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PubMed1997

Activities of various compounds against murine and primate polyomaviruses.

Polyomavirus infections in humans are due to BK virus (BKV) and JC virus (JCV). Diseases associated with human polyomaviruses occur mostly in immunocompromised adults, e.g., progressive multifocal leukoencephalopathy (PML), caused by JCV, in AIDS patients and hemorrhagic cystitis and uretral stenosis, caused by BKV, in transplant recipients. No therapy is available for these diseases, which necessitates the development of chemical entities that are active against polyomaviruses. Several antiviral compounds were evaluated to determine their effects on the in vitro replication of mouse polyomavirus and the primate viruses simian virus 40 (SV40), SV40 PML-1, and SV40 PML-2. The activity of the different compounds was assessed by a cytopathic effect reduction assay and confirmed in a virus yield assay. Cidofovir [HPMPC; (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine] and its cyclic counterpart emerged as the most selective antipolyomavirus agents. The 50% inhibitory concentrations for HPMPC were in the range of 4 to 7 micrograms/ml, and its selectivity index varied from 11 to 20 for mouse polyomavirus and from 23 to 33 for SV40 strains in confluent cell monolayers. Cell cytotoxicity was up to 15-fold greater in growing cells. Other acyclic nucleoside phosphonates (i.e., HPMPA; [(S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine] and PMEG [9-(2-phosphonylmethoxyethyl)-guanine]) also showed some activity but had low selectivity. None of the other drugs tested against these animal viruses (i.e., acyclovir, ganciclovir, brivudine, ribavirin, foscarnet, and cytarabine) showed significant activity. Thus, HPMPC deserves further evaluation as a candidate drug for polyomavirus infections in the immunocompromised host.

Animals↗
PubMed2007

Transcription factor Runx1 recruits the polyomavirus replication origin to replication factories.

Eukaryotic DNA replication takes place in the replication factories, where replication proteins are properly assembled to form replication forks. Thus, recruitment of DNA replication origins to the replication factories must be the key step for the regulation of DNA replication. The transcription factor Runx1 associates with the nuclear matrix, the putative substructure of DNA replication factories. An earlier report from our laboratory showed that Runx1 activates polyomavirus DNA replication, and that this requires its nuclear matrix-binding activity. Here, we show that Runx1 activates polyomavirus DNA replication by stimulating the binding of the viral-encoded replication initiator/helicase, large T antigen, to its replication origin. We found that newly replicated polyomavirus DNA is associated with the nuclear matrix and that large T antigen is targeted to replication factories, suggesting that polyomavirus is replicated in replication factories on the nuclear matrix. Although Runx1 did not co-localize with large T antigen-containing foci by itself, it co-localized with large T antigen-containing replication factories during Runx1-dependent polyomavirus DNA replication. These observations together suggest that Runx1 recruits the polyomavirus replication origin to the replication factory on the nuclear matrix, and that this requires the nuclear matrix-binding activity of Runx1.

Animals↗
PubMed1997

Screening human tumor samples with a broad-spectrum polymerase chain reaction method for the detection of polyomaviruses.

Polyomaviruses induce tumors of different histological types when inoculated into experimental animals. An etiological role for this virus group in the development of malignant tumors in humans remains questionable, despite several reports demonstrating the presence of SV40, JCV, and BKV DNA in human cancers. Only two human polyomavirus types are known to date: JCV, causing progressive multifocal leukoencephalopathy (PML) under severe immunosuppression, and BKV, first isolated from the urine of a renal transplant recipient and associated with hemorrhagic cystitis. We developed a degenerate polymerase chain reaction assay in an attempt to identify additional, presently unknown human polyomavirus types that may be involved in the malignant transformation of human tissues. A large part of the gene coding for the viral capsid protein VP1 is highly conserved in nine polyomavirus types (and their strains) and was therefore selected as most suitable for the primer design. Degenerate oligonucleotide primers were deduced from four different conserved amino acid motifs in this region. Three different sets of primers were included in each test to obtain the highest sensitivity in combination with primers with the lowest degeneracy numbers. The sensitivity obtained ranged from 1 copy/cell for bovine polyomavirus to 100 copies/cell for LPV after ethidium bromide staining and was increased at least 10-fold after hybridization with a radiolabeled probe. A subsequent seminested amplification allowed for the detection of 1 copy/cell for LPV. These degenerate primers were applied to analyze bladder carcinomas, Hodgkin lymphomas, meningiomas, Kaposi tumors, and Kaposi-derived cell lines. No polyomavirus DNA sequences could be detected.

