Isolation of a possible archetypal JC virus DNA sequence from nonimmunocompromised individuals.

We molecularly cloned JC polyomavirus DNAs from urine samples of eight nonimmunosuppressed patients and two healthy individuals. The cloned viral DNAs all contained an archetypal regulatory sequence from which various regulatory sequences of JC polyomavirus isolates derived from patients with progressive multifocal leukoencephalopathy could have evolved by deletion and amplification.     

Novel polyomavirus detected in the feces of a chimpanzee by nested broad-spectrum PCR

In order to screen for new polyomaviruses in samples derived from various animal species, degenerated PCR primer pairs were constructed. By using a nested PCR protocol, the sensitive detection of nine different polyomavirus genomes was demonstrated. The screening of field samples revealed the presence of a new polyomavirus, tentatively designated chimpanzee polyomavirus (ChPyV), in the feces of a juvenile chimpanzee (Pan troglodytes). Analysis of the region encoding the major capsid protein VP1 revealed a unique insertion in the EF loop of the protein and showed that ChPyV is a distinct virus related to the monkey polyomavirus B-lymphotropic polyomavirus and the human polyomavirus JC polyomavirus.     

A Case Control Study Reveals that Polyomaviruria Is Significantly Associated with Interstitial Cystitis and Vesical Ulceration

Objectives

To investigate whether polyomaviruses contribute to interstitial cystitis pathogenesis.

Subjects and Methods

A prospective study was performed with 50 interstitial cystitis cases compared with 50 age-matched, disease-free controls for the frequency of polyomaviruria. Associations between polyomaviruria and disease characteristics were analysed in cases. Polyomavirus in urine and bladder tissue was detected with species (JC virus vs. BK virus) specific, real-time PCR.

Results

Case patients were reflective of interstitial cystitis epidemiology with age range from 26–88 years (median 58) and female predominance (41/50 F). There was a significant increase in the frequency of polyomavirus shedding between cases and controls (p<0.02). Polyomavirus shedding, in particular BK viruria, was associated with vesical ulceration, a marker of disease severity, among interstitial cystitis cases after adjustment for age and sex (OR 6.8, 95% CI 1.89–24.4). There was a significant association among cases between the presence of BK viruria and response to intravesical Clorpactin therapy (OR 4.50, 95% CI 1.17–17.4).

Conclusion

The presence of polyomaviruria was found to be associated with the ulcerative form of interstitial cystitis. Clorpactin, which has anti-DNA virus activity, was more likely to improve symptoms in the presence of BK viruria. These data from this pilot study suggest associations between polyomaviruria and interstitial cystitis warranting further investigation.     

Structural optimization of a retrograde trafficking inhibitor that protects cells from infections by human polyoma- and papillomaviruses

Human polyoma- and papillomaviruses are non-enveloped DNA viruses that cause severe pathologies and mortalities. Under circumstances of immunosuppression, JC polyomavirus causes a fatal demyelinating disease called progressive multifocal leukoencephalopathy (PML) and the BK polyomavirus is the etiological agent of polyomavirus-induced nephropathy and hemorrhagic cystitis. Human papillomavirus type 16, another non-enveloped DNA virus, is associated with the development of cancers in tissues like the uterine cervix and oropharynx. Currently, there are no approved drugs or vaccines to treat or prevent polyomavirus infections. We recently discovered that the small molecule Retro-2^cycl, an inhibitor of host retrograde trafficking, blocked infection by several human and monkey polyomaviruses. Here, we report diversity-oriented syntheses of Retro-2^cycl and evaluation of the resulting analogs using an assay of human cell infections by JC polyomavirus. We defined structure–activity relationships and also discovered analogs with significantly improved potency as suppressors of human polyoma- and papillomavirus infection in vitro. Our findings represent an advance in the development of drug candidates that can broadly protect humans from non-enveloped DNA viruses and toxins that exploit retrograde trafficking as a means for cell entry.     

