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Susceptibility to infection by the human polyomavirus, JCV, is determined by intracellular mechanisms which control transcription and replication. Originally thought to propagate well only in human cells of oligodendroglial lineage, JCV has recently been shown to infect astrocytes, astrogliomas, and a neuroblastoma cell line. The data reported here describe two cell types that have been subcultured from a human neuroblastoma cell line, SK-N-SH. The SH-SY5Y subclone displays neuronal phenotypes and is not susceptible to JCV infection, while the SH-EP subclone displays glial cell phenotypes and is susceptible to infection. Binding of nuclear proteins from the permissive SH-EP cells to the nuclear factor-1 (NF-1) site in the JCV regulatory DNA sequences results in a gel shift pattern that is different from the nonpermissive SH-SY5Y cell proteins. Northern analysis of mRNA for the four classes of NF-1 proteins showed a predominance of the NF-1/X class in SH-EP cells similar to the highly permissive human fetal glial cells. Very low levels of mRNA for NF-1/X were seen in the nonpermissive SH-SY5Y cells, similar to that seen for the nonpermissive HeLa cells. Several other cell lines tested that were permissive for JCV infection also showed synthesis of the NF-1/X class of proteins. SH-EP cells represent a cell line in a glial cell lineage which is susceptible to JCV multiplication. These cells may be a useful cell culture system for the investigation of DNA binding factors which correlates with viral susceptibility.

The effects of the varicella-zoster virus (VZV) OKA vaccine strain in producing morphologic and antigenic changes in dissociated cultures of human fetal brain was investigated. Cultures containing 80% glial acidic fibrillary protein (GFAP), GFAP+ (positive) astrocytes and 20% GFAP- (negative) fibroblastic-like cells were infected with cell-free VZV OKA at a multiplicity of infection of 0.1 plaque-forming units per cell. Cytopathic effects and significant viral antigen labeling with antibodies against VZV gpl and immediate-early (IE) protein 62 were first detected 6 to 7 days postinfection. Several observations indicated that astrocyte GFAP expression was altered and diminished as a result of VZV infection itself, thereby raising doubts about the utility of combining cell markers and viral antigenic labeling in assessing the susceptibility of neural cell types to viral infection. The down-regulation of GFAP expression by VZV appears to be mediated by early rather than late events in the viral replication cycle and may not be the result of virally induced global shut-off of host cell protein synthesis. Similar observations were made using VZV Ellen, a multipassaged, nonvaccine strain. These observations have potential in vivo implications related to histologic analysis of VZV-infected tissues and disease pathogenesis.

Human astrocytes can be infected with HIV-1 both in vivo and in vitro. The amount of HIV-1 p24 structural protein production is low in comparison to that of the macrophage. Several weeks following infection or transfection, however, cocultivation with uninfected lymphocytes or stimulation with the cytokines TNF-alpha and IL 1-beta will increase viral production from this cell type. In the present study we demonstrate that phorbol 12-myristate 13-acetate (PMA) also increases HIV-1 p24 production from the primary human astrocyte. Using electrophoretic mobility shift assay (EMSA) in combination with supershift studies using specific antibodies, we demonstrate that PMA, like TNF-alpha, increases the p50/p65 form of NF-kB. Furthermore we demonstrate that the protein kinase inhibitor H7 inhibits PMA- and TNF-alpha-associated increases in HIV-1 expression at a time when it has little to no inhibitory effect on the associated increases in p50/p65 NF-kB. Thus, unless p50/p65 NF-kB or its binding is affected by H7 in a manner that cannot be resolved by EMSA, an increase in this form of NF-kB is not always sufficient to increase HIV-1 expression from the astrocyte.

The human polyomavirus, JCV, is the causative agent of the central nervous system demyelinating disease progressive multifocal leukoencephalopathy (PML). The principal target of JCV infection in the central nervous system (CNS) is the myelinating oligodendrocyte. However, the site of JCV multiplication outside of the CNS and the mechanism by which virus gains access to the brain are not known. Recently, JCV infected B-lymphocytes have been demonstrated in PML patients in several lymphoid organs, in circulating peripheral lymphocytes, and in brain, suggesting a possible role of B-lymphocytes in the dissemination of virus to the brain. The experiments reported here were undertaken to understand more about the interactions of JCV with human B-lymphocytes. The data show that JCV is able to multiply in either Epstein-Barr virus transformed (EBV) or EBV negative human B cell lines resulting in production of infectious, progeny virions. In addition, nuclear proteins extracted from these B cells bind to similar nucleotides within the JCV regulatory region that are bound by nuclear proteins extracted from human fetal glial cells, the most susceptible host and principal target cell for JCV infection in vitro. It is not known, however, whether these DNA binding proteins from susceptible B cells and glial cells are similar.

