Polyomavirus Replication Research Articles

The Long and Evolving Relationship between Viruses and Multiple Sclerosis

Multiple sclerosis (MS) is a demyelinating disorder of unknown etiology, possibly caused by a virus or is virus-triggered. Several viruses, including herpesviruses, were suggested as etiologic agents or risk factors for exacerbation in the course of illness but none have been shown to be irrefutably linked. Recently the interest of researchers and clinicians in the association between viruses and MS was reawakened by the development of progressive multifocal leukoencephalopathy, a demyelinating and fatal disease caused by JC polyomavirus replication, in natalizumab-treated MS patients. In this review, we will illustrate the evidence underlying the viral hypothesis for MS pathogenesis and will review the main features of the potential viral candidates. We will also describe the risks associated with newer MS therapies and with viral/bacterial vaccinations.

Virion Assembly Factories in the Nucleus of Polyomavirus-Infected Cells

Most DNA viruses replicate in the cell nucleus, although the specific sites of virion assembly are as yet poorly defined. Electron microscopy on freeze-substituted, plastic-embedded sections of murine polyomavirus (PyV)-infected 3T3 mouse fibroblasts or mouse embryonic fibroblasts (MEFs) revealed tubular structures in the nucleus adjacent to clusters of assembled virions, with virions apparently “shed” or “budding” from their ends. Promyelocytic leukemia nuclear bodies (PML-NBs) have been suggested as possible sites for viral replication of polyomaviruses (BKV and SV40), herpes simplex virus (HSV), and adenovirus (Ad). Immunohistochemistry and FISH demonstrated co-localization of the viral T-antigen (Tag), PyV DNA, and the host DNA repair protein MRE11, adjacent to the PML-NBs. In PML−/− MEFs the co-localization of MRE11, Tag, and PyV DNA remained unchanged, suggesting that the PML protein itself was not responsible for their association. Furthermore, PyV-infected PML−/− MEFs and PML−/− mice replicated wild-type levels of infectious virus. Therefore, although the PML protein may identify sites of PyV replication, neither the observed “virus factories” nor virus assembly were dependent on PML. The ultrastructure of the tubes suggests a new model for the encapsidation of small DNA viruses.

High-Throughput Screening Identifies a Bisphenol Inhibitor of SV40 Large T Antigen ATPase Activity

The authors conducted a high-throughput screening campaign for inhibitors of SV40 large T antigen ATPase activity to identify candidate antivirals that target the replication of polyomaviruses. The primary assay was adapted to 1536-well microplates and used to screen the National Institutes of Health Molecular Libraries Probe Centers Network library of 306 015 compounds. The primary screen had an Z value of ~0.68, signal/background = 3, and a high (5%) DMSO tolerance. Two counterscreens and two secondary assays were used to prioritize hits by EC50, cytotoxicity, target specificity, and off-target effects. Hits that inhibited ATPase activity by >44% in the primary screen were tested in dose–response efficacy and eukaryotic cytotoxicity assays. After evaluation of hit cytotoxicity, drug likeness, promiscuity, and target specificity, three compounds were chosen for chemical optimization. Chemical optimization identified a class of bisphenols as the most effective biochemical inhibitors. Bisphenol A inhibited SV40 large T antigen ATPase activity with an IC50 of 41 µM in the primary assay and 6.2 µM in a cytoprotection assay. This compound class is suitable as probes for biochemical investigation of large T antigen ATPase activity, but because of their cytotoxicity, further optimization is necessary for their use in studying polyomavirus replication in vivo.

The Status of BK Polyomavirus Replication in Adult Renal Transplant Recipients in Northeastern Poland

