Identification and characterization of mefloquine efficacy against JC virus in vitro.

Progressive multifocal leukoencephalopathy (PML) is a rare but frequently fatal disease caused by the uncontrolled replication of JC virus (JCV), a polyomavirus, in the brains of some immunocompromised individuals. Currently, no effective antiviral treatment for this disease has been identified. As a first step in the identification of such therapy, we screened the Spectrum collection of 2,000 approved drugs and biologically active molecules for their anti-JCV activities in an in vitro infection assay. We identified a number of different drugs and compounds that had significant anti-JCV activities at micromolar concentrations and lacked cellular toxicity. Of the compounds with anti-JCV activities, only mefloquine, an antimalarial agent, has been reported to show sufficiently high penetration into the central nervous system such that it would be predicted to achieve efficacious concentrations in the brain. Additional in vitro experiments demonstrated that mefloquine inhibits the viral infection rates of three different JCV isolates, JCV(Mad1), JCV(Mad4), and JCV(M1/SVEDelta), and does so in three different cell types, transformed human glial (SVG-A) cells, primary human fetal glial cells, and primary human astrocytes. Using quantitative PCR to quantify the number of viral copies in cultured cells, we have also shown that mefloquine inhibits viral DNA replication. Finally, we demonstrated that mefloquine does not block viral cell entry; rather, it inhibits viral replication in cells after viral entry. Although no suitable animal model of PML or JCV infection is available for the testing of mefloquine in vivo, our in vitro results, combined with biodistribution data published in the literature, suggest that mefloquine could be an effective therapy for PML.

Transforming activities of JC virus early proteins.

Polyomaviruses, as their name indicates, are viruses capable of inducing a variety of tumors in vivo. Members of this family, including the human JC and BK viruses (JCV, BKV), and the better characterized mouse polyomavirus and simian virus 40 (SV40), are small DNA viruses that commandeer a cell's molecular machinery to reproduce themselves. Studies of these virus-host interactions have greatly enhanced our understanding of a wide range of phenomena from cellular processes (e.g., DNA replication and transcription) to viral oncogenesis. The current chapter will focus upon the five known JCV early proteins and the contributions each makes to the oncogenic process (transformation) when expressed in cultured cells. Where appropriate, gaps in our understanding of JCV protein function will be supplanted with information obtained from the study of SV40 and BKV.

Induction of chromosomal instability in colonic cells by the human polyomavirus JC virus.

Most colorectal cancers display chromosomal instability, which is characterized by gross chromosomal rearrangements, loss of heterozygosity and aneuploidy. We have previously demonstrated a link between JC virus strains Mad-1 and Delta98 and colorectal cancer. Others have also associated the virus to the induction of colon cancer and aneuploid brain tumors by producing a highly tumorigenic protein named T antigen (TAg), which binds to beta-catenin and inactivates key proteins such as p53. The aim is to demonstrate that JC virus is capable of inducing chromosomal instability in colonic cells. We used the human colon cancer cell line RKO as a model. The cell line has wild-type p53, wild-type beta-catenin and APC and is diploid. Neuroblastoma JCI cells, which are infected with the virus, VA13 fibroblasts, which are transformed by the SV40 TAg, were used as positive controls. HCT116, which has mutated beta-catenin, and SW480, which is a model of CIN, were also used as controls. The genomes of the Mad-1 and Delta98 strains were transfected into cells. As negative controls we used pUC or no plasmids. Cells were collected at 0, 7, 14, and 21 days after transfection. PCR was used for the detection of TAg and the regulatory region DNA sequences at different time frames and Southern blot of whole genomic extracts for viral DNA integration into the host genome. Immunofluorescence and Western blot were performed for TAg, viral capsid proteins, and nuclear beta-catenin expressions, whereas coimmunoprecipitation was used to detect protein interactions. Karyotype analysis and electron microscopy were performed to seek chromosomal instability and cell abnormalities, respectively. Retention of viral sequences was observed for Mad-1- and Delta98-transfected RKO cells at all time frames with PCR only, whereas Southern blot analysis showed nonintegrated sequences at T7 alone. TAg and capsid protein expressions, as well as increased p53 and nuclear beta-catenin, were observed between T0 and T7 for Mad-1 and Delta98 alone. Also, interaction between TAg and both p53 and beta-catenin was also observed between T0 and T7. Chromosomal instability, characterized by chromosomal breakage, dicentric chromosomes, and increasing ploidy, was observed at all time frames for Mad-1 and Delta98, as well as cell abnormalities. In conclusion, we demonstrate that JC virus Mad-1 and Delta98 are able to induce chromosomal instability in colonic cells with a hit and run mechanism that involves an early interaction with beta-catenin and p53.

A rapid in vitro polyomavirus DNA replication assay.