Animals↗
PubMed2003

Microtubule-dependent intracellular transport of murine polyomavirus.

The mechanisms used by murine polyomavirus for intracellular migration are yet to be clarified. In this work we selectively depolymerized microtubules or actin fibers and then studied the progression of polyomavirus infection in cultured cells. Our results demonstrate that microtubule depolymerization prevents polyomavirus migration toward the nucleus and from the nucleus to the cell surface, being also involved in viral release, while disruption of the actin microfilaments appears to have no detrimental effect on the virus ability to reach the nucleus. The ultrastructural observation of polyomavirus nonenveloped particles interacting with the free end and the lateral sides of microtubules together with the coimmunoprecipitation of tubulin and viral VP-1 further supports the idea that polyomavirus intracellular migration seems to be mediated by the interaction of polyomavirus major capsid protein VP-1 with tubulin.

3T3 Cells↗
PubMed1998

Improvement of retroviral packaging cell lines by introducing the polyomavirus early region.

To obtain high-titer recombinant retroviruses, we constructed plasmid pDL+, which carries the extended Psi region and the polyomavirus early region, including the replication origin and the early gene. Although pDL+ is useful for obtaining high-titer recombinant retroviruses, this vector plasmid is difficult to modify further for tissue-specific expression of foreign genes. To overcome this problem, the coding region of the polyomavirus early gene was expressed in the packaging cell lines. We modified the packaging cell lines, psi2 and PA317, by stably introducing the polyomavirus early gene, and established psiMP34 and psiMP37 from psi2, and PAMP51 from PA317. In the transient expression system using plasmids with and without the polyomavirus replication origin, the titers of recombinant retrovirus produced by these cell lines were 10-100 times higher than produced by the parent cell line and reached levels of 0.5-1.5 x 106 cfu/ml. Expression of the polyomavirus early gene in the packaging cell lines did not stimulate the production of replication-competent retrovirus. We also routinely established stable clones producing retrovirus titers over 1 x 10(7) cfu/ml from psiMP34 and PAMP51. We found that the activity of the long terminal repeat (LTR) promoter is stimulated by the polyomavirus early region protein(s) in these cell lines. Therefore, increases titer can be expected to occur in all the retroviral vectors in which LTR promoter is used to transcribe the retroviral genome.

3T3 Cells↗
PubMed2004

Meningoencephalitis and demyelination are pathologic manifestations of primary polyomavirus infection in immunosuppressed rhesus monkeys.

The human polyomavirus JC (JCV) is the etiologic agent of progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the CNS that occurs in immunosuppressed individuals. Because polyomavirus-induced CNS pathology usually occurs as a result of the reactivation of latent virus, little is known about the disease manifestations of a primary polyomavirus-induced disease in man. To model such a primary infection, SV40-negative rhesus monkeys were immunosuppressed by infection with the virus SHIV-89.6P and then superinfected with the polyomavirus SV40. The animals developed CNS pathology characterized by both demyelination and meningoencephalitis. This observation suggests that a primary polyomavirus infection can be associated with an inflammatory CNS process. These data shed new light on the pathogenic mechanisms of primate polyomaviruses in the immunocompromised host.

Animals↗
PubMed2000

Early steps of polyomavirus entry into cells.