Purpose BK polyomavirus (BKV) infection and BKV-associated nephropathy (BKVAN) are among the most important problems in renal transplantation. We aimed to determine the incidence of BK viruria, viremia, and BKVAN in renal transplant recipients in the northeastern part of Poland. Methods Urine and blood samples from 126 cadaveric renal transplant recipients were analyzed for BK viruria and viremia using quantitative real-time polymerase chain reaction and the patients were followed prospectively. The diagnosis of BKVAN was established on the allograft biopsy. Results Based on the BKV DNA analysis, the patients were divided into three groups: group 1 ( n = 89; 70.6%) without viruria or viremia, group 2 ( n = 24; 19.1%) with isolated viruria, and group 3 ( n = 13; 10.3%) with both viruria and viremia. The presence of BK viremia negatively correlated with time after the transplantation. BK viruria was associated with mycophenolate mofetil daily dose. In group 3 there were four patients (3.2%) with high viremia (>10 4 genome equivalents [gEq]/mL) and viruria (>10 7 gEq/mL) loads. Only one patient from this group developed clinical symptoms and had BKVAN in allograft biopsy. In all four cases, the maintenance immunosuppression therapy was based on tacrolimus and steroids. Conclusion Prevalence of BKV infection in renal transplant recipients in the northeastern part of Poland is similar to that reported by studies from other countries. We confirm that BK viremia could be predicted by the presence of intense viruria. Time after transplantation and the type of immunosuppression strategy are the most important predictors of BK viremia and viruria in patients after renal transplantation.

Cellular and viral factors regulating Merkel cell polyomavirus replication.

Merkel cell polyomavirus (MCV), a previously unrecognized component of the human viral skin flora, was discovered as a mutated and clonally-integrated virus inserted into Merkel cell carcinoma (MCC) genomes. We reconstructed a replicating MCV clone (MCV-HF), and then mutated viral sites required for replication or interaction with cellular proteins to examine replication efficiency and viral gene expression. Three days after MCV-HF transfection into 293 cells, although replication is not robust, encapsidated viral DNA and protein can be readily isolated by density gradient centrifugation and typical ∼40 nm diameter polyomavirus virions are identified by electron microscopy. The virus has an orderly gene expression cascade during replication in which large T (LT) and 57kT proteins are first expressed by day 2, followed by expression of small T (sT) and VP1 proteins. VP1 and sT proteins are not detected, and spliced 57kT is markedly diminished, in the replication-defective virus suggesting that early gene splicing and late gene transcription may be dependent on viral DNA replication. MCV replication and encapsidation is increased by overexpression of MCV sT, consistent with sT being a limiting factor during virus replication. Mutation of the MCV LT vacuolar sorting protein hVam6p (Vps39) binding site also enhances MCV replication while exogenous hVam6p overexpression reduces MCV virion production by >90%. Although MCV-HF generates encapsidated wild-type MCV virions, we did not find conditions for persistent transmission to recipient cell lines suggesting that MCV has a highly restricted tropism. These studies identify and highlight the role of polyomavirus DNA replication in viral gene expression and show that viral sT and cellular hVam6p are important factors regulating MCV replication. MCV-HF is a molecular clone that can be readily manipulated to investigate factors affecting MCV replication.

Inhibition of Human BK Polyomavirus Replication by Small Noncoding RNAs

Small noncoding RNAs regulate a variety of cellular processes, including genomic imprinting, chromatin remodeling, replication, transcription, and translation. Here, we report small replication-regulating RNAs (srRNAs) that specifically inhibit DNA replication of the human BK polyomavirus (BKV) in vitro and in vivo. srRNAs from FM3A murine mammary tumor cells were enriched by DNA replication assay-guided fractionation and hybridization to the BKV noncoding control region (NCCR) and synthesized as cDNAs. Selective mutagenesis of the cDNA sequences and their putative targets suggests that the inhibition of BKV DNA replication is mediated by srRNAs binding to the viral NCCR, hindering early steps in the initiation of DNA replication. Ectopic expression of srRNAs in human cells inhibited BKV DNA replication in vivo. Additional srRNAs were designed and synthesized that specifically inhibit simian virus 40 (SV40) DNA replication in vitro. These observations point to novel mechanisms for regulating DNA replication and suggest the design of synthetic agents for inhibiting replication of polyomaviruses and possibly other viruses.

Chemical methodology as a source of small-molecule checkpoint inhibitors and heat shock protein 70 (Hsp70) modulators.

