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PubMed2003

[Therapeutic possibilities for polyomavirus infections in renal transplantation].

TO IMPROVE THERAPEUTIC MANAGEMENT: The aim is the early detection of polyomavirus infection, before the onset of tubulo-interstitial nephritic lesions, and to reduce viral replication. AT THE STAGE OF POLYOMAVIRUS INFECTION: Treatment relies on the reduction of immunosuppression. Efficacy is controlled by monitoring the decoy cells in the urine and the detection and quantification of the DNA of polyomaviruses in the plasma and urine. AT THE STAGE OF POLYOMAVIRUS DISEASE: The aim is to reduce the viral replication by further decreasing immunosuppression to stabilize renal function and avoid graft rejection. When signs of rejection and viral infection co-exist, cidofovir could be a therapeutic alternative. However, the use of cidofovir remains in the field of clinical research and requires the further development of therapeutic protocols.

Antiviral Agents↗
PubMedDate not supplied

Presence and incidence of DNA sequences of human polyomaviruses BKV and JCV in colorectal tumor tissues.

The human polyomaviruses JCV and BKV are widespread within population, as shown by serological studies. However, exposure to these viruses does not seem to have pathological consequences in immunocompetent individuals, while in immunocompromised or immunosuppressed patients, polyomaviruses can be activated, giving rise to serious pathologies. Viral DNA sequences were also found in cells from a number of human tumors of mesothelial origin, suggesting that activation of BKV and JCV, taking place in genetically predisposed and/or in immunodepressed individuals, might be involved in the mechanisms of tumor transformation. In this study, samples obtained from 18 patients with colon rectal carcinoma were probed for the presence of JCV and BKV by three different techniques: Southern blot, PCR and in situ hybridization. Our results demonstrate that viral DNA sequences were present in 16 out of the 18 cases considered (88.9%). In the large majority of cases, viruses were detected both in the tumor mass and in the surrounding healthy tissues. Lymphocytes in the investigated areas were also found to be infected by polyomaviruses. These data indicate, for the first time, a possible involvement of polyomaviruses in the pathogenesis of tumors of endothelial origin, like the human colon rectal carcinoma.

Adenocarcinoma↗
PubMedDate not supplied

Inhibition of pp60c-src protein kinase by herbimycin A in polyomavirus middle tumor antigen-transformed cells.

The middle T antigen (MTAg) encoded by polyomavirus plays an important role in virus-mediated tumorigenesis. The activated protein kinase activity of MTAg-associated pp60c-src has been shown to be necessary for cell transformation by polyomavirus. In this study, the effects of herbimycin A on the pp60c-src kinase activities in the polyomavirus- and MTAg-transformed cells were studied. Phosphorylation of src and MTAg is reduced in polyomavirus and MTAg- transformed cells pretreated with herbimycin A. Inactivation of the enzymatic activity by herbimycin A was found to be dependent on the time of incubation and the drug concentration. In contrast, src immunoprecipitates from untreated MTAg-transformed cells appeared to be resistant to inhibition by herbimycin A. Herbimycin A does not affect the synthesis of MTAg and pp60c-src in the MTAg-transformed cells. These results suggest that pp60c-src kinase activity in the drug-treated cell lysates is more sensitive to herbimycin A inhibition than the same activity in the immunoprecipitates.

3T3 Cells↗
PubMed1997

The N terminus of hamster polyomavirus middle T antigen carries a determinant for specific activation of p59c-Fyn.

Transformation by rodent polyomaviruses is mediated primarily by middle T antigen, a membrane-bound protein that does not carry an intrinsic enzymatic activity but interacts and subverts the activity of cellular regulators of proliferation. The multiple protein partners of murine polyomavirus (Py) middle T antigen include the tyrosine kinases c-Src and, to a lesser extent, c-Fyn and c-Yes. By contrast, the hamster polyomavirus (HaPV) middle T antigen selectively activates the c-Fyn gene product. This difference may account for the contrasting tumor patterns induced by the two viruses. The sequences of the respective N-terminal and C-terminal functional domains of murine Py and HaPV middle T antigens are highly conserved whereas the intervening stretches are clearly divergent, leading to the speculation that this divergence may direct the specificity for tyrosine kinase activation. We have addressed this issue by constructing a chimera middle T antigen molecule carrying the N-terminal domain from HaPV (exon 1) in phase with the other two domains from murine Py (exon 2). The biological properties of this chimera molecule are indistinguishable from those of HaPV middle T antigen; it specifically activates p59c-Fyn and carries the transforming phenotype of the HaPV middle T antigen on rat fibroblasts.

