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PubMed1995

Intrachromosomal recombination mediated by the polyomavirus large T antigen.

We used a spleen necrosis virus-based retroviral vector to introduce the polyomavirus replication origin into rat cells and developed a system to analyze homologous recombination events that do not reconstitute a selectable marker. Introduction of the gene coding for the polyomavirus large T antigen into the cell lines by DNA transfection promoted high-frequency recombination between the two retroviral LTRs, leading to amplification and excision of DNA sequences. To analyze homology requirements, we constructed cell lines carrying only the replication origin without exogenous repeats. Most of the cell lines sustained high-frequency recombination, presumably by undergoing homologous recombination between repetitive DNA lying in the vicinity of the integrated origin. Our results indicate that homologous recombination promoted by large T antigen does not require recombination hot spots in the viral genome other than the replication origin and they explain the cytotoxicity observed in some cell types when large T antigen is expressed in the presence of a functional origin.

Animals↗
PubMed1996

Polyomavirus large T antigen zinc finger is not required for efficient cellular immortalization of primary rat embryo fibroblasts.

The polyomavirus large T antigen contains a zinc finger domain required for the formation of hexamers involved in viral DNA replication. Since mutations within the zinc finger domains of transforming proteins like SV40 large T antigen and human papilloma virus E7 protein generally decrease their overall transforming activity, we have examined the ability of a mutant polyomavirus large T antigen that harbors a deletion in sequences within the zinc finger motif to immortalize primary rat embryo fibroblasts. In contrast to result obtained with SV40 large T antigen and the human papilloma virus E7 protein we show that deletion of the entire zinc finger motif enhances the immortalization efficiency of this mutant T antigen.

Animals↗
PubMed1997

Polyomavirus large T-antigen binds the "pRb related' protein p130 through sequences in conserved region 2.

The transforming potential of DNA tumor viruses derives mainly from the ability of their encoded oncogene products to interact with cellular proteins. Many of these viral oncoproteins share regions of sequence similarity, designated conserved region 1 and 2, which have been implicated in complex formation with pRb, the product of the retinoblastoma tumor suppressor gene, and related p107 and p130 species. It has now been shown that the EIA protein of adenovirus is able to bind to all three pRb-related proteins through sequences in conserved region 1 and 2. We have shown previously that polyomavirus large T-antigen also interacts with pRb and p107 in vitro. The pRb and p107 binding domains reside between residues 141, 158 which include conserved region 2. In the present study, we demonstrate that polyomavirus large T-antigen also interacted with p130 in vitro through the same sequences in conserved region 2.

Amino Acid Sequence↗
PubMed2003

Genetic immunization is augmented by murine polyomavirus VP1 pseudocapsids.

To improve immune responses induced by DNA immunization, murine polyomavirus major capsid protein (VP1) pseudocapsids were complexed with a DNA plasmid encoding the p37 (p24 and p17) nucleocapsid proteins of the human immunodeficiency virus type 1 (HIV-1). A 10-fold increase in antibody titer was noted in mice given DNA plasmid together with VP1 pseudocapsids in comparison to animals that received DNA plasmid alone. Cell mediated responses to HIV-1 p24 occurred, but were not significantly augmented by delivering the DNA as a VP1 complex. We have consequently for the first time shown a carrier/adjuvant effect of polyomavirus pseudocapsids that strongly increased the humoral immune response in DNA immunization.

Animals↗
PubMed2003

Polyomavirus in kidney and kidney-pancreas transplant recipients.