Frequency and phenotype of JC virus-specific CD8+ T lymphocytes in the peripheral blood of patients with progressive multifocal leukoencephalopathy

JC virus (JCV)-specific CD8+ cytotoxic T lymphocytes (CTL) are associated with a favorable outcome in patients with progressive multifocal leukoencephalopathy (PML) and cross-recognize the polyomavirus BK virus (BKV). We sought to determine the frequency and phenotype in fresh blood of CD8+ T cells specific for two A*0201-restricted JCV epitopes, VP1(p36) and VP1(p100), and assess their impact on JC and BK viremia and viruria in 15 healthy subjects, eight human immunodeficiency virus-positive (HIV+) individuals, and nine HIV+ patients with PML (HIV+ PML patients) classified as survivors. After magnetic pre-enrichment of CD8+ T cells, epitope-specific cells ranged from 0.001% to 0.022% [corrected] by tetramer staining, with no significant difference among the three study groups. By use of seven-color flow cytometry, there was no predominant differentiation phenotype subset among JCV-specific CD8+ T cells in healthy individuals, HIV+ subjects, or HIV+ PML patients. However, in one HIV+ PML patient studied in the acute phase, there was a majority of activated effector memory cells. BKV DNA was undetectable in all blood samples by quantitative PCR, while a low JC viral load was found in the blood of only one HIV+ and two HIV+ PML patients. JCV and BKV DNA were detected in 33.3% and 13.3% of all urine samples, respectively, independent of the presence of JCV-specific CTL. The detection of JCV DNA in the urine was associated with the presence of a JCV VP1(p100) CTL response. Immunotherapies aiming at increasing the cellular immune response against JCV may be valuable in the treatment of HIV+ individuals with PML.     

Analysis of JC virus DNA purified directly from human progressive multifocal leukoencephalopathy brains.

Human polyomavirus JC DNA was purified directly from the diseased brain tissue of two patients with progressive multifocal leukoencephalopathy (PML) by a method employing differential salt precipitation (B. Hirt, J. Mol. Biol. 26:365-369, 1967). Each of the viral genomes (JC-NIH-1 and JC-NIH-2) was molecularly cloned intact in Escherichia coli, using pBR322, at their unique EcoRI (0.00 map unit) and BamHI (0.51 map unit) sites. The JC-NIH-1 genome was approximately 50 base pairs larger and the JC-NIH-2 genome was approximately 50 base pairs smaller than the prototype human polyomavirus JC (Mad-1) DNA. Analysis of the restriction endonuclease cleavage fragments of these two DNAs and the human polyomavirus JC (Mad-1) DNA revealed only slight differences which mapped in a region of the genome extending from 0.67 to 0.74 map unit. From previous homology studies, this region of variance corresponds to the noncoding region to the late side of the origin of DNA replication.     

A virus takes an "L" turn to find its receptor

Virus-receptor interaction represents a crucial step during virus entry. In this issue of Cell Host & Microbe, Neu et?al. (2010) identify a receptor motif that engages JC virus, a human polyomavirus known to cause progressive multifocal leukoencephalopathy in immunocompromised individuals.     

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.     

Derivation and characterization of POJ cells, transformed human fetal glial cells that retain their permissivity for JC virus.

The study of the medically important polyomavirus JC virus is limited to only a few laboratories, primarily because the permissive cell system most often used, primary human fetal glial cells, is difficult to obtain and propagate. We have introduced mutations at the origin of DNA replication of JC virus and transformed glial cells with the replication-defective genomes. Although normal glial cell cultures rapidly lose their permissivity for the virus after subculture, the transformed cells (designated POJ) had a greatly expanded life span and remained permissive for JC virus even after 30 passages in vitro. POJ cells constitutively express a functional T protein that complements the replication defect of lethal early-region mutations in JC virus. We expect that these cells will greatly facilitate the study of this human virus.     

Diagnostic value of JC/BK virus antibody immunohistochemistry staining in urine samples from posttransplant immunosuppressed patients in relation to polyomavirus reactivation

OBJECTIVE: To compare the diagnostic value of cytology and immunohistochemistry staining (IHS) of urine samples for polyomavirus reactivation diagnosis. STUDY DESIGN: Sixty-eight urine samples collected from 18 immunosuppressed patients were analyzed by Papanicolaou and IHS with a JC/BK virus-specific monoclonal antibody. RESULTS: Overall, polyomavirus BK (BKV) was positive in 11 of 18 patients (61.1%) (3 of whom developed hemorrhagic cystitis) and in 23 of 68 urine samples (28%). Of 23 samples, 4 (17%) were positive by 1 of the 2 techniques, only. Of 23 samples, 19 (83%) were positive by both methods. In matching urine samples from the same patient, the number of BKV-infected positive cells detected by IHS in urine slides was higher than those detected by Papanicolaou staining (71.3%). CONCLUSION: The main advantage of LHS is that it allowed confirmation of BKV infection diagnosis in urine samples. IHS detected more BKV-infected cells in samples with few positive urothelial cells, which would have gone undetected if only Papanicolaou staining had been used as the BKV screening method. Urine samples testing for BKV by both techniques will improve diagnosis in asymptomatic patients, allowing early therapeutic intervention and a better clinical outcome.     