Animals↗
PubMed2005

Maternal human polyomavirus infection and risk of neuroblastoma in the child.

To investigate if polyomavirus infection during pregnancy is linked to development of neuroblastoma in the child, serum samples of 115 index mothers from the pregnancy where the child eventually developed neuroblastoma were identified and matched with serum samples from 8 control mothers per index mother. The samples were tested for specific IgG and IgM antibodies to BK and JC virus using enzyme immunoassays based on purified yeast-expressed virus-like particles (VLPs). The serum samples as well as 10 neuroblastoma cell lines were also analyzed using Real Time (TaqMan) PCR for detection and quantification of BK virus DNA. The BK virus IgG seroprevalence was similar among index mothers (80%) and control mothers (83%) [OR 0.8; 95% confidence interval (95% CI): 0.5-1.3]. BK virus IgM was also not associated with neuroblastoma risk (OR was OR = 0.6; 95% with CI, 0.2-1.9). Also JC virus had no association, neither for IgG (OR = 0.9; 95% CI, 0.6-1.4) nor for IgM (OR = 0.9; 95% CI, 0.4-1.9). All serum samples and all neuroblastoma cell lines were negative for BKV DNA. In summary, a comprehensive cohort using both serology and polyomavirus DNA detection found no evidence for association between BKV or JCV polyomaviruses and neuroblastoma.

Adolescent↗
PubMed1991

Activation of polyomavirus DNA replication by yeast GAL4 is dependent on its transcriptional activation domains.

The polyomavirus replication origin contains transcriptional regulatory sequences. To determine how these elements function in DNA replication, and to learn whether a common mechanism underlies the activation of transcription and DNA replication, we tested whether a well-characterized transcriptional activator, yeast GAL4, was capable of stimulating DNA replication and transcription in the same mammalian cell line. We observed that GAL4 activated polyomavirus DNA replication in mouse cells when its binding site was juxtaposed to the late border of the polyomavirus origin core. Synergistic activation of DNA replication was achieved by multimerization of the GAL4 binding site. Analysis of GAL4 mutant proteins, GAL4 hybrid proteins and mutants of the latter revealed that the activation domains of these transcriptional activators were required to stimulate DNA replication. In agreement with previously published data, the activation domains of GAL4 were also required to enhance transcription in the same mouse cell line. These observations implicate transcriptional activators in Py DNA replication and suggest that similar mechanisms govern the activation of transcription and DNA replication.

Animals↗
PubMed1993

Lytic functions of mutant polyomavirus large T-antigen with deletion of retinoblastoma protein-binding motif.

Polyomavirus large T-antigen has a binding site for the retinoblastoma protein. Mutant proteins without this binding site retain their activity in the initiation of viral DNA synthesis, but are not able to induce immortalization of rat embryo fibroblasts. To investigate the function of the retinoblastoma binding site in polyomavirus lytic infection, a set of mutants with small in-frame deletions was constructed. We found that the mutant large T-antigens had partial or complete defects in immortalization and had corresponding disabilities in trans-activation of the adenovirus E2 promoter in secondary rat embryo cells. However, one partially immortalization-defective mutant trans-activated the E2 promoter in HeLa cells. All mutants were normal in trans-activation of the polyomavirus late promoter. The mutant large T-antigens had a decreased activity in viral DNA replication. This phenotype was observed only after transfection of growth-arrested mouse cells. The replication of the mutants could be restored to nearly wild-type levels when middle and small T-antigens were coexpressed in the cells. The same effect was achieved with a 287-amino-acid N-terminal fragment of large T-antigen with an intact retinoblastoma protein-binding motif. This result suggests that the effect on viral DNA synthesis was indirect.

3T3 Cells↗
PubMed2006

Polyomavirus-associated nephropathy in renal transplantation: critical issues of screening and management.

