replication, but whether it interacts with Tag is not known. Affinity chromatography and NMR revealed physical interaction between hRPA32C and the Tag origin DNA-binding domain, and a structural model of the complex was determined. Point mutations were then designed to reverse charges in the binding sites, resulting in substantially reduced binding affinity. Corresponding mutations introduced into intact hRPA impaired initiation of replication and primosome activity, implying that this interaction has a critical role in assembly and progression of the SV40 replisome.

Pairs of Vp1 cysteine residues essential for simian virus 40 infection.

Transient disulfide bonding occurs during the intracellular folding and pentamerization of simian virus 40 (SV40) major capsid protein Vp1 (P. P. Li, A. Nakanishi, S. W. Clark, and H. Kasamatsu, Proc. Natl. Acad. Sci. USA 99:1353-1358, 2002). We investigated the requirement for Vp1 cysteine pairs during SV40 infection. Our analysis identified three Vp1 double-cysteine mutant combinations that abolished viability as assayed by plaque formation. Mutating the Cys49-Cys87 pair or the Cys87-Cys254 pair led to ineffective nuclear localization and diminished accumulation of the mutant Vp1s, and the defect extended in a dominant-negative manner to the wild-type minor capsid proteins Vp2/3 and an affinity-tagged recombinant Vp1 expressed in the same cells. Mutating the Cys87-Cys207 pair preserved the nuclear localization and normal accumulation of the capsid proteins but diminished the production of virus-like particles. Our results are consistent with a role for Cys49, Cys87, and Cys254 in the folding and cytoplasmic-nuclear trafficking of Vp1 and with a role for Cys87 and Cys207 in the assembly of infectious particles. These findings suggest that transient disulfide bond formation between certain Vp1 cysteine residues functions at two stages of SV40 infection: during Vp1 folding and oligomerization in the cytoplasm and during virion assembly in the nucleus.

Factors influencing the production of recombinant SV40 vectors.

Most gene therapy approaches employ viral vectors for gene delivery. Ideally, these vectors should be produced at high titer and purity with well-established protocols. Standardized methods to measure the quality of the vectors produced are imperative, as are techniques that allow reproducible quantitation of viral titer. We devised a series of protocols that achieve high-titer production and reproducible purification and provide for quality control and titering of recombinant simian virus 40 vectors (rSV40s). rSV40s are good candidate vehicles for gene transfer: they are easily modified to be nonreplicative and they are nonimmunogenic. Further, they infect a wide variety of cells and allow long-term transgene expression. We report here these protocols to produce rSV40 vectors in high yields, describe their purification, and characterize viral stocks using quality control techniques that monitor the presence of wild-type SV40 revertants and defective interfering particles. Several methods for reproducible titration of rSV40 viruses have been compared. We believe that these techniques can be widely applied to obtain high concentrations of high-quality rSV40 viruses reproducibly.

Molecular analysis of SV-40-CAL, a new slow growing SV-40 strain from the kidney of a caged New World monkey with fatal renal disease.

A decline of the Callimico goeldii population in American zoos is presently occurring due to glomerulonephritis of unknown etiology. We hypothesized that this emerging idiopathic fatal renal disease (IFRD) was caused by a virus. We therefore attempted to isolate virus from the kidneys three C. goeldi in Illinois that had IFRD. Along with other viruses, Simian virus 40 (SV-40) strain CAL was isolated. SV-40-CAL is currently the slowest-growing natural isolate of SV-40 in CV-1 cells. Inefficient SV-40-CAL growth in CV-1 cells stems from two features: a suboptimal protoarchetypal regulatory region, and a Large tumor antigen gene sequence like that of SV-40 strain T302, previously considered the slow-growing natural isolate of SV-40. To our knowledge, this is the first documented isolation of SV-40 from a New World monkey outside of a laboratory setting. Though SV-40 is renaltropic, the role of SV-40-CAL in IFRD is uncertain. Transmission of SV-40 to C. goeldii through anthropogenic activity is suspected.

DA-125, a new antitumor agent, inhibits topoisomerase II as topoisomerase poison and DNA intercalator simultaneously.

DA-125, a novel derivative of adriamycin, is known for its anti-cancer activity. In this study, the inhibitory mechanism of DA-125 on topoisomerase was investigated in the simian virus 40 (SV40) replicating CV-1 cell by studying the SV40 DNA replication intermediates and DNA-topoisomerase complexes. DNA-protein complexes that were formed in the drug-treated cells were quantitated by using a glass filter assay. SV40 DNA replication intermediates that were accumulated in the drug-treated CV-1 cell were analyzed in a high resolution gel. DA-125 did not accumulate B-dimers of SV40 DNA replication intermediates which were found in the adriamycin-treated CV-1 cells. DA-125 induced a dose-dependent formation of the DNA-protein complexes, while adriamycin did not. When adriamycin and etoposide (VP16) were added to the SV40-infected cells at the same time, adriamycin blocked the formation of the DNA-protein complexes induced by VP16 in a dose-dependent manner. However, DA-125 blocked the formation of the DNA-protein complexes induced by VP16 up to the maximum level of the DNA-protein complexes that were induced by DA-125 alone. Adriamycin and DA-125 did not inhibit the formation of the DNA-protein complexes that were caused by camptothecin, a known topoisomerase I poison. DA-125 is bifunctional in inhibiting topoisomerase II because it simultaneously has the properties of the topoisomerase II poison and the DNA intercalator. As a topoisomerase II poison, DA-125 alone induced dose-dependent formation of the DNA-protein complexes. However, as a DNA intercalator, it quantitatively inhibited the formation of the DNA-protein complexes induced by a strong topoisomerase II poison VP16. Furthermore considering that the levels of the DNA-protein complex induced by VP16 were decreased by DA-125 in terms of the topoisomerase II poison, we suggest that DA-125 has a higher affinity to the drug-binding sites of DNA than VP16 has.