PURPOSE: To report the incidence and clinical characteristics of polyomavirus (PV) nephritis in kidney (KTX) and kidney-pancreas transplant (KPTX) recipients. METHODS: Single center retrospective analysis of all cases of PV nephritis in KTX and KPTX patients transplanted between 1994 and 1999. RESULTS: Thirteen (5 KTX and 8 KPTX) patients (2.1%) had PV nephritis diagnosed on multiple biopsies (n = 22) among 504 KTX and 106 KPTX recipients. The incidence of PV nephritis was higher in cadaver donor transplants (2.6% cadaver vs. 0.7% living donors), after KPTX (1% KTX vs. 7.5% KPTX), in males (3.3% male vs. 0.7% female), and in diabetic patients (4.4% diabetic vs. 0.8% nondiabetic). The mean time to diagnosis of PV nephritis was 18 (range 6-48) months after KTX and 17 (range 9-31) months after KPTX. Three KTX patients and 5 KPTX patients had calcineurin inhibitor toxicity on biopsy prior to developing PV nephritis. Reduction in immunosuppression occurred in 100% of KTX and 63% of KPTX patients. Three patients (23%) developed rejection within 3 months of diagnosis of PV, 1 after a reduction in immunosuppression. Despite multiple antiviral treatment regimens, renal allograft failure requiring dialysis occurred in 60% of KTX and 50% of KPTX patients. All KPTX patients remain insulin independent and 2 were successfully retransplanted with living donor kidneys. 2 patients (15%) died but there was no mortality directly related to the virus. CONCLUSIONS: Polyomavirus nephritis may be increasing in incidence and appears to be unresponsive to either conventional antiviral agents or a reduction in immunosuppression. Most of our cases occurred in male diabetic patients undergoing cadaveric donor transplantation and were preceded by biopsy-proven nephrotoxicity. Further studies are needed to better define the pathogenesis of PV and effective antiviral treatment.

Adult↗
PubMed2003

Renal transplantation and polyomavirus infection: recent clinical facts and controversies.

Although many articles have been published on polyomavirus-induced pathologies in transplant recipients, our knowledge regarding their clinical aspects remains relatively limited. In fact, the number of questions and controversies on the subject seems even to be increasing as new publications continue to appear. This article presents some of these controversies through a brief review of recent clinical facts about the three polyomaviruses that infect humans--JC virus, simian virus 40, and BK virus--as they relate to renal transplantation.

Animals↗
PubMed2004

Pathology of spontaneous and experimental infections by Goose haemorrhagic polyomavirus.

Haemorrhagic nephritis enteritis of geese (HNEG) is a fatal disease of geese aged from 3 to 12 weeks. The causative virus, Goose haemorrhagic polyomavirus (GHPV), is a member of the Polyomaviridae family We examined goslings either spontaneously or experimentally infected with GHPV. Tissues were sampled for histology, GHPV DNA detection and electron microscopy. Clinical signs and gross lesions observed in experimentally infected goslings were largely consistent with those noticed in field cases. Histological examination showed that, in the acute phase of HNEG, GHPV replicates in almost all the tissues with a particular tropism for endothelial and lymphoid cells. Haemorrhagic foci were widespread in many tissues, including brain. Ultrastructural features were largely consistent with other polyomavirus infections, with accumulation of virions in the nucleus. Non-typical, double-membraned organelles were observed in the cytoplasm. GHPV DNA distribution was widespread in tissues of infected birds, from day 5 post-infection. GHPV therefore induces a systemic disease in its host, leading to severe vascular dysfunction and immunosuppressive B-cell depletion.

Animals↗
PubMed2003

Brain tumors and polyomaviruses.

Polyomaviruses, including JC virus (JCV), BK virus (BKV), and simian virus 40 (SV40) have attracted much attention in the past decade due to their repeated isolation from various human tumors, including those originating from the central nervous system (CNS). JCV and BKV are considered to be ubiquitous human pathogens that become reactivated under impaired physiological conditions such as immunosuppression. Productive replication of JCV and BKV induces diseases such as progressive multifocal leukoencephalopathy in the brain and hemorrhagic or nonhemorrhagic cystitis and nephritis in the kidney. JCV DNA sequences have been isolated from a number of human CNS tumors, including medulloblastoma, ependymoma, and a broad range of glial-origin neoplasms. SV40, once believed to be a monkey virus, has now been isolated from a variety of human cancer cells, including mesothelioma, ependymoma, and non-Hodgkin's lymphoma. In this mini-review, the authors focused their attention on the possible involvement of polyomaviruses, such as JCV, BKV, and SV40, with human brain tumors.

Animals↗
PubMed2000

An immunodominant, cross-reactive B-cell epitope region is located at the C-terminal part of the hamster polyomavirus major capsid protein VP1.