Traditionally, the Hirt extraction method, a multi-step, labor-intensive and time-consuming procedure, is employed to extract selectively low-molecular weight DNA for polyomavirus DNA replication analyses. DNA replication results obtained with this approach are often inconsistent between replicate samples. To increase the efficiency and reproducibility of the polyomavirus DNA replication assay, we compared the DNA quality and yield using Qiagen Spin Column technology and the Hirt extraction technique. CV-1 cells transfected with SV40 DNA were harvested at days 2, 4, and 6 post-transfection, and DNA was extracted using the Qiagen Spin Column and the Hirt extraction methods. Southern hybridization was performed using a (32)P-labeled linear full-length SV40 DNA probe. Viral DNA replication was quantitated using a BioRad phosphorimager, and results obtained with the two procedures were compared. Southern blot analysis revealed consistent and enhanced SV40 DNA recovery using the Qiagen Spin Column technology, and viral DNA replication over a 6-day period was reproducible among triplicate samples. In addition, Qiagen Spin Column technology reduced the time required to obtain good quality DNA for polyomavirus replication assays from 24 h to less than 3 h. Adoption of this extraction procedure will improve the determination of polyomavirus DNA replication activity, while reducing the investigator's exposure to and disposal of toxic organic compounds.

Interaction and co-localization of JC virus large T antigen and the F-box protein ß-transducin-repeat containing protein.

Lytic infection and transformation of cultured cells by JC virus (JCV) require five tumor proteins, which interact with factors regulating critical cellular processes. We demonstrate that JCV large T antigen (TAg) binds the F-box proteins ß-transducin-repeat containing protein-1 and 2 (ßTrCP1/2). These interactions involve a phosphodegron (DpSGX(2-4)pS) found in ßTrCP substrates. TAg stability is unaltered, suggesting TAg is a pseudo-substrate. ßTrCP and TAg co-localize in the cytoplasm, and a functional SCF complex is required. We examined whether TAg influences the levels of ß-catenin, a ßTrCP substrate. We were unable to demonstrate that TAg elevates ß-catenin as previously reported, and a mutant TAg unable to bind ßTrCP also had no detectable effect on ß-catenin stability. Results presented in this study link JCV TAg to the cellular degradation complex, SCF(ßTrCP1/2). Proteasomal degradation is essential for proper regulation of cellular functions, and interference with proteasomal pathways highlights possible JCV pathogenic and oncogenic mechanisms.

JC virus small T antigen binds phosphatase PP2A and Rb family proteins and is required for efficient viral DNA replication activity.

BACKGROUND: The human polyomavirus, JC virus (JCV) produces five tumor proteins encoded by transcripts alternatively spliced from one precursor messenger RNA. Significant attention has been given to replication and transforming activities of JCV's large tumor antigen (TAg) and three T' proteins, but little is known about small tumor antigen (tAg) functions. Amino-terminal sequences of tAg overlap with those of the other tumor proteins, but the carboxy half of tAg is unique. These latter sequences are the least conserved among the early coding regions of primate polyomaviruses. METHODOLOGY AND FINDINGS: We investigated the ability of wild type and mutant forms of JCV tAg to interact with cellular proteins involved in regulating cell proliferation and survival. The JCV P99A tAg is mutated at a conserved proline, which in the SV40 tAg is required for efficient interaction with protein phosphatase 2A (PP2A), and the C157A mutant tAg is altered at one of two newly recognized LxCxE motifs. Relative to wild type and C157A tAgs, P99A tAg interacts inefficiently with PP2A in vivo. Unlike SV40 tAg, JCV tAg binds to the Rb family of tumor suppressor proteins. Viral DNAs expressing mutant t proteins replicated less efficiently than did the intact JCV genome. A JCV construct incapable of expressing tAg was replication-incompetent, a defect not complemented in trans using a tAg-expressing vector. CONCLUSIONS: JCV tAg possesses unique properties among the polyomavirus small t proteins. It contributes significantly to viral DNA replication in vivo; a tAg null mutant failed to display detectable DNA replication activity, and a tAg substitution mutant, reduced in PP2A binding, was replication-defective. Our observation that JCV tAg binds Rb proteins, indicates all five JCV tumor proteins have the potential to influence cell cycle progression in infected and transformed cells. It remains unclear how these proteins coordinate their unique and overlapping functions.

Stability and function of JC virus large T antigen and T' proteins are altered by mutation of their phosphorylated threonine 125 residues.

