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PubMed2001

Molecular genotyping of BK and JC viruses in human polyomavirus-associated interstitial nephritis after renal transplantation.

The human polyomaviruses BK virus (BKV) and JC virus (JCV) have been linked to ureteric stenosis and allograft interstitial nephritis, but molecular characterization of the species involved has not been performed. We studied paraffin-embedded renal tissue from 19 cases of allograft viral interstitial nephritis. Histological sections were subjected to polymerase chain reaction amplification using consensus, BKV-, and JCV-specific primers, with subsequent DNA sequencing for strain determination. BKV was present in all (100%) interstitial nephritis kidneys and placed in genotypes corresponding to serological groups I (n = 11), II (n = 1), and IV (n = 5). Fourteen of 17 isolates (82%) showed sequence variations in the viral capsid protein-1 (VP1) capsid region, with predicted changes in the encoded amino acids and sometimes with potential implications for the secondary and tertiary structure of the corresponding protein molecules. An additional case showed a previously reported glutamine-->leucine T-antigen region mutation. JCV was seen in seven interstitial nephritis kidneys (37%), with types 4 (n = 3), 3A (n = 2), and 2A (n = 1) identified. Most white individuals with asymptomatic infection are reported to shed type 1 JCV in the urine. Simian 40 polyomavirus was not identified in any case. These observations may have pathogenic relevance to the development of an extremely refractory form of polyomavirus interstitial nephritis seen after kidney transplantation.

Amino Acid Sequence↗
PubMed2003

Chaperone-mediated in vitro assembly of Polyomavirus capsids.

The polyomavirus coat protein viral protein 1 (VP1) has the intrinsic ability to self-assemble in vitro into polymorphic capsid-like structures on addition of calcium. In contrast, polyomavirus assembly in vivo is rigorously controlled, such that virions of uniform size are formed only in the cell nucleus. During viral infection, the 72 kDa cellular chaperone heat shock cognate protein (hsc70) binds VP1 posttranslation and colocalizes with VP1 to the nucleus, thereby suggesting a role for approximately 70-kDa heat shock protein (hsp70) family chaperones in regulating the quality and location of capsid assembly. We found that, after expression of recombinant VP1 in Escherichia coli, the prokaryotic hsp70 chaperone DnaK copurified with the VP1 C-terminal domain that links pentamers in an assembled capsid. When stably bound to VP1, DnaK inhibited in vitro assembly induced by calcium. However, in the presence of ATP, the hsp70 chaperone system comprised of DnaK, DnaJ, and GrpE assembled VP1 into uniform capsids without requiring calcium. Chaperone-mediated assembly was similarly catalyzed by the eukaryotic hsc70 protein, in combination with the J-domain function of the simian virus 40 large T-antigen protein. Thus, polyomavirus capsid assembly can be recapitulated with high-fidelity in vitro using either prokaryotic or eukaryotic hsp70 chaperone systems, thereby supporting a role for cellular chaperones in the in vivo regulation of virion assembly.

Adenosine Triphosphate↗
PubMed1984

Regulation of polyomavirus transcription by large tumor antigen.

We have analyzed the regulation of viral transcription by the large tumor antigen in cells infected by several viable deletion and insertion mutants of polyomavirus. We find that deletion of the early promoter "TATA box" and associated large tumor antigen binding site has only a small effect on the balance of early and late mRNAs. Furthermore, transcription of a polyomavirus containing a heterologous adenovirus promoter in place of the normal TATA box and cap sites is regulated by the large tumor antigen. We conclude that repression of polyomavirus early transcription cannot occur simply by binding of the large tumor antigen to DNA sequences at the site of transcription initiation and must involve the interaction of the large tumor antigen binding at other sites.

Antigens, Viral, Tumor↗
PubMed1987

A pancreas specificity results from the combination of polyomavirus and Moloney murine leukemia virus enhancer.

An infectious recombinant polyomavirus was constructed in which a regulatory region of its genome, the B enhancer region (nucleotides 5128-5265) has been replaced with the 72- or 73-base-pair repeat enhancer from the Moloney murine leukemia virus genome. We show that this recombinant polyomavirus displays a strong tissue specificity for the pancreas of mice. This organ was not permissive for either the parental polyomavirus, which is predominantly kidney and salivary gland specific, or the Moloney murine leukemia virus, which is lymphotropic. This result indicated that tissue specificity can be achieved by a combination of apparently modular elements. Some of the implications of a modular mechanism of tissue specificity are considered.

Animals↗
PubMed2005

Quantitation of DNA of polyomaviruses BK and JC in human kidneys.