Polyomavirus-associated nephropathy (PVAN) is an emerging disease in renal transplant patients with variable prevalence of 1-10% and graft loss up to 80%. BK virus (BKV) is the primary etiologic agent, but JC virus (JCV) and possibly simian virus SV40 may account for some cases. Intense immunosuppression is viewed as the most important risk factor. However, the preferential manifestation in renal transplants as compared to other allografts or to autologous kidneys of other organ transplants suggests that organ determinants and immunologic factors synergize: Renal tubular epithelial cells and their compensatory proliferation to restore tubular integrity after immunologic, ischemic or toxic injury may provide the critical cellular milieu supporting polyomavirus replication while immune control is impaired due to maintenance immunosuppression, anti-rejection treatment and HLA-mismatches. Patient determinants (older age, male gender, seronegative recipient), and viral factors (genotype, serotype) may have a contributory role. The definitive diagnosis of PVAN requires allograft biopsy which is, however, challenged by (i) limited sensitivity due to (multi-)focal involvement (sampling errors); (ii) varying presentations with cytopathic-inflammatory and/or fibrotic/scarring patterns; (iii) coexisting acute rejection which is difficult to differentiate, but impacts on intervention strategies. Screening for polyomavirus replication in the urine and in the plasma complements allograft biopsy by high sensitivity and allows for noninvasive monitoring. Thus, we suggest a terminology similar to invasive fungal diseases where viruria ("decoy cells") defines patients at risk ("possible PVAN") who should be evaluated for plasma viral load. Increasing BK viremia (>10,000 copies/mL) or urine VP-1 mRNA (>6.5x10(5) copies/ng total RNA) load defines "presumptive PVAN" for which an intervention of reducing immunosuppression should be considered even if the diagnosis could not be confirmed by allograft biopsy ("definitive PVAN"). The response to intervention should be monitored using plasma DNA or urine mRNA load.

Humans↗
PubMed2006

Pharmacotherapeutic options for the management of human polyomaviruses.

Polyomaviruses [BK virus (BKV), JC virus (JCV) and simian virus 40 (SV40)] have been known to be associated with diseases in humans for over thirty years. BKV-associated nephropathy and JCV-induced progressive multifocal leukoencephalopathy (PML) were for many years rare diseases occurring only in patients with underlying severe impaired immunity. Over the past decade, the use of more potent immunosuppression (IS) in transplantation, and the Acquired Immune Deficiency Syndrome (AIDS) epidemic, have coincided with a significant increase in the prevalence of these viral complications. Prophylactic and therapeutic interventions for human polyomavirus diseases are limited by our current understanding of polyomaviral pathogenesis. Clinical trials are limited by small numbers of patients affected with clinically significant diseases, lack of defined risk factors and disease definitions, no proven effective treatment and the overall significant morbidity and mortality associated with these diseases. This chapter will focus on a review of the current and future research related to therapeutic targets and interventions for polyomavirus-associated diseases.

Disease Management↗
PubMed2006

Serological diagnosis of human polyomavirus infection.

Measurement of antibody titres to the human polyomaviruses BK and JC has for many years had to rely on Hemagglutination inhibition. In recent years, viral serology based on virus-like particles (VLPs) in enzyme immunoassays (EIAs) has become widely used for a variety of viruses. We sought to establish a modern method for serological diagnosis of BK and JC viruses, by using purified VLPs containing the VP1 major capsid proteins. Antibody titres in assays based on VLPs of BKV (strain SB) showed no correlation to the titres in similar JCV assays. BKV (SB) seropositivity increases rapidly with increasing age of the children and reaches a 98 % seroprevalence at 7-9 years of age, whereas JCV seroprevalences increase more slowly with increasing age reaching 51 % positivity among children 9-11 years of age. The antibody levels are almost identical in serial samples taken up to 5 years apart, suggesting that both BKV and JCV VLP seropositivitities are usually stable over time and can be used to measure cumulative exposure to these viruses. Serology using SV40 VLPs showed strong cross-reactivity with human polyomaviruses, in particular with BKV strain AS, and establishing a specific VLP-based serology assay for SV40 required blocking with several hyperimmune sera to the human polyomaviruses. SV40-specific seropositivity also increased with increasing age of children, reaching 14% seroprevalence among children 7-9 years of age, but had limited stability over time in serial samples.

Antibodies, Viral↗
PubMed2006

The role of sialic acid in human polyomavirus infections.