Recombinant JC viral DNA: Verification and physical map of prototype

Genomic DNA of the human polyomavirus JC was molecularly cloned from DNA extracted from primary human fetal glial cells infected with prototype (MAD-1) virus and from diseased brain tissue from which JC virus originally was isolated. This report documents that these recombinant MAD-1 DNAs are identical, are prototypical, and may serve as unambiguous references to distinguish the variant DNAs produced by independent isolates of JC virus. Possible ambiguity in other recombinants of MAD-1 DNA is discussed. A new restriction endonuclease cleavage map of MAD-1 DNA was derived for 52 sites cleaved by eight enzymes.     

Cloned human polyomavirus JC DNA can transform human amnion cells.

The genome of the human polyomavirus JC (Mad-1 strain) was molecularly cloned in Escherichia coli by using the plasmid vector pBR322. Recombinant DNA molecules were constructed with the entire JC genome inserted either at its unique EcoRI site at 0.0 map units or at its unique BamHI site at 0.51 map units. Viral DNA from each of these recombinant plasmids was capable of transforming human amnion cells, and cell lines established from transformed foci were positive for JC tumor antigen as assayed by indirect immunofluorescence.     

Inhibition of JC polyomavirus infectivity by the retrograde transport inhibitor Retro-2.1

JC polyomavirus (JCPyV) is a common human pathogen that results in a chronic asymptomatic infection in healthy adults. Under conditions of immunosuppression, JCPyV spreads to the central nervous system and can cause the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML), a disease for which there are no vaccines or antiviral therapies. Retro-2 is a previously identified small molecule inhibitor that was originally shown to block retrograde transport of toxins such as ricin toxin from endosomes to the Golgi apparatus and endoplasmic reticulum (ER), and Retro-2.1 is a chemical analog of Retro-2 that has been shown to inhibit ricin intoxication of cells at low nanomolar concentrations. Retro-2 has previously been shown to prevent retrograde transport of JCPyV virions to the ER, but the effect of Retro-2.1 on JCPyV infectivity is unknown. Here it is shown that Retro-2.1 inhibits JCPyV with an EC₅₀ of 3.9 μM. This molecule inhibits JCPyV infection at dosages that are not toxic to human tissue culture cells. Retro-2.1 was also tested against two other polyomaviruses, the human BK polyomavirus and simian virus 40, and was also shown to inhibit infection at similar concentrations. Viral uncoating studies demonstrate that Retro-2.1 inhibits BKPyV infectivity in a manner similar to Retro-2. These studies demonstrate that improved analogs of Retro-2 can inhibit infection at lower dosages than Retro-2 and further optimization of these compounds may lead to effective treatment options for those suffering from JCPyV infection and PML.     

Specific antibodies reacting to JC polyomavirus capsid protein mimotopes in sera from multiple sclerosis and other neurological diseases-affected patients

Published data support the hypothesis that viruses could be trigger agents of multiple sclerosis onset. This link is based on evidence of early exposure to viral agents in patients affected by this neurologic disease. JC (JC polyomavirus [JCPyV]), BK (BKPyV), and simian virus 40 (SV40) neurotropic polyomavirus footprints have been detected in brain tissue specimens and samples from patients affected by different neurological diseases. In this investigation, serum samples from patients affected by multiple sclerosis and other inflammatory and noninflammatory neurologic diseases, as well as healthy subjects representing the control, were investigated for immunoglobulin G (IgG) antibodies against JCPyV. To this end, an immunologic approach was employed, which consists of employing indirect enzyme-linked immunosorbent assay testing with synthetic peptides mimicking viral capsid protein 1 antigens. A significantly lower prevalence of IgG antibodies against JCPyV VP1 epitopes, with a low titer, was detected in serum samples from patients with multiple sclerosis (MS) and other neurologic diseases than in healthy subjects. Our study indicates that the prevalence of JCPyV antibodies from patients with multiple sclerosis is 50% lower than in healthy subjects, suggesting specific immune impairments. These results indicate that patients affected by neurological diseases, including MS, respond poorly to JCPyV VP1 antigens, suggesting specific immunologic dysfunctions.     

