Complete nucleotide sequence of polyomavirus SA12

The Polyomaviridae have small icosahedral virions that contain a genome of approximately 5,000 bp of circular double-stranded DNA. Polyomaviruses infect hosts ranging from humans to birds, and some members of this family induce tumors in test animals or in their natural hosts. We report the complete nucleotide sequence of simian agent 12 (SA12), whose natural host is thought to be Papio ursinus, the chacma baboon. The 5,230-bp genome has a genetic organization typical of polyomaviruses. Sequences encoding large T antigen, small t antigen, agnoprotein, and the viral capsid proteins VP1, VP2, and VP3 are present in the expected locations. We show that, like its close relative simian virus 40 (SV40), SA12 expresses microRNAs that are encoded by the late DNA strand overlapping the 3' end of large T antigen coding sequences. Based on sequence comparisons, SA12 is most closely related to BK virus (BKV), a human polyomavirus. We have developed a real-time PCR test that distinguishes SA12 from BKV and the other closely related polyomaviruses JC virus and SV40. The close relationship between SA12 and BKV raises the possibility that these viruses circulate between human and baboon hosts.     

Potential transmission of human polyomaviruses through the gastrointestinal tract after exposure to virions or viral DNA

The mechanism of human-to-human transmission of the polyomaviruses JC virus (JCV) and BK virus (BKV) has not been firmly established with regard to possible human exposure. JCV and BKV have been found in sewage samples from different geographical areas in Europe, Africa, and the United States, with average concentrations of 10(2) to 10(3) JCV particles/ml and 10(1) to 10(2) BKV particles/ml. Selected polyomavirus-positive sewage samples were further characterized. The JCV and BKV present in these samples were identified by sequencing of the intergenic region (the region found between the T antigen and VP coding regions) of JCV and the VP1 region of BKV. The regulatory region of the JCV and BKV strains found in sewage samples presented archetypal or archetype-like genetic structures, as described for urine samples. The stability (the time required for a 90% reduction in the virus concentration) of the viral particles in sewage at 20 degrees C was estimated to be 26.7 days for JCV and 53.6 days for BKV. The presence of JCV in 50% of the shellfish samples analyzed confirmed the stability of these viral particles in the environment. BKV and JCV particles were also found to be stable at pH 5; however, treatment at a pH lower than 3 resulted in the detection of free viral DNA. Since most humans are infected with JCV and BKV, these data indicate that the ingestion of contaminated water or food could represent a possible portal of entrance of these viruses or polyomavirus DNA into the human population.     

Hybrid genomes of the polyomaviruses JC virus, BK virus, and simian virus 40: identification of sequences important for efficient transformation.

Hybrid viral genomes were used to investigate the influence of specific polyomavirus sequences on the transforming behavior of JC virus (JCV). One set of chimeric DNAs was made by exchanging the regulatory regions between JCV and simian virus 40 (SV40) or JCV and BK virus (BKV). A second set of constructs was produced that expressed hybrid JCV-BKV T proteins under the control of either JCV or BKV regulatory signals. Transformation of Rat 2 cells with the parental and chimeric DNAs indicated that both the JCV regulatory signals and the sequence encoding the amino terminus of T protein contributed to the restricted transforming behavior of this virus. Analysis of the viral proteins in the transformed rat cells indicated that the large T antigens of JCV and BKV were less stable than their SV40 counterpart, that small t protein was produced in JCV transformants, and that the subpopulation of T antigen that forms a stable complex with cellular p53 protein was smaller in JCV-transformed cells than in SV40- or BKV-transformed cells.     

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.     

Cross-reactivity of T lymphocytes recognizing a human cytotoxic T-lymphocyte epitope within BK and JC virus VP1 polypeptides