Human fetal cell cultures enriched for astrocytes, Schwann cells, or dorsal root ganglia neurons were infected with cell-free varicella-zoster virus (VZV), and the course of these infections was compared with that in fetal fibroblasts. Virus replication was detected in each neural cell type as early as 10-16 hr postinfection. Permissiveness of each cell type was confirmed by electron microscopy. However, the kinetics of virus spread varied between the neural cell types. Moreover, the accumulation, progression, and localization of VZV putative immediate early (IE), early (E), and late proteins was neural cell-type specific. VZV replication was slower in Schwann cells and neurons than in astrocytes. In Schwann cells and neurons VZV E proteins could be detected before IE proteins, a reversal of the order of accumulation noted with astrocytes and fibroblasts. There was also relatively more VZV IE protein in the perinuclear cytoplasm of Schwann cells and neurons, suggesting a delay in the transport of this antigen to its nuclear site. The permissiveness of non-neuronal cell types suggests that they could play a role in the pathogenesis of herpes zoster.

A chimeric polyomavirus genome was constructed by inserting the 72- and the 21-bp repeats of simian virus 40 (SV40) into the JC virus (JCV) regulatory region on the late side of the JCV 98-bp repeats. Although the chimeric polyomavirus DNA was able to replicate very well in human fetal glial cells, deletions were found in sequences of the regulatory region. DNA sequence analysis of a selected clone indicated a 294-bp deletion from the original construction that retained the sequences for the JCV replication origin, 78 bp of one 98-bp repeat, 33 bp of one SV40 72-bp repeat, and one intact 72-bp repeat. Of significant interest was that this genome demonstrated an extended species and cell-type host range, producing infectious virus in human fetal brain and embryonic kidney as well as in rhesus monkey fetal and adult glial cells. However, the species host range was not extended beyond primate cells since the chimeric polyomavirus was unable to multiply in rodent glial cells. Analysis of the viral RNA transcripts from either kidney or glial cells indicated that the major start sites for early RNA mapped within the JCV sequences of the regulatory region and major start sites for late RNA mapped within the JCV and SV40 sequences. This extension of the JC virus host range was most likely attributable to changes in the regulatory region and not the viral T protein since a recombinant DNA clone which placed the coding sequences for the wild-type JCV T protein subject to regulation by the deleted chimeric regulatory region showed a similar extension of host range.

JCV induces glioblastomas in owl monkeys 18-24 months or longer following intracranial inoculation (W. London, S. Houff, D. Madden, D. Fuccillo, M. Gravell, W. Wallen, A. Palmer, J. Sever, B. Padgett, D. Walker, G. Zu Rhein, and T. Ohashi, 1978, Science 201, 1246-1248). Cells from one brain tumor, owl monkey 26, were successfully established in culture and analyzed for phenotypic characteristics generally associated with persistence and expression of the papovavirus early region of its genome. Owl monkey 26 cells demonstrated nuclear JCV T protein detected by either SV40 hamster tumor sera or PAb 108, a monoclonal antibody to SV40 T protein. However, the JCV T protein was not detected in a complex with the host cell p53 protein as judged by immunoprecipitation using PAb 122, a monoclonal antibody directed to the mammalian p53 cellular protein. These cells also demonstrated increased plasminogen activator secretion and actin cable disorganization properties not before reported for JCV-induced tumor or transformed cells. Of the primate papovaviruses, JCV is unique in its ability to induce brain tumors in these primates. JCV early gene expression can be shown to persist in brain tumor cells once established in culture and correlates with cell phenotypes typical of papovavirus malignant transformation even though the time between virus inoculation and tumor development is usually several years.

We have examined the synthesis of the T protein of the human polyomavirus, JCV, during productive infection in primary cultures of human fetal glial and kidney cells. Immunoprecipitation of protein extracts from virus infected cells revealed that the JCV large T protein from both the prototype Mad and HEK adapted strains migrated as a 94-kDa protein in denaturing polyacrylamide gels. Resolution of the JCV T protein in brain cells could best be achieved following alkylation of the immunoprecipitated proteins prior to gel electrophoresis. The small t protein of either strain of JCV, however, could not be detected. In comparative experiments, the large T protein of the simian polyomavirus, SV40, was also identified as a 94-kDa protein in immortalized human fetal glial and kidney cultures. There were also protein complexes between p53 and SV40 T protein in the human glial and kidney cell lines. No evidence for a similar protein complex could be detected in JC virus infected human fetal brain or kidney cells.