The VP1 represents the major capsid protein of the hamster polyomavirus (HaPV). Here we describe the mapping of epitopes along the VP1 using Escherichia coli-expressed VP1-dihydrofolate reductase (DHFR) fusion proteins and PepScan analysis. By use of DHFR fusion proteins an immunodominant region was localized in the C-terminal part of VP1 between amino acids 320-384. Further epitopes are located in the regions amino acids 1-133 and amino acids 133-320, respectively. There were no obvious differences in the reactivity between sera of tumor-bearing and papilloma-free naturally HaPV-infected hamsters. In contrast, PepScan analysis revealed linear epitopes in the regions amino acids 79-97 and amino acids 353-367 for tumor-bearing animals and amino acids 101-113 and amino acids 165-179 for papilloma-free animals. The region between amino acids 320-384 of HaPV-VP1 was found to be involved in cross-reactivity of VP1 from HaPV and other polyomaviruses. Previously we have demonstrated that heterologous expression of HaPV-VP1 allowed the formation of virus-like particles (VLPs). From epitope mapping data and structural predictions it has been suggested that HaPV-VP1-VLPs may tolerate foreign peptides in the region amino acids 81-88 and the C-terminal part of VP1.

Amino Acid Sequence↗
PubMed2005

Polyomavirus DNA and RNA detection in renal allograft biopsies: results from a European multicenter study.

Polyomavirus mediated nephropathy is an increasingly recognized complication in renal transplant recipients. In all, 362 renal biopsies collected from 15 European transplant centers were analyzed for presence of Polyomavirus nucleic acid (BK virus [BKV] and JC virus [JCV]). We evaluated 302 biopsies of patients with renal allograft dysfunction, including three with known BKV allograft nephropathy (BKVAN), and 60 native kidney biopsies. BKV DNA was detected in 8 of the 302 (2.6 %) biopsies obtained for transplant dysfunction, but in none of the controls. BKV RNA, indicating active viral replication, was found in all BKV DNA positive biopsies available for mRNA expression studies. Retrospective immunohistochemical staining was positive for SV40 large T antigen in all seven evaluated biopsies. BKV DNA and RNA were detected in biopsy tissues from patients with inconspicuous light microscopy for BKVAN. Further studies will evaluate the potential of intrarenal viral BKV RNA as an early predictor for BKVAN.

Adult↗
PubMed1992

Bovine polyomavirus, a cell-transforming virus with tumorigenic potential.

The early region of bovine polyomavirus (BPyV) was tested for its cell transformation potential employing an assay of dense focus formation. Dense foci of morphologically transformed cells were observed upon transfection of primary rodent cells with a plasmid construct encoding the complete early region of BPyV under the transcriptional control of the long terminal repeat of Rous sarcoma virus. No transformation of primary rodent cells was observed upon transfection of these cells with a plasmid encoding the complete early region of BPyV under the control of its own transcriptional regulatory sequences. In BPyV-transformed cells, the viral sequences had become integrated into the cellular genome, and expression of large T antigen could be detected in a high percentage of cells. The transformed cells were demonstrated to be capable of anchorage-independent growth and to be oncogenic in immunocompromised newborn rats. Therefore BPyV should be considered as a potentially tumorigenic polyomavirus. Since many commercial batches of calf serum have been shown to be contaminated with BPyV, our observations may have implications for the use of calf serum in cell culture.

Amino Acid Sequence↗
PubMed2005

Detection of beak and feather disease virus (BFDV) and avian polyomavirus (APV) DNA in psittacine birds in Italy.

Beak and feather disease (psittacine circovirus) and Budgerigar fledgling disease (avian polyomavirus) are viral diseases that can frequently affect captive psittacine birds. We designed the first survey to investigate the presence of beak and feather disease virus (BFDV) and Avian polyomavirus (APV) inside the population of captive psittacine birds in Italy. Samples were collected in 18 Italian psittacine breeding centres and four trade centres over a 4-year period. A total of 1516 birds were tested for BFDV and 877 birds were tested for APV by means of a polymerase-chain-reaction (PCR) assay. BFDV was found in 122 (8.05%) and APV in 7 (0.79%) birds. No significant difference in infection rate was found between imported and locally raised parrots. We report the first BFDV DNA isolation in wild birds imported to Italy from Papua New Guinea.

