Lymphocyte gene expression and JC virus noncoding control region sequences are linked with the risk of progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML)-derived noncoding control region (NCCR) sequences permitted greater early viral gene expression than kidney-associated NCCR sequences. This was driven in part by binding of the transcription factor Spi-B to unique PML-associated Spi-B binding sites. Spi-B is upregulated in developing B cells in response to natalizumab therapy, a known risk factor for PML. Naturally occurring JCV sequence variation, together with drug treatment-induced cellular changes, may synergize to create an environment leading to an increased risk of PML.

Differentiation of human fetal multipotential neural progenitor cells to astrocytes reveals susceptibility factors for JC virus.

Viral infections of the central nervous system (CNS) are of increasing concern, especially among immunocompromised populations. Rodent models are often inappropriate for studies of CNS infection, as many viruses, including JC virus (JCV) and HIV, cannot replicate in rodent cells. Consequently, human fetal brain-derived multipotential CNS progenitor cells (NPCs) that can be differentiated into neurons, oligodendrocytes, or astrocytes have served as a model in CNS studies. NPCs can be nonproductively infected by JCV, while infection of progenitor-derived astrocytes (PDAs) is robust. We profiled cellular gene expression at multiple times during differentiation of NPCs to PDAs. Several activated transcription factors show commonality between cells of the brain, in which JCV replicates, and lymphocytes, in which JCV is likely latent. Bioinformatic analysis determined transcription factors that may influence the favorable transcriptional environment for JCV in PDAs. This study attempts to provide a framework for understanding the functional transcriptional profile necessary for productive JCV infection.

Molecular biology, epidemiology, and pathogenesis of progressive multifocal leukoencephalopathy, the JC virus-induced demyelinating disease of the human brain.

Progressive multifocal leukoencephalopathy (PML) is a debilitating and frequently fatal central nervous system (CNS) demyelinating disease caused by JC virus (JCV), for which there is currently no effective treatment. Lytic infection of oligodendrocytes in the brain leads to their eventual destruction and progressive demyelination, resulting in multiple foci of lesions in the white matter of the brain. Before the mid-1980s, PML was a relatively rare disease, reported to occur primarily in those with underlying neoplastic conditions affecting immune function and, more rarely, in allograft recipients receiving immunosuppressive drugs. However, with the onset of the AIDS pandemic, the incidence of PML has increased dramatically. Approximately 3 to 5% of HIV-infected individuals will develop PML, which is classified as an AIDS-defining illness. In addition, the recent advent of humanized monoclonal antibody therapy for the treatment of autoimmune inflammatory diseases such as multiple sclerosis (MS) and Crohn's disease has also led to an increased risk of PML as a side effect of immunotherapy. Thus, the study of JCV and the elucidation of the underlying causes of PML are important and active areas of research that may lead to new insights into immune function and host antiviral defense, as well as to potential new therapies.

Activities of ICP0 involved in the reversal of silencing of quiescent herpes simplex virus 1.

ICP0 is a transcriptional activating protein required for the efficient replication and reactivation of latent herpes simplex virus 1 (HSV-1). Multiple regions of ICP0 contribute its activity, the most prominent of which appears to be the RING finger, which confers E3 ubiquitin ligase activity. A region in the C terminus of ICP0 has also been implicated in several activities, including the disruption of a cellular repressor complex, REST/CoREST/HDAC1/2/LSD1. We used quiescent infection of MRC-5 cells with a virus that does not express immediate-early proteins, followed by superinfection with various viral mutants to quantify the ability of ICP0 variants to reactivate gene expression and alter chromatin structure. Superinfection with wild-type virus resulted in a 400-fold increase in expression from the previously quiescent d109 genome, the removal of heterochromatin and histones from the viral genome, and an increase in histone marks associated with activated transcription. RING finger mutants were unable to reactivate transcription or remove heterochromatin from d109, while mutants that are unable to bind CoREST activate gene expression from quiescent d109, albeit to a lesser degree than the wild-type virus. One such mutant, R8507, resulted in the partial removal of heterochromatin. Infection with R8507 did not result in the hyperacetylation of H3 and H4. The results demonstrate that (i) consistent with previous findings, the RING finger domain of ICP0 is required for the activation of quiescent genomes, (ii) the RF domain is also crucial for the ultimate removal of repressive chromatin, (iii) activities or interactions specified by the carboxy-terminal region of ICP0 significantly contribute to activation, and (iv) while the effects of the R8507 on chromatin are consistent with a role for REST/CoREST/HDAC1/2/LSD1 in the repression of quiescent genomes, the mutation may also affect other activities involved in derepression.

Reversal of heterochromatic silencing of quiescent herpes simplex virus type 1 by ICP0.

