The overall goal of the proposed research is to elucidate the molecular basis of virus-cell interactions in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease. Myelin breakdown has been shown to be immune-mediated rather than due to a cytolytic effect of the virus and virus persistence is required to perpetuate the demyelinating process. A predominant viral antigen burden resides in macrophages which we believe to be the preferential site of TMEV persistence. Virus replication in CNS macrophages is highly restricted. Characteristic of this disease are chronic elevated levels of virus-specific T cell responses directed at viral epitopes rather than host neuroantigens, at least early in the infection. A central role for virus-specific DTH mediated by major histocompatibility class (MHC) Il-restricted CD4+ Th1 T cells in demyelination has been proposed. Less is known about the role of antibody responses in TMEV infection. We plan to: (1) Identify the cellular receptor for TMEV using recently developed MAbs blocking virus infection which will be used to isolate the gene encoding the receptor from a lambdagt11 cDNA expression library made from BHK-21 cell or mouse intestinal brush border (IBBM) mRNA. Another cloning technique, CELICS, will be used as an alternative method of receptor identification. The cloned receptor gene will be expressed in a mammalian expression system, and the receptor characterized in terms of its homology with other known proteins, biologic function and distribution in mouse tissues and CNS cells. (2) Characterize TMEV-induced programmed cell death (apoptosis) in macrophage cell lines representing different states of activation/differentiation; determine a role, if any, of the bcl-2 oncogene or a bcl-2 homologue in inhibition of apoptosis in macrophages; and map this determinant within the capsid using a panel of TMEV recombinant viruses which have already been constructed. (3)Map the neutralizing immunogenic sites (nlMs) on the Theiler's virion using neutralizing mAbs (nmAb) to select neutralizing escape mutants, the RNAs of which will be sequenced to identify the mutated amino acid sequence(s); and analyze the kinetics of neutralization in vitro and the ability to protect in vivo for nmAb representing different nlMs. and (4) Finish ongoing studies of site-specific mutagenesis of selected virion surface amino acids to demonstrate a role in virus attachment to the cell receptor.
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