Animals↗
PubMed2006

New commercially available PCR and microplate hybridization assay for detection and differentiation of human polyomaviruses JC and BK in cerebrospinal fluid, serum, and urine samples.

JC and BK human polyomaviruses (family Polyomaviridae) may cause severe neurological or urinary tract pathologies in immunocompromised hosts. In the present study, we evaluated a new commercially available PCR and microplate colorimetric hybridization assay for the standardized differential detection of JC virus (JCV) and BK virus (BKV) genomes in clinical samples. This JC/BK Consensus test was first evaluated by testing serial dilutions of JCV or BKV plasmid DNA standards and was then compared with an in-house reference PCR assay for the detection of JC and BK virus genomes in 70 cerebrospinal fluid (CSF) samples of patients with neurological disorders and in 75 serum or plasma samples and 125 urine samples of renal graft recipients. This new test allowed a limit of detection of 10 copies and 1 copy of JC and BK virus genomes, respectively, and was able to differentiate various levels of JCV, BKV, and mixed JCV and BKV DNA genomes in a single reaction tube. Our results showed 100% specificity and sensitivity for the JC/BK Consensus test with CSF samples. With serum or plasma samples, this test had a sensitivity and a specificity of 100% for both JCV and mixed JCV and BKV DNA detection and a sensitivity and a specificity of 100 and 97.8% for BKV DNA detection, respectively. With urine samples, the sensitivity and specificity were 100 and 96.6%, respectively, for JCV DNA detection; 100 and 89.4%, respectively, for BKV DNA detection; and 44.4 and 100%, respectively, for mixed JCV and BKV DNA detection. In conclusion, our data indicate that this new test, the JC/BK Consensus test, is valuable for the sensitive and specific differential detection of single JCV and BKV infections in CSF, serum or plasma, and urine samples. The use of this reliable PCR assay would improve the routine virological diagnosis as well as the clinical care of immunocompromised patients with polyomavirus-related pathologies.

Adult↗
PubMed2006

Independent contributions of polyomavirus middle T and small T to the regulation of early and late gene expression and DNA replication.

We previously showed that murine polyomavirus mutants that lack both middle T (MT) and small T (ST) functions have a severe pleiotropic defect in early and late viral gene expression as well as genome amplification. The respective contribution of MT and ST to this phenotype was unclear. This work separates the roles of MT and ST in both permissive mouse cells and nonpermissive rat cells. It demonstrates for the first time a role for both proteins. To gain insight into the signaling pathways that might be required, we focused on MT and its mutants. The results show that each of the major MT signaling connections, Shc, phosphatidylinositol 3'-kinase, and phospholipase C gamma1, could contribute in an additive way. Unexpectedly, a mutant lacking all these connections because the three major tyrosines had been converted to phenylalanine retained some activity. A mutant in which all six MT C-terminal tyrosines had been mutated was inactive. This suggests a novel signaling pathway for MT that uses the minor tyrosines. What is common to ST and the individual MT signaling pathways is the ability to signal to the polyomavirus enhancer, in particular to the crucial AP-1 and PEA3/ets binding sites. This connection explains the pleiotropy of MT and ST effects on transcription and DNA replication.

Animals↗
PubMed1984

Deletion of the origin of replication impairs the ability of polyomavirus DNA to transform cells and to form tandem insertions.

We examined the transforming properties of polyomavirus DNA molecules which can produce a functional large T-antigen but which are cis defective for viral DNA replication. The inability of these molecules to replicate results from the deletion of sequences comprising the viral replication origin. We found that even in the presence of a functional large T-antigen, transformation of rat cells by these viral DNAs was greatly reduced when compared with replication-competent parental DNA, and cells transformed by origin-minus mutants generally contained the integrated viral DNA in a nontandem arrangement. Therefore, polyomavirus large T-antigen promotes the establishment of transformation and tandem integration by interacting with the viral origin of DNA replication. This indicates that viral DNA synthesis is directly involved in these processes.

Animals↗
PubMed1984

Activities of polyomavirus large-T-antigen proteins expressed by mutant genes.

