A cell-free replication system for human polyomavirus JC DNA.

The human polyomavirus JC virus (JCV) establishes persistent infections in most individuals and is the etiologic agent of progressive multifocal leukoencephalopathy. In this report, we describe the establishment of a soluble cell-free system that is capable of replicating exogenous plasmid DNA containing the JCV origin of replication. Replication in this system is completely dependent on the addition of JCV large T antigen (TAg). To prepare JCV TAg for replication analysis, a recombinant baculovirus containing the JCV TAg-coding sequence was generated. TAg expressed in insect cells was purified by metal chelate chromatography. JCV TAg supported initiation of JCV DNA replication in the presence of DNA polymerase alpha-primase, replication protein A, and topoisomerase I in a dose-dependent manner and was also capable of supporting DNA replication in crude human cell extracts. Point mutation of TAg-binding site I strongly diminished TAg binding and concomitantly reduced JCV DNA replication in vivo and in vitro by approximately 50%. Point mutation of TAg-binding site II or deletion of the early palindrome completely abolished replication of JCV origin-containing plasmid DNA in vivo and in vitro, marking these sequences as essential components of the JCV core origin. A comparison of several TAgs showed that simian virus 40 TAg, but not mouse polyomavirus (PyV) TAg, supported replication of a plasmid containing a JCV origin. These findings provide evidence that replication in the cell-free system faithfully mimics JCV DNA replication in vivo. Therefore, it may be a useful tool for future analysis of interactions between JCV and its host cell.     

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.     

Human polyomavirus JC virus genome.

The complete DNA sequence of the human JC virus, which was found to consist of 5,130 nucleotide pairs, is presented. The amino acid sequence of six proteins could be deduced: the early, nonstructural proteins, large T and small t antigens; the late capsid proteins, VP1, VP2, and VP3; and the agnogene product encoded within the late leader sequence, called the agnoprotein in simian virus 40. The extent of homology between JC virus DNA and the genomes of simian virus 40 (69%) and BK virus (75%) confirmed the close evolutionary relationship of these three polyomaviruses. The sequences showing the greatest divergence in these viral DNAs occurred within the tandem repeats located to the late side of the replication origins.     

Human polyomavirus JC variants in Papua New Guinea and Guam reflect ancient population settlement and viral evolution

The peopling of the Pacific was a complex sequence of events that is best reconstructed by reconciling insights from various disciplines. Here we analyze the human polyomavirus JC (JCV) in Highlanders of Papua New Guinea (PNG), in Austronesian-speaking Tolai people on the island of New Britain, and in nearby non-Austronesian-speaking Baining people. We also characterize JCV from the Chamorro of Guam, a Micronesian population. All JCV strains from PNG and Guam fall within the broad Asian group previously defined in the VP1 gene as Type 2 or Type 7, but the PNG strains were distinct from both genotypes. Among the Chamorro JCV samples, 8 strains (Guam-1) were like the Type 7 strains found in Southeast Asia, while nine strains (Guam-2) were distinct from both the mainland strains and most PNG strains. We identified three JCV variants within Papua New Guinea (PNG-1, PNG-2 and PNG-3), but none of the Southeast Asian (Type 7) strains. PNG-1 strains were present in all three populations (Highlanders and the Baining and Tolai of New Britain), but PNG-2 strains were restricted to the Highlanders. Their relative lack of DNA sequence variation suggests that they arose comparatively recently. The single PNG-3 strain, identified in an Austronesian-speaking Tolai individual, was closely related to the Chamorro variants (Guam-2), consistent with a common Austronesian ancestor. In PNG-2 variants a complex regulatory region mutation inserts a duplication into a nearby deletion, a change reminiscent of those seen in the brains of progressive multifocal leukoencephalopathy patients. This is the first instance of a complex JCV rearrangement circulating in a human population.     

Establishment of COS‐JC cells persistently producing archetype JC polyomavirus

JC polyomavirus (JCPyV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system in immunocompromised patients. Archetype JCPyV circulates in the human population. There have been several reports of archetype JCPyV replication in cultured cells, in which propagation was not enough to produce high titers of archetype JCPyV. In this study, we carried out cultivation of the transfected cells with archetype JCPyV DNA MY for more than 2 months to establish COS‐7 cells (designated COS‐JC cells) persistently producing archetype JCPyV. Moreover, JCPyV derived from COS‐JC cells was characterized by analyzing the viral propagation, size of the viral genome, amount of viral DNA, production of viral protein, and structure of the non‐coding control region (NCCR). Southern blotting using a digoxigenin‐labeled JCPyV probe showed two different sizes of the JCPyV genome in COS‐JC cells. For molecular cloning, four of five clones showed a decrease in the size of complete JCPyV genome. Especially, clone No. 10 was generated the large deletion within the Large T antigen. On the other hand, the archetype structure of the NCCR was maintained in COS‐JC cells, although a few point mutations occurred. Quantitative PCR analysis of viral DNA in COS‐JC cells indicated that a high copy number of archetype JCPyV DNA was replicated in COS‐JC cells. These findings suggest that COS‐JC cells could efficiently propagate archetype JCPyV MY and offer a useful tool to study persistent infection of archetype JCPyV in a kidney‐derived system.