The mechanism by which murine polyomavirus penetrates cells and arrives at the nucleus, the site of viral replication, is not well understood. Simian virus 40 and JC virus, two closely related members of the polyomavirus subfamily, use caveola- and clathrin-mediated uptake pathways for entry, respectively. The data presented here indicate that compounds that block endocytosis of both caveola- and clathrin-derived vesicles have no effect on polyomavirus infectivity. Polyomavirus does not appear to colocalize with either clathrin light chain or caveolin-1 by immunofluorescence microscopy. Additionally, expression of a dominant-negative form of dynamin I has no effect on polyomavirus uptake and infectivity. Therefore, polyomavirus uptake occurs through a class of uncoated vesicles in a clathrin-, caveolin-1-, and dynamin I-independent manner.

3T3 Cells↗
PubMed2003

Mouse polyomavirus utilizes recycling endosomes for a traffic pathway independent of COPI vesicle transport.

Mouse polyomavirus enters host cells internalized, similar to simian virus 40 (SV40), in smooth monopinocytic vesicles, the movement of which is associated with transient actin disorganization. The major capsid protein (VP1) of the incoming polyomavirus accumulates on membranes around the cell nucleus. Here we show that unlike SV40, mouse polyomavirus infection is not substantially inhibited by brefeldin A, and colocalization of VP1 with beta-COP during early stages of polyomavirus infection in mouse fibroblasts was observed only rarely. Thus, these viruses obviously use different traffic routes from the plasma membrane toward the cell nucleus. At approximately 3 h postinfection, a part of VP1 colocalized with the endoplasmic reticulum marker BiP, and a subpopulation of virus was found in perinuclear areas associated with Rab11 GTPase and colocalized with transferrin, a marker of recycling endosomes. Earlier postinfection, a minor subpopulation of virions was found to be associated with Rab5, known to be connected with early endosomes, but the cell entry of virus was slower than that of transferrin or cholera toxin B-fragment. Neither Rab7, a marker of late endosomes, nor LAMP-2 lysosomal glycoprotein was found to colocalize with polyomavirus. In situ hybridization with polyomavirus genome-specific fluorescent probes clearly demonstrated that, regardless of the multiplicity of infection, only a few virions delivered their genomic DNA into the cell nucleus, while the majority of viral genomes (and VP1) moved back from the proximity of the nucleus to the cytosol, apparently for their degradation.

Animals↗
PubMed1984

Isolation of large T antigen-producing mouse cell lines capable of supporting replication of polyomavirus-plasmid recombinants.

Construction of polyomavirus vectors, analysis of mutant viruses, and rescue of integrated polyomavirus genomes would be considerably aided by the availability of transformed, permissive mouse cell lines capable of producing the viral tumor antigens. To isolate such cell lines, we constructed a hybrid transcription unit composed of the simian virus 40 early promoter fused to the coding region for the polyomavirus tumor antigens. This hybrid transcription unit was used to transform NIH 3T3 cells. Independent foci of transformed cells were isolated, recloned, and characterized. Among 10 lines initially analyzed, 7 supported the replication of origin-bearing plasmid DNAs. Three cell lines were characterized in greater detail. Each line contained one or two independent insertions of polyomavirus DNA and synthesized all three viral tumor antigens. Moreover, the large tumor antigen in two of three lines bound with specificity to sequences about the polyomavirus origin and early promoter. These cell lines should prove useful for studying not only the replication of polyomavirus but also the expression of foreign genes in a mouse cell environment.

Animals↗
PubMedDate not supplied

Cytologic and biomolecular diagnosis of polyomavirus infection in urine specimens of HIV-positive patients.