A transgenic mouse model was used to identify an HLA-A*02-restricted epitope within the VP1 polypeptide of a human polyomavirus, BK virus (BKV), which is associated with polyomavirus-associated nephropathy in kidney transplant patients. Peptide stimulation of splenocytes from mice immunized with recombinant modified vaccinia virus Ankara expressing BKV VP1 resulted in expansion of cytotoxic T lymphocytes (CTLs) recognizing the sequence LLMWEAVTV corresponding to amino acid residues 108 to 116 (BKV VP1p108). These effector T-cell populations represented functional CTLs as assessed by cytotoxicity and cytokine production and were cross-reactive against antigen-presenting cells pulsed with a peptide corresponding to the previously described JC virus (JCV) VP1 homolog sequence ILMWEAVTL (JCV VP1p100) (I. J. Koralnik et al., J. Immunol. 168:499-504, 2002). A panel of 10 healthy HLA-A*02 human volunteers and two kidney transplant recipients were screened for T-cell immunity to this BK virus VP1 epitope by in vitro stimulation of their peripheral blood mononuclear cells (PBMC) with the BKV VP1p108 peptide, followed by tetramer labeling combined with simultaneous assays to detect intracellular cytokine production and degranulation. PBMC from 4/10 subjects harbored CTL populations that recognized both the BKV VP1p108 and the JCV VP1p100 peptides with comparable efficiencies as measured by tetramer binding, gamma interferon production, and degranulation. CTL responses to the JCV VP1p100 epitope have been associated with prolonged survival in progressive multifocal leukoencephalopathy patients (R. A. Du Pasquier et al., Brain 127:1970-1978, 2004; I. J. Koralnik et al., J. Immunol. 168:499-504, 2002). Given that both human polyomaviruses are resident in a high proportion of healthy individuals and that coinfection occurs (W. A. Knowles et al., J. Med. Virol. 71:115-123, 2003), our findings suggest a reinterpretation of this protective T-cell immunity, suggesting that the same VP1 epitope is recognized in HLA-A*02 persons in response to either BK or JC virus infection.     

Small tumor antigen of polyomaviruses: role in viral life cycle and cell transformation

The regulatory proteins of polyomaviruses, including small and large T antigens, play important roles, not only in the viral life cycle but also in virus-induced cell transformation. Unlike many other tumor viruses, the transforming proteins of polyomaviruses have no cellular homologs but rather exert their effects mostly by interacting with cellular proteins that control fundamental processes in the regulation of cell proliferation and the cell cycle. Thus, they have proven to be valuable tools to identify specific signaling pathways involved in tumor progression. Elucidation of these pathways using polyomavirus transforming proteins as tools is critically important in understanding fundamental regulatory mechanisms and hence to develop effective therapeutic strategies against cancer. In this short review, we will focus on the structural and functional features of one polyomavirus transforming protein, that is, the small t-antigen of the human neurotropic JC virus (JCV) and the simian virus, SV40.     

Nucleotide sequence of the human polyomavirus AS virus, an antigenic variant of BK virus.

The complete DNA sequence of the human polyomavirus AS virus (ASV) is presented. Although ASV can be differentiated antigenically from the other human polyomaviruses (BK and JC viruses), it shares 94.9% homology at the nucleotide level with the Dunlop strain of BK virus. Differences found in ASV relative to BK virus include the absence of tandem repeats in its regulatory region, the deletion of 32 nucleotides in the late mRNA leader region (altering the initiation codon for the agnoprotein), the presence of a cluster of base pair substitutions within the coding region of the major capsid protein, VP1, and the absence of 4 amino acids in the carboxy-terminal region of the early protein, T antigen. The 43 nucleotides deleted in the Dunlop strain of BK virus relative to the Gardner prototype strain of BK virus are present in ASV. Possible reasons for the distinct antigenicity of the ASV capsid, given the high degree of nucleotide homology with BK virus, are discussed. To reflect the high degree of sequence homology between ASV and BK virus, we suggest ASV be renamed BKV(AS).     

Interplay of cellular and humoral immune responses against BK virus in kidney transplant recipients with polyomavirus nephropathy