We constructed a set of polyomavirus mutants with alterations in the DNA sequences encoding large T-antigen. The mutant genomes were cloned and propagated as recombinants of plasmid pBR322, and the presence of the mutations was confirmed by nucleotide sequence analysis. To facilitate the analysis of defects in the function of large T-antigen, the dl1061 deletion was introduced into the mutant genomes. This deletion restricts the early gene expression to the synthesis of large T-antigen (Nilsson and Magnusson, EMBO J. 2:2095-2101, 1983). The mutant large T-antigens were identified after radioactive labeling. Their functional characterization was based on analysis of DNA binding, activity in the replication of viral DNA, and cellular localization. The native large T-antigen, which is 785 amino acid residues long, binds specifically to the regulatory region of polyomavirus DNA. This binding was significantly reduced by the deletion of amino acid residues 136 to 260. Nevertheless, this mutant large T-antigen was active in the initiation of viral DNA replication. Conversely, all of the mutants in this study that produced large T-antigens with alterations in the carboxy-terminal 146 amino acid residues had normal DNA-binding properties. However, these mutants were inactive in viral DNA synthesis and also inhibited the replication of wild-type DNA in cotransfected cells. The analysis of mutant dl2208 (Nilsson et al., J. Virol. 46:284-287, 1983) led to unexpected results. Its large T-antigen, missing amino acid residues 191 to 209, was overproduced. Although the protein had normal DNA-binding properties, it was not entering the cell nucleus normally. Furthermore, the dl2208 DNA replication was extremely low in the absence of small and middle T-antigens but was normal in the presence of these proteins.

Amino Acid Sequence↗
PubMed1984

The major site of tyrosine phosphorylation in polyomavirus middle T antigen is not required for transformation.

The induction of tumors and cellular transformation mediated by polyomavirus requires the action of middle T antigen. Accordingly, we have begun to define the domains of the viral protein important for these processes to learn more about its site and mechanism of action. One of the domains of middle T antigen which is thought to be important for its function includes a stretch of acidic amino acids and a vicinal tyrosine residue (tyrosine 315), the major site of tyrosine phosphorylation in vitro. To determine whether these acidic amino acids and tyrosine 315 are required to maintain the transforming activity of middle T antigen, we constructed deletions within the DNA sequences encoding these amino acids and measured the capacity of the resulting mutants to transform Rat-1 cells in culture. This was accomplished by using in vitro mutagenesis techniques with molecularly cloned polyomavirus DNA. Seven mutants were isolated. Five of these proved incapable of transforming Rat-1 cells and were found to contain deletions which altered the reading frame for middle T antigen. However, two mutants, pPdl1-4 and pPdl2-7, retained the capacity to transform Rat-1 cells at high frequencies. The middle T antigen encoded by one of these mutants, pPdl1-4, lacks part of the acidic string of amino acids but not tyrosine 315 (amino acids 304 through 310 are deleted), whereas the middle T antigen encoded by the other mutant, pPdl2-7, lacks the entire acidic amino acid stretch as well as tyrosine 315 (amino acids 285 through 323 are deleted). Rat-1 cells transformed by one or the other mutant DNA displayed a fully transformed phenotype, including the capacity to form tumors in animals. These results prove that the major site of tyrosine phosphorylation in middle T antigen and the acidic amino acids which precede it are not essential for its transforming activity.

Amino Acid Sequence↗
PubMed1986

Mutation in the polyomavirus genome that activates the properties of large T associated with neoplastic transformation.

We have constructed a polyomavirus mutant genome which exhibits an increased immortalization potential when transfected into primary rat embryo fibroblasts. The mutation is a 30-base-pair deletion (nucleotides 1367 through 1396) that inactivates the transforming potential of middle T but activates some of the properties of large T associated with neoplastic transformation. Unlike the wild-type large T, the mutant large T can fully complement polyoma middle T in the tumorigenic process in vivo as well as in the transformation of primary cells in vitro. The activity of the mutant can be explained by its inability to replicate in cells and, hence, its inability to exert a cytopathic effect after gene transfer at high multiplicity. A recombinant which encodes the middle and small T antigens, but not the large T antigen, can also elicit a fully transformed phenotype when introduced into primary rat fibroblasts. These results confirm previous observations from this laboratory indicating that two, and not three, viral gene functions are required for polyomavirus-mediated oncogenic transformation.

Animals↗