Periodic assessment of urine and serum by cytology and molecular biology as a diagnostic tool for BK virus nephropathy in renal transplant patients

OBJECTIVE: To investigate the significance of polyomavirus (PV) viruria and viremia by morphologic, immunohistochemical and molecular analysis (multiplex nested-polymerase chain reaction) in renal transplant patients. STUDY DESIGN: Urine (n=328), serum (n= 53) and renal biopsies (n=24) from renal transplant patients (n=106) were studied. RESULTS: Decoy cells were found in 53 samples (16%) from 19 patients (18%); viral DNA was amplified in all urinary samples and disclosed BK virus (BKV) (n=24), JC virus (JCV) (n=16), and JCV and BKV DNA (n=13). BKV was the prevailing genotype in patients with a high frequency of decoy cell excretion (p = 0.001). JCV excretion correlated with a low number (p = 0.01) and BKV with a high number of decoy cells (p=0.003). PV DNA was amplified from 30/53 serum samples (56.6%); BKV was the prevailing genotype (p = 0.04). On 24 renal biopsies (18 from the decoy cell-negative and 6 from the decoy cell-positive group) PV nephropathy (PVN) was identified and BKV DNA amplified in 4 biopsies, all from the group with a high frequency of decoy cell excretion. PVN was not identified in renal biopsies from the decoy cell-negative group. CONCLUSION: PV infection is frequent in renal transplant patients. The BKV genotype in urine and serum is significantly related to a high frequency and high number of decoy cells. PVN occurs only in patients with BKV viremia and a high number and frequency of decoy cell excretion in urine. In the absence of decoy cells, PVN can be excluded. Cytologic analysis of urine is an important diagnostic tool for screening renal transplant patients at risk of PVN.     

JC virus as a marker of human migration to the Americas

JC virus is a ubiquitous human polyomavirus present in populations worldwide. Seven genotypes differing in DNA sequence by approximately 1-3% characterize three Old World population groups (African, European and Asian) as well as Oceania. It is possible to follow Old World populations into the New World by the JC virus genotypes they carried. The first population to settle in the Americas, the Native Americans, brought with them type 2A from northeast Asia. European settlers arriving after Columbus carried primarily type 1 and type 4. Africans brought by the slave trade carried type 3 and type 6.     

Restriction endonuclease cleavage map of the DNA of JC virus.

A physical map of the sites cleaved by the following restriction endonucleases was derived for the DNA of JC virus, a human polyomavirus: EcoRI, HpaI, and PstI (one site each); HindII (four sites); and HindIII (three sites). By agarose gel electrophoresis of fragmented DNA, the size of full-length DNA of JC virus was estimated to be 5,125 +/- 105 base pairs (98 +/- 2% of the length of simian virus 40 DNA).     

Inhibition of virus production in JC virus-infected cells by postinfection RNA interference

RNA interference has been applied for the prevention of virus infections in mammalian cells but has not succeeded in eliminating infections from already infected cells. We now show that the transfection of JC virus-infected SVG-A human glial cells with small interfering RNAs that target late viral proteins, including agnoprotein and VP1, results in a marked inhibition both of viral protein expression and of virus production. RNA interference directed against JC virus genes may thus provide a basis for the development of new strategies to control infections with this polyomavirus.     

The T Antigen Locus of Merkel Cell Polyomavirus Downregulates Human Toll-Like Receptor 9 Expression

Establishment of a chronic infection is a key event in virus-mediated carcinogenesis. Several cancer-associated, double-stranded DNA (dsDNA) viruses act via their oncoproteins to downregulate Toll-like receptor 9 (TLR9), a key receptor in the host innate immune response that senses viral or bacterial dsDNA. A novel oncogenic virus, Merkel cell polyomavirus (MCPyV), has been recently identified that causes up to 80% of Merkel cell carcinomas (MCCs). However, it is not yet known whether this oncogenic virus also disrupts immune-related pathways. We find that MCPyV large T antigen (LT) expression downregulates TLR9 expression in epithelial and MCC-derived cells. Accordingly, silencing of LT expression results in upregulation of mRNA TLR9 levels. In addition, small T antigen (sT) also appears to inhibit TLR9 expression, since inhibition of its expression also resulted in an increase of TLR9 mRNA levels. LT inhibits TLR9 expression by decreasing the mRNA levels of the C/EBPβ transactivator, a positive regulator of the TLR9 promoter. Chromatin immunoprecipitation reveals that C/EBPβ binding at a C/EBPβ response element (RE) in the TLR9 promoter is strongly inhibited by expression of MCPyV early genes and that mutation of the C/EBP RE prevents MCPyV downregulation of TLR9. A survey of BK polyomavirus (BKPyV), JC polyomavirus (JCPyV), KI polyomavirus (KIPyV), MCPyV, simian virus 40 (SV40), and WU polyomavirus (WUPyV) early genes revealed that only BKPyV and MCPyV are potent inhibitors of TLR9 gene expression. MCPyV LT targeting of C/EBP transactivators is likely to play an important role in viral persistence and potentially inhibit host cell immune responses during MCPyV tumorigenesis.