BACKGROUND: Renal allograft recipients can be monitored for polyomavirus-associated nephropathy (PVAN) using urine samples. Because virus in urine can be derived from the kidneys, ureter, or urinary bladder, we evaluated whether measurement of intrarenal concentrations of viral DNA might serve as a more reliable monitoring tool. METHODS: Real-time quantitative polymerase chain reaction was used to quantitate DNA of polyomaviruses BK (BKV) and JC (JCV) in renal tissue obtained from various clinical settings. RESULTS: Renal biopsy samples from 28 nonimmunosuppressed patients contained very low viral copy numbers. Minimally higher BKV loads (mean +/- SE, 3.4 +/- 1.7 copies/cell) were observed in 74 renal biopsy samples from renal allograft recipients with BKV viruria. The BKV DNA concentration was approximately 10-fold higher in renal allograft recipients with BKV viruria, but 58 (50.4%) of 115 renal biopsy samples tested negative for BKV DNA, reflecting the focal nature of infection. JCV DNA was found in only 2 renal biopsy samples. CONCLUSIONS: The BKV load is better measured in urine than in tissue, because a urine sample represents material from the entire kidney. An increase in the BKV load is usually not accompanied by a proportional increase in the JCV load, which indicates that these 2 related polyomaviruses are subject to different mechanisms regulating viral replication.

BK Virus↗
PubMed1991

Amplification of polyomavirus DNA sequences stably integrated in rat cells.

To investigate the mechanism by which the polyomavirus large T antigen (T-Ag) promotes amplification of integrated viral sequences, we constructed a rat cell line, Hy2-ts5, carrying two different inserts of polyomavirus DNA. The first insert, designated the middle T (pmt) locus, was devised to analyze homologous recombination between two defective copies of pmt lying 3.3 kb apart on the same chromosome. Reconstitution of a functional pmt by spontaneous recombination occurred at a rate of about 2 x 10(-7) per cell generation. The second locus contained the polyomavirus large T (plt) gene carrying a temperature-sensitive mutation and producing a nonfunctional large T-Ag at 39 degrees C. A shift to the permissive temperature for as little as 24 h induced the production of a functional large T-Ag which, in turn, promoted homologous recombination in the pmt locus at a rate close to 1.0 per cell generation. The particularity of this system is that it allowed recombination products to be analyzed as early as a single cell doubling following the initial recombinational event. Amplification occurred by successive duplications of a discrete sequence in the viral insert. Unequal sister chromatid exchange was ruled out as the recombination mechanism promoted by large T-Ag. Instead, we proposed a model of nonconservative recombination involving mispairing between homologous sequences.

Animals↗
PubMed1991

Replication dependent and cell specific activation of the polyomavirus early promoter.

The relationship of viral DNA replication to the activation of viral gene expression is usually considered with respect to late genes. In this report we examine the replication activation of the polyomavirus early promoter. Using origin active and inactive mutants to drive luciferase gene expression from the polyomavirus early promoter, we show that the early promoter is also subjected to a replication dependent activation. The degree of activation can be up to a hundred fold greater than that seen without replication and is about 13 fold on a per template basis. This replication based activation is, however, cell type dependent and was seen in FOP cells but not in 3T6 cells. Analysis of the requirements of cis acting DNA show that these enhancer elements affect early transcription predominantly through the activation of replication, although some replication independent stimulation can also be seen. The implications of this result for the regulation of polyomavirus early gene regulation are considered.

Base Sequence↗
PubMed2005

Polyomavirus-associated nephropathy in renal transplantation: interdisciplinary analyses and recommendations.

Polyomavirus-associated nephropathy (PVAN) is an emerging cause of kidney transplant failure affecting 1-10% of patients. As uncertainty exists regarding risk factors, diagnosis, and intervention, an independent panel of experts reviewed the currently available evidence and prepared this report. Most cases of PVAN are elicited by BK virus (BKV) in the context of intense immunosuppression. No specific immunosuppressive drug is exclusively associated with PVAN, but most cases reported to date arise while the patient is on triple immunosuppressive combinations, often comprising tacrolimus and/or mycophenolate mofetil plus corticosteroids. Immunologic control of polyomavirus replication can be achieved by reducing, switching, and/or discontinuing components of the immunosuppressive regimen, but the individual's risk of rejection should be considered. The success rate of this intervention is increased with earlier diagnosis. Therefore, it is recommended that all renal transplant recipients should be screened for BKV replication in the urine: 1) every three months during the first two years posttransplant; 2) when allograft dysfunction is noted; and 3) when allograft biopsy is performed. A positive screening result should be confirmed in <4 weeks and assessed by quantitative assays (e.g. BKV DNA or RNA load in plasma or urine). Definitive diagnosis of PVAN requires allograft biopsy. If PVAN and concurrent acute rejection is diagnosed, antirejection treatment should be considered, coupled with subsequently reducing immunosuppression. The antiviral cidofovir is not approved for PVAN, but investigational use at low doses (0.25-0.33 mg/kg intravenously biweekly) without probenicid should be considered for refractory cases. Retransplantation after renal allograft loss to PVAN remains a treatment option for patients clearing polyomavirus replication.

