Lewis rat T cells can reutilize, process, and present myelin basic protein to antigen-specific T cell lines.

MHC class-II-negative astrocytes prevented from intracellular antigen (Ag) processing induce myelin basic protein (MBP)-specific short-term T cell lines to proliferate. This process results from the ability of the T cells themselves to take up, process, and present Ag to each other. The Ag-presenting function of the T cells occurred in the absence of any conventional antigen-presenting cell (APC), was independent of their T cell receptor specificity, was sensitive to chloroquine, and was prevented by anti-class-II MHC antibody. Both native and HPLC-purified MBP were effective in stimulating T cell lines, and there was no obvious benefit in using either enzymatically digested or synthetic peptide preparations of the Ag. Furthermore, the Ag-presenting T cells could take up, reutilize, and re-present Ag adsorbed to the surface of histoincompatible astrocytes. Responding T cells activated by Ag-presenting T cells in the absence of other conventional APC were fully encephalitogenic upon transfer to syngeneic recipients. These results have relevance for understanding pathogenetic mechanisms in T cell-mediated autoimmunity in the central nervous system.

Distribution and trafficking of JHM coronavirus structural proteins and virions in primary neurons and the OBL-21 neuronal cell line.

The neurotropic murine coronavirus JHM is capable of inducing various forms of neurologic diseases, including demyelination. Neurons have been shown to act as a repository site at the early stages of the disease process (O. Sorensen and S. Dales, J. Virol. 56:434-438, 1985). JHM virus (JHMV) replication and trafficking of viral proteins and virions in cultured rat hippocampal neurons and a neuronal cell line, OBL-21, were examined, with an emphasis placed on the role of the microtubular network. We show here that JHMV spread within the central nervous system occurs transneuronally and that virus protein trafficking was dependent upon microtubules. Viral trafficking occurred asymmetrically, involving both the somatodendritic and the axonal domains. Thus coronavirus can be disseminated from neurons at either the basolateral or the apical domains. A specific interaction between antibodies derived against the microtubule-associated protein tau and JHMV nucleocapsid protein (N) was observed, which can presumably be explained by an overall amino acid similarity of 44% and an identity of 20% between proteins N and tau, with optimal alignment at the microtubule binding domain of tau. Collectively, our data suggest an important role of the microtubule network in viral protein trafficking and distribution. They also draw attention to protein sequence mimicry of a cell component by this coronavirus as one strategy for making use of the host's functions on behalf of the virus.

SJL/J resistance to mouse hepatitis virus-JHM-induced neurologic disease can be partially overcome by viral variants of S and host immunosuppression.

The basis of the resistance of SJL/J mice to various strains of mouse hepatitis virus (MHV) has been the subject of some debate, especially as it relates to the number and nature of the determinants involved. Our previous work demonstrated that resistance by primary SJL/J glial cultures may involve events subsequent to viral gene expression, possibly due to a defect in cell-to-cell spread of the infection. Since S, the virion's major spike glycoprotein, is known to facilitate the spread of infection due to its syncytiogenic properties, we decided to investigate the role of this viral structural protein in resistance by primary SJL/J glial cells. Variants possessing deletions within the S coding region were able to grow in SJL/J glial cells 10-100 times easier and fuse five-times more efficiently than wt virus. Induction of neurologic disease in SJL/J mice following intracranial inoculation with either wt JHMV or the S deletion variant, AT11f cord, was age-dependent, occurring only in animals inoculated under 4 weeks of age. Resistance in older animals to wt and variant viruses could be abrogated by immunosuppression with cyclosporin A. However, both disease incidence and viral brain titers were higher in animals receiving the JHM variant AT11f cord virus, suggesting that SJL/J resistance to neurologic disease may manifest itself through interactions between inefficient cell-to-cell spread of the infection and protective aspects of the immune response.

Infection by coronavirus JHM of rat neurons and oligodendrocyte-type-2 astrocyte lineage cells during distinct developmental stages.

Primary telencephalic cultures derived from neonatal Wistar Furth rats were able to support the growth of coronavirus JHM if a viable neuronal population was maintained. This occurred under serum-free defined, but not serum-supplemented, growth conditions. The importance of neurons in establishing infections in mixed cultures was confirmed by immunocytochemical and electron microscopic studies. Glia, although more abundant than neurons in these cultures, were less frequently infected during the initial 48 h postinoculation. The two glial lineages present in mixed telencephalic cultures were separated into type-1 astrocytes and oligodendrocyte-type-2 astrocyte (O-2A) lineage cells and individually assessed for their ability to support virus growth. Infection could not be established in type-1 astrocytes regardless of the culture conditions employed, consistent with our previous study (S. Beushausen and S. Dales, Virology 141:89-101, 1985). In contrast, infections could be initiated in selected O-2A lineage cells grown in serum-free medium. Virus multiplication was however significantly reduced by preconditioning the medium with mixed telencephalic or enriched type-1 astrocyte cultures, suggesting that intercellular interactions mediated by soluble factor(s) can influence the infectious process in O-2A lineage cells. This presumption was supported by eliciting similar effects with basic fibroblast growth factor and platelet-derived growth factor, two central nervous system cytokines known to control O-2A differentiation. The presence of these cytokines, which synergistically block O-2A cells from differentiating into oligodendrocytes was correlated with specific and reversible resistance to JHM virus (JHMV) infection. These data, combined with our finding that accelerated terminal differentiation of the oligodendrocyte phenotype confers resistance to JHMV (Beushausen and Dales, Virology, 1985), suggest that the permissiveness of O-2A cells for JHMV is restricted to a discrete developmental stage.

Regulated expression of stably transfected herpes simplex virus thymidine kinase genes in continuous cell lines expressing a temperature-sensitive mutant form of the immediate-early protein ICP4.

We stably transfected the herpes simplex virus type 1 thymidine kinase gene into a continuous cell line expressing a temperature-sensitive form of the viral immediate-early protein ICP4. In these cells, expression of the thymidine kinase gene was regulated in a temperature-sensitive manner, partially reproducing the controls that operate during a viral infection.