     

Differences in regulatory sequences of naturally occurring JC virus variants.

The regulatory region was sequenced for DNAs representative of seven independent isolates of JC virus, the probable agent of progressive multifocal leukoencephalopathy. The isolates included an oncogenic variant (MAD-4), an antigenic variant (MAD-11), and two different isolates derived from the urine (MAD-7) and from the brain (MAD-8) of the same patient. The representative DNAs were molecularly cloned directly from diseased brain tissue and from human fetal glial cells infected with the corresponding isolated viruses. The regulatory sequences of these DNAs were compared with those of the prototype isolate, MAD-1, sequenced previously (R. J. Frisque, J. Virol. 46:170-176, 1983). We found that the regulatory region of JC viral DNA is highly variable due to complex alterations of the previously described 98-base-pair repeat of MAD-1 DNA. On the basis of these alterations, there are two general types of JC virus. There were no consistent alterations in regulatory sequences which could distinguish brain tissue DNAs from tissue culture DNAs. Furthermore, for each isolate except MAD-1 (R. J. Frisque, J. Virol. 46:170-176, 1983), the regulatory regions of brain tissue and tissue culture DNAs were not identical. The arrangement, sequence, or both of potential regulatory elements (TATA sequence, GGGXGGPuPu, tandem repeats) of JC viral DNAs are sufficiently different from those in other viral and eucaryotic systems that they may effect the unique properties of this slow virus.     

Preferred crossover sites on polyomavirus DNA.

RmI is a hybrid replicon consisting of polyomavirus (Py) and mouse sequences that yields unit-length polyomavirus DNA via recombination between two directly repeated viral sequences of 182 base pairs (S repeats). To define the contribution of the S repeats in this intramolecular recombination, we derived from RmI a series of replicons containing the original S repeats as well as additional direct viral repeats which were 1 to 2 kilobases in length (L repeats). After mouse 3T6 cells were transfected with these constructs, recombination products that displayed the physical properties of homologous recombinants were detected. The structures of these recombinants indicated that whereas repeat length influences the likelihood of recombination, crossover occurs preferentially near the S repeats, provided that one of them is proximal to the viral origin of replication. This finding suggests that recombination near the S repeats depends on a process initiated near the viral origin of replication.     

Molecular basis of organ-specific selection of viral variants during chronic infection.

Viral variants of different phenotypes are present in the central nervous system (CNS) and lymphoid tissues of carrier mice infected at birth with the Armstrong strain of lymphocytic choriomeningitis virus. The CNS isolates are similar to the parental virus and cause acute infections in adult mice, whereas the lymphoid isolates cause chronic infections associated with suppressed T-cell responses. In this study, we provide a molecular basis for this organ-specific selection and identify a single amino acid change in the viral glycoprotein that correlates with the tissue specific selection and the persistent and immunosuppressive phenotype of the variants. This phenylalanine (F)-to-leucine (L) change at position 260 of the viral glycoprotein was seen in the vast majority (43 of 47) of the lymphoid isolates, and variants with L at this residue were selected in spleens of persistently infected mice. In striking contrast, isolates with the parental sequence (F at residue 260) predominated (48 of 59 isolates) in the CNS of the same carrier mice. Complete nucleotide sequence analysis of the major structural genes of several independently derived (from different mice) spleen isolates showed that these variants were greater than 99.8% identical to the parental virus. In fact, the only common change among these spleen isolates was the F----L mutation at residue 260 of the glycoprotein. These results show that an RNA virus can exhibit minimal genetic drift during chronic infection in its natural host, and yet a single or few mutations can result in the organ-specific selection of variants that are markedly different from the parental virus.     