Humans↗
PubMed2001

Avian polyomavirus agnoprotein 1a is incorporated into the virus particle as a fourth structural protein, VP4.

Agnoproteins, encoded by the 5'-region of the late bicistronic mRNA of some polyomaviruses, are small proteins with largely unknown functions. In avian polyomavirus (APV)-infected cells, mRNAs of seven putative agnoproteins have been observed. Recently, it has been shown that agnoprotein 1a and its truncated variant agnoprotein 1b, encoded by the predominant mRNA species, are essential for APV replication. Here, the presence of agnoprotein 1a is demonstrated in the nucleus of APV-infected cells and in purified APV particles. Interaction between agnoprotein 1a and the major structural protein, VP1, was demonstrated by co-immunoprecipitation experiments using lysates of recombinant baculovirus-infected insect cells. With proteins expressed in E. coli, binding to double-stranded DNA in a sequence-unspecific manner was shown for agnoprotein 1a, whereas agnoprotein 1b failed to bind. A leucine zipper-like motif present in agnoprotein 1a is considered to be involved in DNA binding. Due to the absence of any structural or functional homologies between APV agnoprotein 1a and the agnoproteins of mammalian polyomaviruses, it is suggested that this protein should be renamed VP4, indicating its function as a fourth structural protein of APV.

Amino Acid Sequence↗
PubMed2006

Polyomavirus-associated nephropathy: update of clinical management in kidney transplant patients.

Over the last decade, polyomavirus-associated nephropathy (PVAN) has occurred with increasing frequency after renal transplantation, leading to significant renal dysfunction and graft loss. More than 95% of all cases are caused by the human polyomavirus type 1 called the BK virus. The primary treatment for PVAN is immunosuppression reduction, which must be carefully balanced against increased risks of rejection. Although no validated protocols exist, a first step commonly involves reduction of calcineurin inhibitors with antiproliferative agents by more than one-third, e.g., reaching trough levels of tacrolimus <6 ng/mL, of cyclosporine <150 ng/mL, dosing of mycophenolate mofetil to <1 g/day, and azathioprine <75 mg/day. When rejection is diagnosed together with PVAN, a transient pulse treatment is recommended before subsequent reduction in immunosuppression. No antiviral treatments for PVAN have been approved by the United States Food and Drug Administration. The antiviral drug cidofovir has shown in vitro activity against murine polyomaviruses, and has been used in some patients in lower doses in an effort to minimize the nephrotoxic effects of cidofovir while treating PVAN. Small series of PVAN patients treated with leflunomide, intravenous immune globulin therapy, and fluoroquinolones have also been reported recently.

BK Virus↗
PubMed2000

Documenting the epidemiologic patterns of polyomaviruses in human populations by studying their presence in urban sewage.

This is the first description, to our knowledge, of the distribution of human polyomavirus and simian virus 40 (SV40) in urban sewage. Using a nested-PCR procedure, we report the detection of human polyomaviruses JC virus (JCV) and BK virus (BKV) but not SV40 in a high percentage of urban sewage samples obtained from widely divergent geographical areas in Europe and Africa. For a total of 28 samples analyzed, JCV was detected in 26, BKV was detected in 22, and none was positive for SV40. All geographical areas showed a high prevalence of these viruses with mean estimated values of JC viral particles per ml on the order of 10(3) in Barcelona (Spain) and Nancy (France) and 10(2) in Pretoria (South Africa) and Umeå (Sweden) and mean values of BK viral particles on the order of 10(2) in Pretoria and Barcelona and 10(1) in Nancy and Umeå. This compares with estimated mean values of 10(2) to 10(3) for human adenovirus that was evaluated as a control. Nucleotide sequence analysis of the amplified DNA from some of the samples is also presented and represents the sequence of the most abundant JC and BK viral strains in these samples. The nucleotide sequence of the JCV detected was also analyzed in a phylogenetic study and for genomic characterization in the regulatory region. This study has shown that human polyomaviruses are spread in high concentrations in the sewage of different geographical areas and are present in contaminated environments. The frequency and concentration of JCV detected in the environment and the absence of described animal hosts suggest that JCV may be useful as a marker for fecal pollution of anthropogenic origin. The results also support the idea previously described that the strains of JCV are closely related to the ethnic origin of the population studied. The procedure applied should also be useful in future studies of population patterns of viral excretion and as a tool in epidemiological studies for the detection of changes in the prevalence of specific viral pathogens.