OBJECTIVE: To evaluate the frequency of human polyomavirus reactivation in urine specimens from HIV-positive patients; compare the sensitivity of cytology, immunohistochemistry and molecular biology; differentiate viral genotypes; and correlate the results with urinary cytologic abnormalities. STUDY DESIGN: Urine specimens from 78 unselected HIV-positive patients were evaluated by means of cytology, immunohistochemistry and nested polymerase chain reaction (n-PCR) to evaluate the presence of polyomaviruses. Restriction fragment length polymorphism (RFLP) was carried out in positive cases in order to differentiate BK virus (BKV) from JC virus (JCV). CD4 cells and serum creatinine levels were evaluated as indices of immune status and renal function, respectively, whereas the presence of red blood cells was used as an index of urogenital damage. RESULTS: Cytologic evidence of polyomavirus infection was found in 17 samples and immunohistochemically confirmed in 9; another 6 cytologically negative cases were detected by means of immunohistochemistry. In all cases, only one or two cells showed typical viral inclusions or positive staining. n-PCR identified 44 positive samples, thus confirming all of the cytologically and immunohistochemically positive cases and detecting polyomavirus genome in a further 21. RFLP detected 39 JCV, 1 BKV and 4 JCV-BKV infections. No correlation was found between the presence or type of polyomavirus and immune status, but red blood cells were found more frequently in the positive than in the negative samples. Serum creatinine levels fell within the normal range in all cases. CONCLUSION: Molecular biology is the most sensitive tool for detecting polyomavirus urinary infection in HIV-positive patients and the only reliable method of differentiating JCV and BKV viral genotypes.

Adult↗
PubMed2002

Medulloblastomas and primitive neuroectodermal tumors rarely contain polyomavirus DNA sequences.

To address the hypothesis that medulloblastoma or supratentorial primitive neuroectodermal tumor (sPNET) can arise through infection by polyomaviruses, we examined genomic DNA isolated from 15 primary medulloblastoma and 5 sPNET biopsy specimens and from 2 medulloblastoma cell lines for the presence of DNA sequences from the polyomaviruses simian virus 40 (SV40), JC virus, and BK virus. These polyomaviruses have oncogenic potential in animals, and their DNA sequences have been detected in other surveys of various solid tumors, including childhood brain tumors. The tumor DNA samples were analyzed by Southern blot hybridization of polymerase chain reaction products that employed probes designed to detect specific polyomavirus sequences. Neither JC virus nor BK virus DNA sequences were detected in any of the specimens. None of the primary medulloblastoma or sPNET specimens contained SV40 sequences. However, SV40 DNA coding and noncoding sequences were detected in the D283-Med (medulloblastoma) cell line. Immunocytochemical studies of D283-Med revealed nuclear expression of SV40 large T antigen. In contrast to childhood ependymomas and choroid plexus tumors, medulloblastomas and sPNETs infrequently express evidence of polyomavirus infection.

Antigens, Neoplasm↗
PubMed1994

Infection of human polyomaviruses JC and BK in peripheral blood leukocytes from immunocompetent individuals.

Widely disseminated infection with the human polyomaviruses JC and BK is followed by lifelong asymptomatic viral persistence that can be reactivated under prolonged immunosuppression to fatal CNS and urogenital disease. In an attempt to understand the pathogenesis of polyomavirus diseases, we asked whether leukocytes are involved in polyomavirus infection in the immunocompetent host. Peripheral blood leukocytes from 29 immunocompetent individuals and umbilical cord blood from 10 newborn children were analyzed for the presence of polyomavirus DNA. Southern blot analysis demonstrated the presence of JCV-specific full-length virus genomes and indicated involvement of the second human polyomavirus BK. In contrast to specimens from newborn children, PCR amplification of target DNA in the adult age group followed by species-specific hybridization provided evidence of concomitant JCV and BKV infection in almost all specimens. Nucleotide sequencing of virus-specific products representing DNA segments essential for virus multiplication confirmed presence of both virus species in leukocytes. The detection of a new virus subtype and single base changes or deletions in the noncoding DNA region from individual cases suggested widespread heterogeneity in the circulating virus population, although the structure of the transcriptional control elements in all cases was comparable to highly active elements found in lytically infected cells. Those findings and the localization of both virus types in the nuclei of blood cells by in situ hybridization demonstrate that JCV and BKV frequently infect peripheral leukocytes and give strong evidence that leukocytes are common sites of polyomavirus persistence in healthy individuals.

BK Virus↗
PubMed2003

VP1 pseudocapsids, but not a glutathione-S-transferase VP1 fusion protein, prevent polyomavirus infection in a T-cell immune deficient experimental mouse model.