JC virus (JCV), a human polyomavirus, exhibits oncogenic activity in rodents and primates. The large tumor antigens (TAgs) of the polyomaviruses play key roles in viral replication and oncogenic transformation. Analyses of JCV TAg phosphorylation mutants indicated that the amino-terminal phosphorylation site at threonine 125 (T125) is critical to TAg replication function. This site is also conserved in the TAg splice variants T'(135), T'(136), and T'(165). By constructing stable cell lines expressing JCV T125A and T125D mutants, we show that mutation of this phosphorylation site to alanine generates an unstable TAg; however, the stability of the three T' proteins is unaffected. JCV T125A mutant proteins bind the retinoblastoma protein (RB) family members p107 and p130 with slightly reduced efficiencies and fail to induce the release of transcriptionally active E2F from RB-E2F complexes. On the other hand, cell lines expressing JCV T125D mutant proteins produce stable TAg and T' proteins which bind p107 and p130 more efficiently than do the wild-type proteins. In addition, T125D mutant proteins efficiently induce the release of E2F from RB-E2F complexes. T125D mutant cell lines, unlike the T125A mutant lines, continue to grow under conditions of low serum concentration and anchorage independence. Finally, both T125A and T125D mutant viruses are replication defective. Phosphorylation of the T125 site is likely mediated by a cyclin-cyclin-dependent kinase, suggesting that JCV TAg and T' protein functions that mediate viral replication and oncogenic transformation events are regulated in a cell cycle-dependent manner.

JC virus T'135, T'136 and T'165 proteins interact with cellular p107 and p130 in vivo and influence viral transformation potential.

The JC virus (JCV) regulatory proteins, large T antigen, small t antigen, T'135, T'136, and T'165, are encoded by five transcripts alternatively spliced from the viral early precursor mRNA. T antigen and the T' proteins share N-terminal amino acid sequences that include the L x CxE and J domains, motifs in SV40 T antigen known to mediate binding to the retinoblastoma (Rb) proteins and Hsc70, respectively. In this study, G418-resistant cell lines were created that express wild-type or mutant JCV T antigen and T' proteins individually or in combination. These cell lines were used to evaluate the ability of each viral protein to bind p107 and p130 in vivo, and to influence cellular growth characteristics. Differences were observed in the abilities of individual T' proteins to bind p107 and p130 and to alter their phosphorylation status. The T' proteins were also found to localize to the cell's nucleus and to be phosphorylated in a cell cycle-dependent manner. JCV T antigen and T' proteins expressed from a cytomegalovirus promoter failed to induce dense focus formation in Rat2 cells, but they did cooperate with a mutant Ras protein to overcome cellular senescence and immortalize rat embryo fibroblasts. These data indicate that, despite their sequence similarities, JCV early proteins exhibit unique activities that, in combination, effect the inactivation of cell cycle regulators, a requirement for polyomavirus-induced transformation.

JC virus induces altered patterns of cellular gene expression: interferon-inducible genes as major transcriptional targets.

Human polyomavirus JC (JCV) infects 80% of the population worldwide. Primary infection, typically occurring during childhood, is asymptomatic in immunocompetent individuals and results in lifelong latency and persistent infection. However, among the severely immunocompromised, JCV may cause a fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Virus-host interactions influencing persistence and pathogenicity are not well understood, although significant regulation of JCV activity is thought to occur at the level of transcription. Regulation of the JCV early and late promoters during the lytic cycle is a complex event that requires participation of both viral and cellular factors. We have used cDNA microarray technology to analyze global alterations in gene expression in JCV-permissive primary human fetal glial cells (PHFG). Expression of more than 400 cellular genes was altered, including many that influence cell proliferation, cell communication and interferon (IFN)-mediated host defense responses. Genes in the latter category included signal transducer and activator of transcription 1 (STAT1), interferon stimulating gene 56 (ISG56), myxovirus resistance 1 (MxA), 2'5'-oligoadenylate synthetase (OAS), and cig5. The expression of these genes was further confirmed in JCV-infected PHFG cells and the human glioblastoma cell line U87MG to ensure the specificity of JCV in inducing this strong antiviral response. Results obtained by real-time RT-PCR and Western blot analyses supported the microarray data and provide temporal information related to virus-induced changes in the IFN response pathway. Our data indicate that the induction of an antiviral response may be one of the cellular factors regulating/controlling JCV replication in immunocompetent hosts and therefore constraining the development of PML.

T' proteins influence JC virus biology.

The JC virus early mRNA is alternatively spliced to yield five transcripts that encode large T antigen, small t antigen, T'(135), T'(136), and T'(165). The splicing process is regulated differentially in transformed versus lytically infected cells and temporally during the course of a productive infection. The authors have identified a potential exonic splicing enhancer near the 3' end of the early viral mRNA that, when mutated, results in altered splice site usage. The authors have only recently begun investigating the function of the three T' proteins using genetic and biochemical approaches. These studies indicate that the T' proteins enhance viral DNA replication and bind differentially to the pRB family of cellular tumor suppressor proteins in vitro. Using a G418 selection scheme, the authors have created cell lines that express either T antigen or each of the T' proteins individually. Preliminary analyses of these lines suggest that T antigen may induce apoptosis in rodent cells, an activity that may be blocked by other JC virus early proteins. Furthermore, examination of protein-protein interactions within the G418-selected cells reveal differences in binding of the viral proteins to the pRB family members relative to that seen in vitro.