BK Virus↗
PubMed1991

Polymerase chain reaction assay for avian polyomavirus.

A polymerase chain reaction assay was developed for detection of budgerigar fledgling disease virus (BFDV). The assay used a single set of primers complementary to sequences located in the putative coding region for the BFDV VP1 gene. The observed amplification product had the expected size of 550 bp and was confirmed to derive from BFDV DNA by its restriction digestion pattern. This assay was specific for BFDV and highly sensitive, being able to detect as few as 20 copies of the virus. By using the polymerase chain reaction, BFDV was detected in adult, nestling, and embryo budgerigar (Melopsitticus undulatus) tissue DNAs and in sera from adult and nestling budgerigars. These results suggest the possibility of persistent infections in adult birds and lend further support to previously described evidence of possible in ovo transmission. BFDV was also detected in chicken embryo fibroblast cell cultures and chicken eggs inoculated with the virus. A 550-bp product with identical restriction enzyme sites was amplified from a suspected polyomavirus isolated from a peach-faced lovebird (Agapornis pesonata) and from tissue DNA from a Hahn's macaw (Ara nobilis) and a sun conure (Aratinga solstitialis) with histological lesions suggestive of polyomavirus infection. These fragments also hybridized with a BFDV-derived probe, proving that they were derived from a polyomavirus very similar, if not identical, to BFDV.

Animals↗
PubMed1983

Chemical cleavage of polyomavirus major structural protein VP1: identification of cleavage products and evidence that the receptor moiety resides in the carboxy-terminal region.

As a first step toward identifying the various functional regions of the polyomavirus major capsid protein VP1, we used recently developed methods for the chemical cleavage of proteins and the available polyomavirus sequence data to devise a scheme to produce large, identifiable peptides and generate a cleavage map of VP1. Formic acid (75%) was found to cleave VP1 at only two sites, producing three peptides of apparent molecular weights of 29,000, 16,000, and 2,000. The order of peptides in intact VP1 was determined by recleavage of partial products and was found to be 29,000, 16,000, and 2,000. Two-dimensional peptide mapping studies of 125I-labeled VP1 formic acid peptides established that the limit products of formic acid digestion contained mutually exclusive sets of labeled peptides when either trypsin or chymotrypsin was used and that together the formic acid peptides contained all of the 125I-labeled tryptic and chymotryptic peptides found in VP1. Iodosobenzoic acid (IBA) digestion produced four peptides separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with apparent molecular weights of 12,000, 8,000, 7,000, and 5,000. The approximate positions of the IBA peptides in the VP1 sequence were determined by cleavage of formic acid fragments with IBA. The number of peptides produced, their respective sizes, and their order in the intact VP1 molecule agree with predictions made from available sequence data, both for formic acid cleavage and IBA cleavage. In addition, the numbers of 125I-labeled tryptic peptides produced from digestion of VP1 formic acid peptides also agree with predictions made from the sequence information. These data establish with reasonable certainty that the peptides produced by formic acid cleavage and IBA cleavage of VP1 are indeed those predicted. Antibodies raised against spontaneously produced, previously undefined polypeptides resulting from degradation of VP1 reacted exclusively with the formic acid peptides derived from the C-terminal portion of VP1. These antibodies inhibited hemagglutination and neutralized polyomavirus virions. We interpret this to mean that at least some of the antigenic determinants of the receptor moiety reside in this portion of the VP1 sequence.

Amino Acid Sequence↗
PubMed1984

Essential nucleotides in the polyomavirus origin region.

We have isolated a series of point mutants in the polyomavirus origin-intergenic control region by using a procedure which exploits the single-stranded nature of DNAs cloned into M13 phage both for the generation of mutants and for their analysis by DNA sequencing. In this report we describe the effects of cytosine X guanine----thymine X adenine base-pair substitutions in the polyomavirus origin region upon replication in mouse 3T6 cells of the M13-polyomavirus constructs. Our results indicate that sequences near the center of a 34-base-pair sequence with dyad symmetry are important for replication, whereas specific nucleotides near the ends of the dyad symmetry are not important. Furthermore, a putative large T antigen-binding site at nucleotides 60 to 80 can be mutated without affecting replication function as measured in this assay.

Animals↗
PubMed1986

Polyomavirus small t antigen: overproduction in bacteria, purification, and utilization for monoclonal and polyclonal antibody production.