The ability to vaccinate against polyomavirus infection in a T-cell deficient as well as a normal immune context was studied using polyomavirus major capsid protein (VP1) pseudocapsids (VP1-ps) or a glutathione-S-transferase-VP1 (GST-VP1) fusion protein. VP1-ps (1 or 10 microg) were administered subcutaneously, alone or together with Freund's complete and incomplete adjuvant, to CD4(-/-)8(-/-) T-cell deficient or normal C57Bl/6 mice on four occasions. Alternatively, CD4(-/-)8(-/-) and normal mice were inoculated with either GST-VP1 or Py-VP1-ps (5 microg). Following immunisation, antibody titres were tested by ELISA to VP1-ps or GST-VP1 or by haemagglutination inhibition (HAI). Mice were then infected with polyomavirus. Three weeks post-infection, the mice were killed and examined for the presence of polyomavirus DNA by PCR. Viral DNA was not detected in CD4(-/-)8(-/-) mice immunised with either VP1-ps alone or in combination with Freund's complete and incomplete adjuvant, or in any of the normal mice immunised with VP1-ps or GST-VP1. However, viral DNA was detected in 2/5 of the CD4(-/-)8(-/-) mice immunised with GST-VP1 and in non-immunised controls. Greater antibody titres were observed to VP1-ps than to GST-VP1 in CD4(-/-)8(-/-) mice after VP1-ps compared to GST-VP1 immunisation and antibody responses were better in normal than in immune-deficient mice. Only immunisation with VP1-ps resulted in haemagglutination inhibition. Complete protection against polyomavirus infection in the T-cell deficient context was obtained with VP1-ps, but not with GST-VP1, immunisation using the present vaccination protocol.

Animals↗
PubMed2006

Immunity and autoimmunity induced by polyomaviruses: clinical, experimental and theoretical aspects.

In this chapter, polyomaviruses will be presented in an immunological context. Principal observations will be discussed to elucidate humoral and cellular immune responses to different species of the polyomaviruses and to individual viral structural and regulatory proteins. The role of immune responses towards the viruses or their proteins in context of protection against polyomavirus induced tumors will be described. One central aspect of this presentation is the ability of polyomaviruses, and particularly large T-antigen, to terminate immunological tolerance to nucleosomes, DNA and histones. Thus, in the present chapter we will focus on clinical, experimental and theoretical aspects of the immunity to polyomaviruses.

Autoimmunity↗
PubMed1988

The genome of budgerigar fledgling disease virus, an avian polyomavirus.

Budgerigar fledgling disease virus (BFDV) represents the first avian member of the Polyomavirus family. In contrast to mammalian polyomaviruses BFDV exhibits unique biological properties, in particular it is able to cause an acute disease with distinct organ manifestations in affected birds. Here we present the complete nucleotide sequence of the BFDV genome, consisting of 4980 bp. When compared to published nucleotide sequences of other polyomaviruses, the BFDV genome exposes a number of very similar structural features, and undoubtedly qualifies as a member of that family of viruses. The most important differences include a large T antigen remarkably reduced in size, and an origin of replication region with fundamental deviations from the origin structure of all other polyomaviruses. The specific characteristics of the BFDV genome may be used to place this virus into a distinct subgroup within the Polyomavirus family and may give a clue to the elucidation of its extraordinary biological properties.

Amino Acid Sequence↗
PubMed2001

The hamster polyomavirus--a brief review of recent knowledge.