Unique chemical methodology enables the synthesis of innovative and diverse scaffolds and chemotypes and allows access to previously unexplored "chemical space." Compound collections based on such new synthetic methods can provide small-molecule probes of proteins and/or pathways whose functions are not fully understood. We describe the identification, characterization, and evolution of two such probes. In one example, a pathway-based screen for DNA damage checkpoint inhibitors identified a compound, MARPIN (ATM and ATR pathway inhibitor) that sensitizes p53-deficient cells to DNA-damaging agents. Modification of the small molecule and generation of an immobilized probe were used to selectively bind putative protein target(s) responsible for the observed activity. The second example describes a focused library approach that relied on tandem multicomponent reaction methodologies to afford a series of modulators of the heat shock protein 70 (Hsp70) molecular chaperone. The synthesis of libraries based on the structure of MAL3-101 generated a collection of chemotypes, each modulating Hsp70 function, but exhibiting divergent pharmacological activities. For example, probes that compromise the replication of a disease-associated polyomavirus were identified. These projects highlight the importance of chemical methodology development as a source of small-molecule probes and as a drug discovery starting point.

MAP kinase activation increases BK polyomavirus replication and facilitates viral propagation in vitro

BK polyomavirus causes disease in immunosuppressed patients. BK virus replication was augmented in HEL-299 cells cultured in conditions that activated the MAP kinase, ERK1/2. To determine if MAP kinase activation increased BK virus replication, cells were treated with serum and phorbol 12-myristate 13-acetate (PMA). Serum and PMA stimulated large T-antigen expression and increased BK virus DNA replication. The effects of serum/PMA were directly related to MAP kinase signal activation since viral replication was reduced by the MEK1/2 inhibitor U0126. PMA also increased cyclin D1 expression and inhibition of cyclin D1/CDK4 complex and the cell cycle reduced BK virus infection. The PMA effect occurred independent of direct transcriptional activation of the viral NCCR. In HEL-299 cells, virus infection in high serum and PMA accelerated viral replication that resulted, within 7 days, in the production of high titer infectious BK virus. These results show that MAP kinase signal activation increases BK virus replication.

Polyoma Virus Infection, Replication, and Disease after Kidney Transplantation

Polyoma Virus Infection, Replication, and Disease after Kidney Transplantation

Abl Family Tyrosine Kinases Regulate Sialylated Ganglioside Receptors for Polyomavirus

Sialylated lipids serve as cellular receptors for polyomaviruses. Using pharmacological inhibitors and cell lines derived from knockout mice, we demonstrate that Abl family tyrosine kinases are required for replication of mouse polyomavirus and BK virus, a human polyomavirus associated with allograft failure following kidney transplantation. We show that decreasing Abl family kinase activity results in low levels of cell surface ganglioside receptors for mouse polyomavirus and that inhibition of sialidase activity promotes virion binding in the absence of Abl family kinase activity. These data provide evidence that Abl family kinases reduce ganglioside turnover in the plasma membrane by inhibiting host cell sialidase activity. Thus, Abl family kinases regulate the susceptibility of cells to polyomavirus infection by modulating gangliosides required for viral attachment.

Large T Antigen Promotes JC Virus Replication in G2-arrested Cells by Inducing ATM- and ATR-mediated G2 Checkpoint Signaling

Large T antigen (TAg) of the human polyomavirus JC virus (JCV) possesses DNA binding and helicase activities, which, together with various cellular proteins, are required for replication of the viral genome. We now show that JCV-infected cells expressing TAg accumulate in the G(2) phase of the cell cycle as a result of the activation of ATM- and ATR-mediated G(2) checkpoint pathways. Transient transfection of cells with a TAg expression vector also induced G(2) checkpoint signaling and G(2) arrest. Analysis of TAg mutants with different subnuclear localizations suggested that the association of TAg with cellular DNA contributes to the induction of G(2) arrest. Abrogation of G(2) arrest by inhibition of ATM and ATR, Chk1, and Wee1 suppressed JCV genome replication. In addition, abrogation of the G(2)-M transition by Cdc2 depletion disabled Wee1 depletion-induced suppression of JCV genome replication, suggesting that JCV replication is facilitated by G(2) arrest resulting from G(2) checkpoint signaling. Moreover, inhibition of ATM and ATR by caffeine suppressed JCV production. The observation that oligodendrocytes productively infected with JCV in vivo also undergo G(2) arrest suggests that G(2) checkpoint inhibitors such as caffeine are potential therapeutic agents for JCV infection.