Reactivation of the polyomavirus BK (BKV) causes polyomavirus nephropathy (PVN) in kidney transplant (KTx) recipients and may lead to loss of the renal allograft. We have identified two HLA-A*0201-restricted nine-amino-acid cytotoxic T lymphocyte (CTL) epitopes of the BKV major capsid protein VP1, VP1(p44), and VP1(p108). Using tetramer staining assays, we showed that these epitopes were recognized by CTLs in 8 of 10 (VP1(p44)) and 5 of 10 (VP1(p108)) HLA-A*0201+ healthy individuals, while both epitopes elicited a CTL response in 10 of 10 KTx recipients with biopsy-proven PVN, although at variable levels. After in vitro stimulation with the respective peptides, CTLs directed against VP1(p44) were more abundant than against VP1(p108) in most healthy individuals, while the converse was true in KTx recipients with PVN, suggesting a shift in epitope immunodominance in the setting of active BKV infection. A strong CTL response in KTx recipients with PVN appeared to be associated with decreased BK viral load in blood and urine and low anti-BKV antibody titers, while a low or undetectable CTL response correlated with viral persistence and high anti-BKV antibody titers. These results suggest that this cellular immune response is present in most BKV-seropositive healthy individuals and plays an important role in the containment of BKV in KTx recipients with PVN. Interestingly, the BKV CTL epitopes bear striking homology with the recently described CTL epitopes of the other human polyomavirus JC (JCV), JCV VP1(p36) and VP1(p100). A high degree of epitope cross-recognition was present between BKV and corresponding JCV-specific CTLs, which indicates that the same population of cells is functionally effective against these two closely related viruses.     

Detection and typing of BKV,JCV, and SV40 by multiplex nested polymerase chain reaction

A multiplex nested polymerase chain reaction (PCR) method was developed for detecting and differentiating simultaneously the DNA of polyomaviruses JC, BK, and SV40 in a single tube. In the first amplification step the same set of primers was used to amplify a conserved DNA region of the large T antigen gene of JCV, BKV, and SV40. The second round was carried out using a set of primers designed to obtain products of different size for each related virus. Subsequently, the sensitivity of the multiplex nested PCR was maximized by optimizing parameters such as primer, magnesium, and dNTP concentrations. The sensitivity of the method ranged between 1 and 10 copies of the polyomavirus genome. The assay was then used for detecting polyomavirus DNA in urine, serum, and biopsy specimens from renal transplant recipients. Based on the results obtained, the multiplex nested PCR developed in our study represents a useful tool for supporting the diagnosis of polyomavirus infection and could be used for epidemiological purposes and to better define the role of polyomaviruses in human pathology.     

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.     

Polyomaviruses of Nonhuman Primates: Implications for Research

Polyomaviruses are a family of small nonenveloped DNA viruses that infect birds and mammals. At least 7 nonhuman primate polyomaviruses that occur in macaques, African green monkeys, marmosets baboons, and chimpanzees have been described, as well as 4 polyomaviruses that occur in humans. Simian virus 40 (SV40), which infects macaques, was the first nonhuman primate polyomavirus identified as a contaminant of early polio vaccines. Primate polyomaviruses cause inapparent primary infections but persist in the host and can cause severe disease in situations of immunocompromise. This review describes the primate polyomaviruses, and the diseases associated with the viruses of macaques. In macaques, the greatest current concerns are the potential confounding of study results by polyomavirus infections and the zoonotic potential of SV40.Abbreviations: PML, progressive multifocal leukoencephalopathy; SV40, Simian virus 40Polyomaviruses were previously members of the family Papovaviridae, which included (and derived its name from) rabbit papilloma virus (pa), mouse polyoma virus (po), and simian vacuolating virus (va). Papovaviruses are nonenveloped viruses, with double-stranded circular DNA and an icosahedral capsule. Since the 1980s, studies of Simian virus 40 (SV40) and mouse polyomavirus have demonstrated that these viruses have smaller capsids (45 nm versus 50 nm), smaller genomes (5 kb versus 8 kb), and a different genomic organization than those of papillomaviruses. SV40 and mouse polyomavirus now form an independent family, Polyomaviridae.¹⁸More than 13 members of Polyomaviridae infect mammals and birds. The first polyomavirus was discovered in 1953 in mice²⁸ and was so named because it caused tumors at multiple sites in neonatal mice. Indeed oncogenicity is a common feature of polyomaviruses, particularly tumor production in non-native hosts. Various members of the group transform cell lines and immortalize primary cell cultures as well as induce tumors in susceptible animals. SV40 was identified in 1960 in primary macaque kidney cell cultures, as a contaminant of polio vaccines.⁶⁸ In 1971, the human polyomaviruses BKV²³ and JCV⁵⁴ were identified (both are named after the initials of the patients in which they were first recognized). JCV was discovered in the brain of a patient with progressive multifocal leukoencephalopathy, and BKV was found in the urine of a renal transplant patient. Recently, 2 additional polyomaviruses of the nasopharynx of humans, KIV and WUV, have been identified^2,25 through the use of molecular techniques. KIV was found in nasopharyngeal samples from patients with respiratory disease, and WUV initially was detected in a child with pneumonia. KIV and WUV are closely related genetically and may form a new subfamily of polyomaviruses: their early coding regions (T antigens) are similar to those of other primate polyomaviruses, but their late regions (structural proteins) differ.^7,25 Both KIV and WUV appear to be geographically widespread.The capsids of the polyomaviruses contain 3 structural proteins: VP1, the major capsid protein, and VP2 and VP3, which enclose a single molecule of viral DNA. The viruses also encode regulatory proteins, the T (tumor) antigens. SV40 and other primate polyomaviruses encode 2 T antigens, large T and small t, whereas mouse polyomavirus and some of the other family members have a third, middle T antigen. The T antigens of SV40, BKV, and JCV have about 75% amino-acid homology.⁵⁸ The T antigen of SV40 is essential for initiation of viral DNA replication and promotes transformation and immortalization of host cells, partially through binding to and inhibiting tumor suppressor proteins p53, p107, p130 (pRb2), and pRb (reviewed in reference 10).     