Suppressive effect of topoisomerase inhibitors on JC polyomavirus propagation in human neuroblastoma cells

JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system, in immunocompromised patients. Because no drugs have been approved for treating PML, many antiviral agents are currently being investigated for this purpose. The inhibitory effects of the topoisomerase I inhibitors topotecan and β‐lapachone were assessed by investigating viral replication, propagation and viral protein 1 (VP1) production in cultured cells. JCPyV replication was assayed using the human neuroblastoma cell line IMR‐32 transfected with the JCPyV plasmid and RT‐ PCR combined with Dpn I treatment. Dpn I digests the input plasmid DNA containing methylated adenosine, but not newly replicated JCPyV DNA, in IMR‐32 cells. It was found that JCPyV replicates less in IMR‐32 cells treated with topotecan or β‐lapachone than in untreated cells. Moreover, drug treatment of JCI cells, which are IMR‐32 cells persistently infected with JCPyV, led to a reduction in the amount of JCPyV DNA and population of VP1‐positive cells. These results demonstrate that topotecan and β‐lapachone affects JCPyV propagation in human neuroblastoma cell lines, suggesting that topotecan and β‐lapachone could potentially be used to treat PML.     

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.     

CPT11 prevents virus replication in JCI cells persistently infected with JC polyomavirus

JC polyomavirus (JCPyV) is the causative agent of the demyelinating disease of the central nervous system known as progressive multifocal leukoencephalopathy (PML), which occurs in immunocompromised patients. Moreover, patients treated with natalizumab for multiple sclerosis or Crohn disease can develop PML, which is then termed natalizumab‐related PML. Because few drugs are currently available for treating PML, many antiviral agents are being investigated. It has been demonstrated that the topoisomerase I inhibitors topotecan and β‐lapachone have inhibitory effects on JCPyV replication in IMR‐32 cells. However, both of these drugs have marginal inhibitory effects on virus propagation in JC1 cells according to RT‐PCR analysis. In the present study, the inhibitory effect of another topoisomerase I inhibitor, 7‐ethy‐10‐[4‐(1‐piperidino)‐1‐piperidino] carbonyloxy camptothecin (CPT11), was assessed by investigating viral replication, propagation, and viral protein 1 (VP1) production in cultured cells. JCPyV replication was assayed using real‐time PCR combined with Dpn I treatment in IMR‐32 cells transfected with JCPyV DNA. It was found that JCPyV replicates less in IMR‐32 cells treated with CPT11 than in untreated cells. Moreover, CPT11 treatment of JCI cells persistently infected with JCPyV led to a dose‐dependent reduction in JCPyV DNA and VP1 production. Additionally, the inhibitory effect of CPT11 was found to be stronger than those of topotecan and β‐lapachone. These findings suggest that CPT11 may be a potential anti‐JCPyV agent that could be used to treat PML.

     

T-antigen of the human polyomavirus JC attenuates faithful DNA repair by forcing nuclear interaction between IRS-1 and Rad51

JC polyomavirus (JCV), which infects 90% of the human population, is detectable in human tumors. Its early protein, JCV T-antigen, transforms cells in vitro and is tumorigenic in experimental animals. Although T-antigen-mediated transformation involves genetic alterations of the affected cells, the mechanism underlying this genomic instability is not known. We show that JCV T-antigen inhibits homologous recombination DNA repair (HRR), which results in an accumulation of mutations. T-antigen does not operate directly but utilizes a cytosolic molecule, insulin receptor substrate 1 (IRS-1). Following T-antigen-mediated nuclear translocation, IRS-1 binds Rad51 at the site of damaged DNA. This T-antigen-mediated inhibition of HRR does not function in cells lacking IRS-1, and can be reproduced in the absence of T-antigen by IRS-1 with artificial nuclear localization signal. Our observations define a new mechanism by which viral protein utilizes cytosolic molecule to inhibit faithful DNA repair, and suggest how polyomaviruses could compromise stability of the genome. (c) 2005 Wiley-Liss, Inc.     

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro.

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.     

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.     

Unique morphogenesis in Saccharomyces cerevisiae strain GS1731

During the lag and early exponential phase of growth, 50–60% of budded cells of Saccharomyces cerevisiae strain GS1731 were multiply budded. During subsequent culture growth, the frequency of multiply budded cells decreased until by stationary phase multiply budded cells were rare. Data from renewed growth of a culture after hydroxyurea treatment indicated that GS1731 mother cells could assemble up to three pre-bud sites and begin bud growth and development in each. Light and scanning electron microscopy showed two or three very small buds emerging simultaneously on a mother cell and either reaching full size at the same time or enlarging sequentially. Immunofluorescence studies revealed that these multiply budded cells had multiple bundles of cytoplasmic microtubules. DAPI staining of nuclei revealed that some of the unbudded mother cells were multinucleate and completed cytokinesis giving rise to normal daughter cells.