Prevalence of Polyomavirus BK and JC Infection and Replication in 400 Healthy Blood Donors

The replication of BK virus (BKV) and JC virus (JCV) is linked to polyomavirus-associated nephropathy, hemorrhagic cystitis, and multifocal leukoencephalopathy in immunodeficient patients, but the behavior of these viruses in immunocompetent individuals has hardly been characterized. We used EIA to study samples obtained from 400 healthy blood donors aged 20-59 years for BKV- and JCV-specific antibodies against virus-like particles. We also studied BKV and JCV loads in plasma and urine among these individuals by use of real-time polymerase chain reaction. IgG seroprevalence was 82% (328 of 400 donors) for BKV and 58% (231 of400) for JCV. As age increased (age groups were divided by decade), the seroprevalence of BKV decreased from 87% (87 of 100) in the youngest group (aged 20-29 years) to 71% (71 of 100) in the oldest group (aged 50-59 years) (P = .006), whereas the seroprevalence of JCV increased from 50% (50 of 100) in the youngest group to 68% (68 of 100) in the oldest group (P = .06). Asymptomatic urinary shedding of BKV and JCV was observed in 28 (7%) and 75 (19%) of 400 subjects, respectively, with median viral loads of 3.51 and 4.64 log copies/mL, respectively (P < .001). Unlike urinary BKV loads, urinary JCV loads were positively correlated with IgG levels. The shedding of JCV was more commonly observed among individuals who were seropositive only for JCV, compared with individuals who were seropositive for both BKV and JCV, suggesting limited cross-protection from BKV immunity. Noncoding control regions were of archetype architecture in all cases, except for 1 rearranged JCV variant. Neither BKV nor JCV DNA was detected in plasma. Our study provides important data about polyomavirus infection and replication in healthy, immunocompetent individuals. These data indicate significant differences between BKV and JCV with respect to virus-host interaction and epidemiology.

Inhibition of Simian Virus 40 replication by targeting the molecular chaperone function and ATPase activity of T antigen

Polyomaviruses such as BK virus and JC virus have been linked to several diseases, but treatments that thwart their propagation are limited in part because of slow growth and cumbersome culturing conditions. In contrast, the replication of one member of this family, Simian Virus 40 (SV40), is robust and has been well-characterized. SV40 replication requires two domains within the viral-encoded large tumor antigen (TAg): The ATPase domain and the N-terminal J domain, which stimulates the ATPase activity of the Hsp70 chaperone. To assess whether inhibitors of polyomavirus replication could be identified, we examined a recently described library of small molecules, some of which inhibit chaperone function. One compound, MAL2-11B, inhibited both TAg's endogenous ATPase activity and the TAg-mediated activation of Hsp70. MAL2-11B also reduced SV40 propagation in plaque assays and compromised DNA replication in cell culture and in vitro. Furthermore, the compound significantly reduced the growth of BK virus in a human kidney cell line. These data indicate that pharmacological inhibition of TAg's chaperone and ATPase activities may provide a route to combat polyomavirus-mediated disease.

Mutations in the BC‐loop of the BKV VP1 region do not influence viral load in renal transplant patients

The reactivation and replication of the BK polyomavirus (BKV) leading to BKV-associated nephropathy (BKVAN) is one of the major complications in renal transplantation patients. BKV isolates were classified into four subtypes (I-IV) based on genotype variations within the VP1-coding region. The type-specific amino acid differences cluster within the BC-loop of the major capsid protein VP1. As demonstrated in vitro, mutations in this region also play a role in the infectivity, attachment and stability of viral particles. Therefore, we analyzed the prevalence of BC-loop mutations in isolates of kidney transplant patients and compared their viral load in the urine. The VP1 subtyping regions of BKV isolates obtained from urine samples of 45 renal transplant patients were sequenced. The phylogenetic analysis of these sequences revealed that subtype I (66.67%) is the most prevalent genotype. The remaining isolates belong to subtype IV (33.33%). A high frequency of changes to specific amino acids within the BC-loop was identified among the BKV isolates from renal transplant patients. Patients with BKVAN exhibited a higher viral replication than patients without nephropathy. Although titers of isolates of subtype I were higher than titers of subtype IV isolates, the difference did not reach statistical significance. In addition, amino acid changes in the BC-loop did not influence the viral load and the incidence of BKVAN. These in vivo results demonstrate that high replication rates which serve as a predictive marker for BKVAN are not caused by altered receptor binding or affinity via mutated BC-loops.