Persisting latent herpes simplex virus genomes are to some degree found in a heterochromatic state, and this contributes to reduced gene expression resulting in quiescence. We used a relatively long-term quiescent infection model in human fibroblasts, followed by provision of ICP0 in trans, to determine the effects of ICP0 on the viral chromatin state as gene expression is reactivated. Expression of ICP0, even at low levels, results in a reduction of higher-order chromatin structure and heterochromatin on quiescent viral genomes, and this effect precedes an increase in transcription. Concurrent with transcriptional activation, high levels of ICP0 expression result in the reduction of the heterochromatin mark trimethylated H3K9, removal of histones H3 and H4 from the quiescent genome, and hyperacetylation of the remaining histones. In contrast, low levels of ICP0 did not appreciably change the levels of histones on the viral genome. These results indicate that ICP0 activity ultimately affects chromatin structure of quiescent genomes at multiple levels, including higher-order chromatin structure, histone modifications, and histone association. Additionally, the level of ICP0 expression affected its ability to change chromatin structure but not to reactivate gene expression. While these observations suggest that some of the effects on chromatin structure are possibly not direct, they also suggest that ICP0 exerts its effects through multiple mechanisms.

Epigenetic modulation of gene expression from quiescent herpes simplex virus genomes.

The ability of herpes simplex virus to persist in cells depends on the extent of viral-gene expression, which may be controlled by epigenetic mechanisms. We used quiescent infection with the viral mutants d109 and d106 to explore the effects of cell type and the presence of the viral protein ICP0 on the expression and chromatin structure of the human cytomegalovirus (HCMV) tk and gC promoters on the viral genome. Expression from the HCMV promoter on the d109 genome decreased with time and was considerably less in HEL cells than in Vero cells. Expression from the HCMV promoter in d106 was considerably more abundant than in d109, and this increased with time in both cell types. The same pattern of expression was seen on the tk and gC genes on the viral genomes, although the levels of tk and gC RNA were approximately 10(2)- and 10(5)-fold lower than those of wild-type virus in d106 and d109, respectively. In micrococcal-nuclease digestion experiments, nucleosomes were evident on the d109 genome, and the amount of total H3 as determined by chromatin immunoprecipitation was considerably greater on d109 than d106 genomes. The acetylation of histone H3 on the d106 genomes was evident at early and late times postinfection in Vero cells, but only at late times in HEL cells. The same pattern was observed for H3 acetylated on lysine 9. Trimethylation of H3K9 on d109 genomes was evident only at late times postinfection in Vero cells, while it was observed both early and late in HEL cells. Heterochromatin protein 1gamma (HP1gamma) was generally present only on d109 genomes at late times postinfection of HEL cells. The observations of chromatin structure correlate with the expression patterns of the three analyzed genes on the quiescent genomes. Therefore, several mechanisms generally affect the expression and contribute to the silencing of persisting genomes. These are the abundance of nucleosomes, the acetylation state of the histones, and heterochromatin. The extents to which these different mechanisms contribute to repression vary in different cell types and are counteracted by the presence of ICP0.

Clonal immortalized human glial cell lines support varying levels of JC virus infection due to differences in cellular gene expression.

JC virus (JCV) is a ubiquitous human polyomavirus that causes the demyelinating disease Progressive Multifocal Leukoencephalopathy (PML). JCV replicates in limited cell types in culture, predominantly in human glial cells. Following introduction of a replication defective SV40 mutant that expressed large T protein into a heterogeneous culture of human fetal brain cells, multiple phenotypes became immortalized (SVG cells). A subset of SVG cells could support JCV replication. In the current study, clonal cell lines were selected from the original SVG cell culture. The 5F4 clone showed low levels of viral growth. The 10B1 clone was highly permissive for JCV DNA replication and gene expression and supported persistent and stable JCV infection over months in culture. Microarray analysis revealed that viral infection did not significantly change gene expression in these cells. More resistant 5F4 cells expressed high levels of transcription factors known to inhibit JCV transcription. Interestingly, 5F4 cells expressed high levels of RNA of markers of radial glia and 10B1 cells had high expression of markers of immature glial cells and activation of transcription regulators important for stem/progenitor cell self-renewal. These SVG-derived clonal cell lines provide a biologically relevant model to investigate cell type differences in JCV host range and pathogenesis, as well as neural development. Several transcription regulators were identified which may be targets for therapeutic modulation of expression to abrogate JCV replication in PML patients. Additionally, these clonal cell lines can provide a consistent culture platform for testing therapies against JCV infection of the central nervous system.

Prophylactic vaccine strategies and the potential of therapeutic vaccines against herpes simplex virus.

Herpes Simplex Virus Type 1 (HSV-1) infection is widespread and causes significant disease. A number of prophylactic vaccine strategies have elicited protective immunity in animal models, but no human vaccine has yet been effective. Asymptomatic HSV-1 infection is common, demonstrating that the immune system is able to control infection, despite failure to clear the virus. Therefore, therapeutic vaccination may be a viable strategy against HSV-1. This review will discuss the epidemiology, molecular biology, and immune response to HSV-1, prophylactic and therapeutic vaccine strategies, and the potential of future therapeutic HSV-1 vaccines to reduce or eliminate HSV-1 pathology.