The archetype enhancer of simian virus 40 DNA is duplicated during virus growth in human cells and rhesus monkey kidney cells but not in green monkey kidney cells.

Archetype SV40, obtained directly from its natural host, is characterized by a single 72-bp enhancer element. In contrast, SV40 grown in cell culture almost invariably exhibits partial or complete duplication of the enhancer region. This distinction has been considered important in studies of human tumor material, since SV40-associated tumor isolates have been described having a single enhancer region, suggesting natural infection as opposed to possible contamination by laboratory strains of virus. However, the behavior of archetypal SV40 in cultured cells has never been methodically studied. In this study we reengineered nonarchetypal 776-SV40 to contain a single 72-bp enhancer region and used this reengineered archetypal DNA to transfect a number of simian and human cell lines. SV40 DNA recovered from these cells was analyzed by restriction endonuclease analysis, PCR, and DNA sequencing. Reengineered archetype SV40 propagated in green monkey TC-7 or BSC-1 kidney cells remained without enhancer region duplication even after extensive serial virus passage. Archetype SV40 grown in all but one of the rhesus or human cell lines initially appeared exclusively archetypal. However, when virus from these cell types was transferred to green monkey cells, variants with partial enhancer duplication appeared after as little as a single passage. These findings suggest (1) that virus with a single 72-bp enhancer may persist in cultured cells of simian and human origin; (2) that variants with partially duplicated enhancer regions may arise within cell lines in quantities below limits of detection; (3) that these variants may enjoy a selective advantage in cell types other than those from which they arose (e.g., green monkey kidney cells); and (4) that certain cell lines may support a selective growth advantage for the variants without supporting their formation. Our data indicate that enhancer duplication may also occur in human as well as rhesus kidney cells. Thus, detection of enhancer region duplication may not, a priori, indicate laboratory contamination, nor does detection of a single 72-bp enhancer exclude the possibility that contamination may have occurred. These findings may be of relevance to studies attempting to detect SV40 DNA in human tumors or other clinical specimens.

In vitro assessment of the cytotoxicity of nisin, pediocin, and selected colicins on simian virus 40-transfected human colon and Vero monkey kidney cells with trypan blue staining viability assays.

Gram-positive bacterial bacteriocins (nisin and pediocin) and gram-negative bacterial bacteriocins (colicins [Col] E1, E3, E6, E7, and K) were evaluated for cytotoxicity against cultured simian virus 40-transfected human colon (SV40-HC) and Vero monkey kidney (Vero) cells. Bacteriocin-treated cells were assessed for viability by trypan blue staining. Monolayers of SV40-HC and Vero cells were cultured in tissue culture plates (35 degrees C, 10% CO2 in humidified air) with the use of Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol) calf serum. Actively growing cells in the log phase (ca. 10(4) cells per ml) were treated with individual partially purified bacteriocin preparations at 170, 350, and 700 activity units per ml. Duplicate culture plates for each bacteriocin treatment and untreated controls were withdrawn after 16, 32, and 48 h of incubation. Cells were dissociated with trypsin and treated with trypan blue and were then counted in a hemocytometer with the use of a phase-contrast microscope. Viability assays indicated dose-dependent toxicity for some bacteriocins. Nisin, pediocin, and Col E6 were the most cytotoxic bacteriocins; SV40-HC cells demonstrated greater sensitivity than Vero cells did. Some bacteriocins can be toxic to mammalian cells; therefore, bacteriocins intended for use as biopreservatives must be evaluated for toxicity to mammalian cells and for other toxicities. Col E1, Col E3, Col E7, and Col K demonstrated little toxicity at the activities tested, indicating that they are safe and thus have potential for use as food biopreservatives.

The abundant nuclear enzyme PARP participates in the life cycle of simian virus 40 and is stimulated by minor capsid protein VP3.

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.

Molecular identification of SV40 infection in human subjects and possible association with kidney disease.

Simian virus 40 (SV40), a monkey polyomavirus that is believed to have entered the human population through contaminated vaccines, is known to be renotropic in simians. If indeed SV40 is endemic within the human population, the route of transmission is unknown. It was therefore hypothesized that SV40 might be renotropic in humans and be detected more frequently in samples obtained from patients with kidney diseases. This study found that typical polyomavirus cytopathic effects (CPE) were present and SV40 T antigen was detected in CV-1 cells cultured with peripheral blood mononuclear cells (PBMC) or urinary cells obtained from patients with kidney disease and healthy volunteers. DNA sequences homologous to the SV40 viral regulatory genome were detected by PCR in urinary cells from 15 (41%) of 36 patients with focal segmental glomerulosclerosis (FSGS), 2 (10%) of 20 patients with other kidney diseases, and 1 (4%) of 22 healthy volunteers (FSGS compared with other glomerular disease, P < 0.02; FSGS compared with healthy volunteers, P = 0.003). SV40 viral regulatory region genome was detected from PBMC at similar frequencies in patients with FSGS (35%), other glomerular diseases (20%), and healthy volunteers (22%). SV40 genome was detected by PCR in kidney tissues from 17 (56%) of 30 of patients with FSGS and 4 (20%) of 20 patients with minimal change disease and membranous nephropathy (P < 0.01). Considerable genetic heterogeneity of the viral regulatory region was detected, which argues against laboratory contamination. SV40 genome was localized to renal tubular epithelial cell nuclei in renal biopsies of patients with FSGS by in situ hybridization. This study demonstrates for the first time that human kidney can serve as a reservoir for SV40 replication and that SV40 may contribute to the pathogenesis of kidney disease, particularly FSGS.