JC virus (JCV) and BK virus (BKV) are human polyomaviruses that infect approximately 85% of the population worldwide [1,2]. JCV is the underlying cause of the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML), a condition resulting from JCV induced lytic destruction of myelin producing oligodendrocytes in the brain [3]. BKV infection of kidneys in renal transplant recipients results in a gradual loss of graft function known as polyomavirus associated nephropathy (PVN) [4]. Following the identification of these viruses as the etiological agents of disease, there has been greater interest in understanding the basic biology of these human pathogens [5,6]. Recent advances in the field have shown that viral entry of both JCV and BKV is dependent on the ability to interact with sialic acid. This review focuses on what is known about the human polyomaviruses and the role that sialic acid plays in determining viral tropism.

Humans↗
PubMed1992

Stalling by RNA polymerase II in the polyomavirus intergenic region is dependent on functional large T antigen.

RNA polymerase II encounters an elongation block and stalls in vivo during transcription of the late strand of polyomavirus DNA. In this study, we performed transcriptional run-on assays and localized the stalling site to a 164-nucleotide region (nt 11-175) that contains specific binding sites for polyomavirus large T antigen. The effect of large T antigen on elongation by RNA polymerase II through this region was examined in cells infected with a mutant polyomavirus (AT3-ts25E) which encodes a thermolabile large T antigen. Removal of functional large T antigen by shifting to the nonpermissive temperature (39 degrees) eliminated stalling by RNA polymerase in this region, although RNA polymerases transcribing other regions of the viral genome were unaffected. RNA polymerase resumed stalling when functional large T antigen was again allowed to accumulate by shifting back to the permissive temperature (32 degrees). We conclude that stalling by RNA polymerase II in vivo is dependent on the presence of functional large T antigen.

Animals↗
PubMed2006

Generation of virus-like particles consisting of the major capsid protein VP1 of goose hemorrhagic polyomavirus and their application in serological tests.

Goose hemorrhagic polyomavirus (GHPV) is the causative agent of hemorrhagic nephritis and enteritis of geese (HNEG), a fatal disease of young geese with high mortality rates. GHPV cannot be efficiently propagated in tissue culture. To provide antigens for diagnostic tests and vaccines, its major structural protein VP1 was recombinantly expressed in Sf9 insect cells and in the yeast Saccharomyces cerevisiae. As demonstrated by density gradient centrifugation and electron microscopy, GHPV-VP1 expressed in insect cells formed virus-like particles (VLPs) with a diameter of 45 nm indistinguishable from infectious polyomavirus particles. However, efficiency of VLP formation was low as compared to the monkey polyomavirus SV-40-VP1. In yeast cells, GHPV-VP1 alone formed smaller VLPs, 20 nm in diameter. Remarkably, co-expression of GHPV-VP2 resulted in VLPs with a diameter of 45 nm. All three types of GHPV-VLPs were shown to hemagglutinate chicken erythrocytes. ELISA and hemagglutination inhibition tests using the VLPs as antigen detected GHPV-specific antibodies in up to 85.7% of sera derived from flocks with HNEG but in none of the sera of a clinically healthy flock. However, GHPV-specific antibodies were also detected in sera from two other flocks without HNEG indicating a broad distribution of GHPV due to subclinical or unrecognised infections.

Animals↗
PubMed1997

Production of active polyomavirus large T antigen in yeast Pichia pastoris.

The coding region of polyomavirus large T antigen was engineered into the genome of the methylotrophic yeast Pichia pastoris by use of the vector pHIL-D2. Expression of large T antigen was induced by methanol under the control of the strong alcohol oxidase (AOX1) promoter. Large T antigen was purified by immunoaffinity chromatography. We showed that yeast-derived large T antigen bound specifically to a DNA fragment that contains the polyomavirus replication origin, protected the four known major binding sites in the origin against DNase I digestion, and could unwind the strands of an origin-containing DNA fragment in an ATP-dependent manner. This system therefore provides a convenient and inexpensive source of biologically active polyomavirus large T antigen for in vitro studies.

Alcohol Oxidoreductases↗
PubMed2002

Immunization of T-cell deficient mice against polyomavirus infection using viral pseudocapsids or temperature sensitive mutants.