Prospective study of urine cytology screening for BK polyoma virus replication in renal transplant recipients

BK virus (BKV) may be associated with interstitial nephritis in renal transplant recipients and this can lead to irreversible chronic allograft dysfunction. Early diagnosis of BKV nephropathy determines its progress because no specific antiviral therapy exists. Urine cytology, detection of viral DNA in urine or blood and renal biopsy are the main diagnostic tools. The purpose of this study was to evaluate the use of urine cytology for diagnosis of BKV replication in renal graft recipients. We studied 32 de novo renal transplant recipients prospectively with sequential urine samples for a period of 1 year. Thin-Prep methodology was used to prepare the slides. Cytology results were correlated with polymerase chain reaction (PCR) in urine and blood. Decoy cells indicative of BKV infection were detected in 14 (7.3%) of the 190 urine samples derived from 11 recipients. In three cases with positive decoy cells, BK viraemia and viruria were simultaneously identified. In a further three cases, BKV active replication was confirmed in urine by both cytology and PCR. Urine cytology is an easy and rapid method of detecting decoy cells in cases where renal biopsy is not possible. However, the low incidence of detection of decoy cells in the present study, together with poor correlation with PCR results, questions its sensitivity and specificity in diagnosing BKV reactivation.

In vitro effect of cyclosporin A on primary and chronic BK polyoma virus infection in Vero E6 cells

Cyclosporin A (CsA) is known to possess antiviral activity against several viruses in vitro, but the effect of CsA on BK polyoma virus (BKV) replication has not been examined. We investigated the impact of CsA on primary, chronic, and high-level BKV infection using a cell system of kidney cell origin (Vero E6 cells). During the first 2 h post infection, cells treated with CsA up to 3200 microg/L showed a near-identical BK viral load to untreated cells, with only a very minor reduction in the CsA-treated cells observed at 4 h. In chronic culture, CsA completely suppressed the primary BKV infection peak in a non-dose-dependent manner within the dose range of 200-12,800 microg/L (P<0.05). BKV reactivation was also inhibited in the presence of CsA at doses of 200-3200 microg/L: the mean number of BKV DNA copies/mL remained stable or even decreased slightly compared with a 7-log increase in the non-CsA group (P<0.01). CsA did not influence BKV DNA copies/mL in Vero E6 cells with high-level infection (>10(9) copies/mL). Cellular protein measurements indicated that the antiviral effect of CsA was not a result of cytotoxicity. These findings from a relevant in vitro kidney cell system indicate that CsA suppresses the primary BKV infection peak and inhibits escape to BKV reactivation; these effects are dose independent and not related to cytotoxicity. The intracellular antiviral mode of action of CsA against BKV does not appear to be via inhibition of viral cell entry pathways.

Impact of prophylactic administration of ciprofloxacin on BK polyoma virus replication

Impact of prophylactic administration of ciprofloxacin on BK polyoma virus replication

Suppression of early and chronic BK polyoma virus replication by mycophenolic acid in Vero cells

Consensus is lacking about which immunosuppressant agents potentiate BK virus infection. The effects of mycophenolic acid (MPA) were investigated in BK virus (BKV)-infected Vero E6 cells. MPA (1-16 mg/l) exhibited a dose-dependent anti-viral effect (10(1)-10(4) fold reduction in BKV DNA copies/ml) in supernatant, similar to cidofovir (2.5-25 mg/l). This effect was observed for early and persistent infection, and infection with noncoding control region (NCCR) rearranged BKV. MPA reduced BKV DNA copies/ml by >1 log after day 14 in three patient isolates before and after NCCR rearrangement, and in cells. MPA reduced total cellular protein levels, consistent with an anti-metabolite effect without increased cytopathic activity. BKV infection was associated with a transient, significant reduction of collagen 1A1 on day 7 but not on days 14, 21, and 28 or in the presence of MPA. Reduction of alpha smooth muscle actin mRNA was observed only in the BKV + MPA group, and only on day 7. There was no significant alteration of heat shock protein 47 or transforming growth factor-beta mRNA expression. These in vitro data suggest that MPA may have a protective, anti-viral effect in BKV-infected renal tubular cells with an anti-viral response. Maintaining, or even increasing, the MPA dose should be evaluated for reduction of BKV viremia levels.