Role of JC virus agnoprotein in virion formation

JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and causes progressive multifocal leukoencephalopathy in humans. JCV encodes early proteins (large T antigen, small T antigen, and T' antigen) and four late proteins (agnoprotein, and three viral capsid proteins, VP1, VP2, and VP3). In the current study, a novel function for JCV agnoprotein in the morphogenesis of JC virion particles was identified. It was found that mature virions of agnoprotein-negative JCV are irregularly shaped. Sucrose gradient sedimentation and cesium chloride gradient ultracentrifugation analyses revealed that the particles of virus lacking agnoprotein assemble into irregularly sized virions, and that agnoprotein alters the efficiency of formation of VP1 virus-like particles. An in vitro binding assay and immunocytochemistry revealed that agnoprotein binds to glutathione S-transferase fusion proteins of VP1 and that some fractions of agnoprotein colocalize with VP1 in the nucleus. In addition, gel filtration analysis of formation of VP1-pentamers revealed that agnoprotein enhances formation of these pentamers by interacting with VP1. The present findings suggest that JCV agnoprotein plays a role, similar to that of SV40 agnoprotein, in facilitating virion assembly.     

Cellular immunotherapy for patients with reactivation of JC and BK polyomaviruses after transplantation

Immunosuppression of patients after hematopoietic stem cell or kidney transplantation potentially leads to reactivation of JC and BK polyomaviruses. In hematopoietic stem cell transplantation, the reactivation rate of BKV can be up to 60%, resulting in severe complications of the urogenital tract, particularly hemorrhagic cystitis and renal dysfunction. After kidney transplantation, BKV reactivation can cause a loss of the graft. JCV can cause progressive multifocal leukoencephalopathy, a lethal disease. Adoptive transfer of donor-derived polyomavirus-specific T cells is an attractive and promising treatment that restores virus-specific cellular immunity. Pioneering work in the early 1990s on the reconstitution of cellular immunity against cytomegalovirus and recent development in the field of monitoring and isolation of antigen-specific T cells paved the way toward a personalized T-cell therapy. Multimer technology and magnetic beads are available to produce untouched T cells in a single-step, good manufacturing practice–compliant procedure. Another exciting aspect of T-cell therapy against polyomaviruses is the fact that both JCV and BKV can be targeted simultaneously because of their high sequence homology. Finally, “designer T cells” can be redirected to recognize polyomavirus antigens with high-affinity T-cell receptors. This review summarizes the state-of-the art technologies and gives an outlook of future developments in the field.     