A murine experimental model system aimed at developing potential vaccines to papovavirus infection in immunosuppressed individuals was explored. A VP1-pseudocapsid based on the major capsid protein of the murine polyomavirus A2 strain and a mutant, M17-pseudocapsid as well as four temperature sensitive (ts)-mutants were used as immunogens. T-cells deficient CD4-/-8-/- mice were immunized four times with each immunogen and then together with non-immunized control mice challenged with polyomavirus. In contrast to all control mice, only half of the immunized mice exhibited presence of polyoma DNA when assayed by PCR. The results indicate that pseudocapsids and ts-mutant immunization may potentially protect mice with an impaired T-cell function from polyomavirus infection.

Animals↗
PubMed1987

Regulation of c-myc and c-fos mRNA levels by polyomavirus: distinct roles for the capsid protein VP1 and the viral early proteins.

The levels of c-myc, c-fos, and JE mRNAs accumulate in a biphasic pattern following infection of quiescent BALB/c 3T3 mouse cells with polyomavirus. Maximal levels of c-myc and c-fos mRNAs were seen within 1 hr and were nearly undetectable at 6 hr after infection. At 12 hr after infection mRNA levels were again maximal and remained elevated thereafter. Empty virions (capsids) and recombinant VP1 protein, purified from Escherichia coli, induced the early but not the late phase of mRNA accumulation. Virions, capsids, and recombinant VP1 protein stimulated [3H]thymidine nuclear labeling and c-myc mRNA accumulation in a dose-responsive manner paralleling their affinity for the cell receptor for polyoma. The second phase of mRNA accumulation is regulated by the viral early gene products, as shown by polyomavirus early gene mutants and by a transfected cell line (336a) expressing middle tumor antigen upon glucocorticoid addition. These results suggest that polyomavirus interacts with the cell membrane at the onset of infection to increase the levels of mRNA for cellular genes associated with cell competence for DNA replication, and subsequently these levels are maintained by the action of the early viral proteins.

Animals↗
PubMedDate not supplied

Negative-staining electron microscopy of the urine for the detection of polyomavirus infections.

Negative-staining electron microscopy (EM) has played a pivotal role in diagnostic virology. It is a rapid technique for viral detection in the urine and can provide an easy means for monitoring viral activity and productive infections. EM of urine for the detection of polyomaviruses has hitherto not been systematically evaluated as a screening tool for renal transplant patients at risk for BK polyomavirus nephropathy (BKN). Here, the authors discuss technical aspects of negative-staining EM of urine (n = 76 samples) and present a simple and rapid protocol for the semiquantitative evaluation of patient samples. In two patient populations (either with (n = 15 samples) or without (n = 15 samples) an established diagnosis of BKN), EM results were compared with two previously established techniques for monitoring polyomavirus activation: (1) cytology for the quantitation of decoy cells, and (2) quantitative PCR assays for the detection of BK virus DNA load levels. In both patient groups, the dynamics of decoy cell shedding by urine cytology closely paralleled free viral particle shedding by EM, and viral load levels as measured by PCR. A trend toward higher readings was observed in patients with BKN (median values, control versus BKN groups: decoy cells 21 versus 50/slide; free virions by EM: 32 versus 66 viral particles/10 high-power fields; PCR: 3.5 x 10(8) versus 5.4 x 10(8) BK virus copies/ml; all differences not statistically significant). The authors conclude that negative-staining EM and the semiquantitative assessment of free viral particles in the urine can be a useful clinical method to identify patients at increased risk for BKN. EM can be used alone or in combination with urine cytology or PCR assays.

Cytodiagnosis↗
PubMed1987

Regulation of viral and cellular promoter activity by polyomavirus early proteins.

The chloramphenicol-acetyl-transferase (CAT) expression system has been utilized to study the ability of the polyomavirus (Py) early proteins, the 100K large T, the 55K middle T and 22K small T-antigens, to activate a variety of eukaryotic promoters (the SV40 early, the alpha 2(1) collagen, the rabbit beta-globin, the polyomavirus early and the H-2 class I) in both transient and stable expression assays. We have found that either the complete polyomavirus early region or a plasmid expressing only the 55K middle T-antigen are capable of stimulating the expression of all the promoter-CAT plasmids in transient co-transfection experiments in both NIH-3T3 and Rat-2 cells. Conversely, the Py early proteins do not stimulate the transcription of most of the promoter-CAT genes stably introduced in the cell chromosomes, with the exception of H-2 class I promoter, when stimulation of transcription has been observed upon infection with recombinant retrovirus encoding the Py middle T-antigen.

Acetyltransferases↗