Polyomavirus small t antigen was purified from genetically engineered Escherichia coli and used as the immunogen for the production of polyclonal and monoclonal antibodies. A new series of plasmids for increased expression of polyomavirus T antigens or a T antigen-beta-galactosidase fusion protein was constructed by replacing sequences coding for the ribosome-binding site of previously published plasmids with a chemically synthesized sequence that has a higher degree of complementarity to the 3' end of the 16S rRNA. Cells expressing the fusion protein from the plasmid with the synthetic sequence contained 5- to 10-fold more fusion protein after a 3-h induction than did control cells. Pulse-labeling of cells bearing the new plasmids revealed that the T antigens were synthesized at high levels after induction: 10% of total synthesis for small t; 15% for Py-1387T middle T, a truncated mutant of middle T; and probably 1 to 5% for middle T. Small t and Py-1387T middle T, but not wild-type middle T, were seen as minor bands in total cell protein analyzed on sodium dodecyl sulfate-polyacrylamide gels stained with Coomassie blue. A simple, rapid procedure for purification of bacterial small t from the pellet of sonicated bacteria yielded 1 to 2 mg of small t per liter of bacterial culture at 80 to 90% homogeneity. High-titer polyclonal rabbit antisera raised against purified small t recognized all three T antigens and were suitable for immunoaffinity purification of middle T. Mouse monoclonal antibodies raised against bacterial small t were of four classes, immunoprecipitating either all three polyomavirus T antigens, small t and middle T only, primarily small t, or middle T and large T in preference to small t. One of the latter monoclonal antibodies also immunoprecipitated large T but not small t of simian virus 40, suggesting that the site recognized by this antibody may be functionally important. None of the monoclonal antibodies yielded an immunoprecipitate active in phosphorylating middle T in vitro.

Antibodies, Monoclonal↗
PubMed1987

Expression of polyomavirus virion proteins by a vaccinia virus vector: association of VP1 and VP2 with the nuclear framework.

The polyomavirus proteins VP1, VP2, and VP3 move from their cytoplasmic site of synthesis into the nucleus, where virus assembly occurs. To identify cellular or viral components which might control this process, we determined the distribution of VP1, VP2, and VP3 in a soluble fraction, a cytoplasmic cytoskeleton fraction, and a nuclear framework fraction of infected cells. All three proteins were detected in a detergent-extractable form immediately after their synthesis in polyomavirus-infected cells. Approximately 50, 25, and 40% of pulse-labeled VP1, VP2, and VP3, respectively, associated with the skeletal framework of the nucleus within 10 min after their synthesis. The remaining portion of each labeled protein failed to accumulate on the nuclear framework during a 40-min chase and was degraded. When expressed separately by recombinant vaccinia viruses, VP1 and VP2, but not VP3, accumulated on the nuclear framework. This association was not dependent on other polyomavirus proteins or viral DNA. The amount of total VP1 and VP2 which was bound to the nuclear framework approximated 45 and 20%, respectively. Indirect immunofluorescence demonstrated an exclusive nuclear localization of VP1 in situ. In coinfection experiments, a greater percentage of total VP2 and VP3 was bound to the nuclear framework of cells which cosynthesized VP1. These results indicate that although VP1 and VP2 can bind independently to the insoluble nuclear framework, the association of VP3 with this nuclear structure is promoted by the presence of VP1.

Animals↗
PubMed1987

Replicative cis-advantage of polyomavirus regulatory region mutants in different murine cell lines.

To determine the relative growth advantages of polyomavirus regulatory region mutants selected from Friend leukemic and neuroblastoma cells persistently infected with polyomavirus A2 wild type, different murine cell lines, some of which are capable of further differentiation in vitro, were used as hosts in mixed infection and transfection. The tests allowed the determination, through the measurement of cis-advantage in replication, of the most effective polyomavirus regulatory region constitutions for a given cell line and, in some cases, for specific stages of cell differentiation. Furthermore, different domains of the viral regulatory region were shown to have different effects--positive, neutral, or negative--depending on the host analyzed.

Animals↗
PubMed1987

Expression of polyomavirus large T antigen by using a baculovirus vector.

A gene encoding the large T antigen of polyomavirus was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus so that gene expression was under the control of the strong, very late polyhedrin gene promoter. Significantly more large T antigen was produced in recombinant virus-infected insect cells than was observed in polyomavirus-transformed mouse cells. The insect-derived T antigen exhibited polyomavirus origin-specific DNA binding. The baculovirus expression system provides a convenient source of T antigen for in vitro studies.

Animals↗