Modeling a sialic acid binding pocket in the external loops of JC virus VP1

JC virus (JCV) is a common human polyomavirus that infects over 70% of the population worldwide. JCV has a restricted cell tropism that is caused partly by the initial interaction between the virus and sialic acid-containing host cell receptors. To identify the molecular interactions between the virus and its cellular receptor, we used a combined approach of site-directed mutagenesis and homology-based molecular modeling. A model of the major viral capsid protein VP1 based on sequence alignment with other closely related polyomaviruses allowed us to target specific amino acids in the extracellular loops of VP1 for mutagenesis. An analysis of the growth rates of 17 point mutants led to the identification of VP1 amino acids that are critical in virus-host cell receptor interactions. Molecular dynamics simulations were then used to build and confirm a model of the interaction between VP1 and the sialic acid component of the JCV receptor.     

Prevalence of BK virus and human papillomavirus in human prostate cancer

Polyomaviruses such as the BK virus (BKV), JC virus (JCV) and SV40, as well as the human papillomaviruses (HPV) are frequently detected throughout human populations, causing subclinical persistent infections and inducing oncogenesis in human and other cell lines. To test the involvement of these viruses in prostate tumorigenesis, we investigated the prevalence of BKV, JCV and HPV in a series of human prostatic malignancies. Forty-two samples of diagnosed prostatic malignancies were tested using standard polymerase chain reaction (PCR) protocols. Differentiation between BKV and JCV among the polyomavirus-positive samples was achieved after sequencing analysis of the PCR products. Reconstitution of BKV in vitro was performed and indirect immunofluorescence for the large T-antigen of the virus was applied to confirm the production of progeny virus. Detection and typing of HPV was carried out by PCR. The overall prevalence of polyomaviruses was 19% in the prostate cancer cases. Sequencing analysis of the polyomavirus-positive specimens revealed the presence of BKV in all samples. Reconstitution of the BKV from the BKV-positive prostate samples was successfully achieved in cell culture and progeny viral particles were obtained, confirming the presence of the virus in the human biopsies. HPV was detected in 4.8% of the samples, however, no HPV-11, HPV-16, HPV-18 or HPV-33 types were identified. BKV was frequently detected and could play a relevant role in the development and progression of human prostate cancer, whereas HPV does not seem to be implicated in this type of human neoplasia.     

Identification of MW Polyomavirus, a Novel Polyomavirus in Human Stool

We have discovered a novel polyomavirus present in multiple human stool samples. The virus was initially identified by shotgun pyrosequencing of DNA purified from virus-like particles isolated from a stool sample collected from a healthy child from Malawi. We subsequently sequenced the virus' 4,927-bp genome, which has been provisionally named MW polyomavirus (MWPyV). The virus has genomic features characteristic of the family Polyomaviridae but is highly divergent from other members of this family. It is predicted to encode the large T antigen and small T antigen early proteins and the VP1, VP2, and VP3 structural proteins. A real-time PCR assay was designed and used to screen 514 stool samples from children with diarrhea in St. Louis, MO; 12 specimens were positive for MWPyV. Comparison of the whole-genome sequences of the index Malawi case and one St. Louis case demonstrated that the two strains of MWPyV varied by 5.3% at the nucleotide level. The number of polyomaviruses found in the human body continues to grow, raising the question of how many more species have yet to be identified and what roles they play in humans with and without manifest disease.     

JC polyomavirus infection in candidates for kidney transplantation living in the Brazilian Amazon Region

This study evaluated the relative occurrences of BK virus (BKV) and JC virus (JCV) infections in patients with chronic kidney disease (CKD). Urine samples were analysed from CKD patients and from 99 patients without CKD as a control. A total of 100 urine samples were analysed from the experimental (CKD patients) group and 99 from the control group. Following DNA extraction, polymerase chain reaction (PCR) was used to amplify a 173 bp region of the gene encoding the T antigen of the BKV and JCV. JCV and BKV infections were differentiated based on the enzymatic digestion of the amplified products using BamHI endonuclease. The results indicated that none of the patients in either group was infected with the BKV, whereas 11.1% (11/99) of the control group subjects and 4% (4/100) of the kidney patients were infected with the JCV. High levels of urea in the excreted urine, low urinary cellularity, reduced bladder washout and a delay in analysing the samples may have contributed to the low prevalence of infection. The results indicate that there is a need to increase the sensitivity of assays used to detect viruses in patients with CDK, especially given that polyomavirus infections, especially BKV, can lead to a loss of kidney function following transplantation.