The hamster polyomavirus (HaPV) was first described in 1967 as a virus associated with skin epithelioma of the Syrian hamster. The tumors appear spontaneously in a hamster colony bred in Berlin-Buch (HaB). Virus particles isolated from skin epitheliomas cause lymphoma and leukemia when injected into newborn hamsters from a distinct colony bred in Potsdam, Germany (HaP). The viral genome has been totally sequenced and the overall genetic organization establishes HaPV as a member of the polyomaviruses. HaPV is a second example of an middle T (MT) antigen encoding polyomavirus and nucleotide sequence homologies designates the mouse polyomavirus (Py) as the closest relative. Lymphomas induced by HaPV in HaP hamsters do not contain virus particles but instead accumulate different amounts of nonrandomly deleted free and/or integrated viral genomes. Transgenic mice produced by microinjection of HaPV DNA into the pronucleus of fertilized eggs of Gat: NMRI mice developed both, epitheliomas and lymphomas. Both tumor types contain extrachromosomal DNA. HaPV DNA was found to replicate in hamster lymphoid and fibroblast cell lines. Fully reproductive cycles could be detected only in GD36 lymphoblastic leukemia cells. HaPV carries the full transforming properties of a polyomavirus in vitro. Immortalization of primary rat cells is essentially carried out by the HaPV large T (LT) antigen and coexpression of HaPV MT and HaPV small T (ST) antigen is required for full transformation of rat fibroblasts. The preferential binding of HaPV MT to c-Fyn, a Src family kinase, has been proposed as a mechanism leading to lymphoid malignancies. Heterologous expression of HaPV-VP1 allowed the formation of virus like particles (VLPs) resembling HaPV particles. The high flexibility of HaPV-VP1 for insertion of foreign peptides offers a broad range of potential applications, especially in vaccine development.

Animals↗
PubMed2004

Constraints imposed by supercoiling on in vitro amplification of polyomavirus DNA.

Previous attempts to identify oncogenic polyomaviruses in human cancers have yielded conflicting results, even with the application of PCR technology. Here, it was considered whether the topological features of the polyomavirus genome interfere with efficient PCR amplification. Plasmid and SV40 DNAs were used as a model system for comparing the amplification efficiency of supercoiled, circular relaxed and linear templates. It was found that detection of circular templates required 10 times more molecules than detection of identical but linear templates. Supercoiling hindered the in vitro amplification of SV40 circles by a factor of 10, and erratic amplification of supercoiled SV40 occurred with subpicogram amounts of template. Accordingly, topoisomerase I treatment of DNA improved the PCR detection of supercoiled SV40, significantly decreasing the number of false-negative samples. Previously described, yet controversial, polyomavirus presence in human tissues should be reconsidered and topoisomerase I-sensitive polyomavirus amplification might help to detect polyomavirus genomes in mammalian tissues.

Cell Line, Tumor↗
PubMed1987

Overproduction of polyomavirus middle T antigen in mammalian cells through the use of an adenovirus vector.

To overproduce biologically active polyomavirus middle T antigen, we used an adenovirus vector and human 293 cells as hosts. Two helper-independent recombinant adenoviruses were isolated that contain a hybrid transcription unit, in differing orientations, at a site in the adenovirus genome from which the E1a and most of the E1b transcription units have been deleted. The hybrid transcription unit consists of the adenovirus type 2 major late promoter and tripartite leader and a cDNA segment capable of encoding polyomavirus middle T antigen and accompanying 3' RNA-processing signals. Both recombinant viruses were stable and replicated to high titers in human 293 cells. The polyomavirus sequences were expressed, predominantly at late times after infection of 293 cells, to yield mRNAs that encoded middle T antigen. One of the recombinant viruses also expressed a middle T antigen-related protein in 293 cells. The latter was translated from one of several novel mRNA species that resulted from aberrant splicing and incomplete RNA processing of precursor RNA transcripts. Comparison of the amount of middle T antigen produced in 3T6 cells infected with polyomavirus with that in 293 cells infected with either of the recombinant adenoviruses, under optimal conditions for each system, revealed at least a 10-fold greater yield of the protein on a per-cell basis in the latter system than in the former. The recombinant-virus-encoded middle T antigen was biologically active, as evidenced by its ability to associate with and serve as a substrate for human pp60c-src. The functionality of the middle T antigen was further confirmed by demonstrating that both recombinant viruses efficiently transformed Rat-1 cells. These recombinant viruses will be useful to overproduce middle T antigen and to introduce the polyomavirus oncogene into a wide variety of mammalian cells.

Adenoviruses, Human↗