Association of treatment with 15‐deoxyspergualin and BK virus nephropathy in kidney allograft recipients

BK virus nephropathy (BKVN) has been proposed as an important cause of allograft dysfunction and loss in kidney allograft recipient over the last decade. Intense immunosuppression and tubular injury have been shown to promote the replication of polyomavirus. 15-deoxyspergualin (DSG), an effective immunosuppressive agent, is used as a rescue drug for acute rejection in clinical renal transplantation in Japan. To determine whether DSG is a risk factor for BKVN and outline the relationship among BKVN, DSG, and other risk factors, we analyzed 88 patients who received living-related renal transplantation between January 1999 and April 2003. A total of 114 biopsy specimens from 88 living-related kidney transplantation recipients (performed between January 1999 to April 2003) were retrospectively analyzed. Patients received immunosuppression therapy based on calcineurin inhibitors and corticosteroid [tacrolimus (TAC) 33 and cyclosporin (CyA) 55]. Additionally, mycophenolate mofeteil (MMF) was used in 21 patients; DSG was used in seven patients; and anti-CD3 monoclonal antibody was used in 16 patients. We analyzed the degree of donor/recipient human leucocyte antigen (HLA) compatibility assessed by the number of HLA-A, -B, and -DR-mismatched antigens in 88 patients. The diagnosis of BKVN was made by the light microscopic examination and a positive immunohistochemical staining of anti-40 antibody in biopsy specimens. Patients were divided into two groups of group A (mild histological change) and group B (moderate or severe histological change) to determine the risk factors in different histological staging. The clinical course of two typical patients in different histological stage is described briefly to outline the risk factors of BKVN. We identified seven cases of BKVN (8.0%) from 88 transplanted patients. Significantly higher incidence of combination regimen consisting of TAC and MMF in BKVN group was noticed compared with non-BKVN group (57.1% vs. 9.9%; p = 0.003). BKVN was associated with a significantly higher incidence of DSG administration compared with non-BKVN group (57.1% vs. 3.7%; p </= 0.001). No difference was found in HLA mismatch between BKVN and non-BKVN group. Additionally, a significantly higher incidence of acute rejection episode prior to BKVN diagnosis was found in group B compared with group A (100% vs. 0%; p = 0.002), and the same statistical difference was shown in the number of anti-rejection therapy between group B and group A (100% vs. 0%; p = 0.002). We recognized a significant difference between group B and group A in terms of the combine regimen therapy of TAC with MMF (p = 0.002) and no difference in graft loss rate (p = 0.092), even though it was 50% in group B and was 0% in group A. In this study, our data indicated excessive intense immunosuppression with TAC-MMF is related to the development of BKVN. In addition, we found that anti-rejection therapy, especially with DSG, may accelerate the development of BKVN.

Antivirals for the treatment of polyomavirus BK replication

Antiviral drugs with specific activity against polyomavirus replication have not been developed in the past. This deficiency has become fully apparent with the emergence of polyomavirus-associated nephropathy in kidney-transplant recipients, with a prevalence rate of up to 10%. In most cases, high BK virus replication in tubular epithelial cells causes significant cytopathology, leading to permanently impaired renal allograft function and return to hemodialysis within 6–60 months. In 5–10% of allogenic bone marrow/ hematopoietic stem cell transplant recipients, high-level BK virus replication in the ureter/bladder mucosa has been associated with postengraftment hemorrhagic cystitis, which appears to involve significant immunopathology. Thus, in view of the increasing clinical need, a number of drugs have been studied in small case series. We review the antiviral strategies explored to date and specifically discuss available in vivo and in vitro data on cidofovir, leflunomide, fluoroquinolones and intravenous immunoglobulins, regarding mechanism, administration, dosing and outcome and provide a perspective on future therapy options.