The overall goal of the proposal is to identify and elucidate the role of virus-cell co-receptor and receptor interactions in Theiler's murine encephalomyelitis virus (TMEV) persistence, leading to demyelinating disease. The evidence clearly indicates that TMEV-induced demyelination in susceptible strains of mice is driven by persistent central nervous system (CNS) infection, largely but not exclusively in macrophages, recruited into the CNS. In contrast to persistence of non-cytolytic RNA viruses in which there is host cell survival, persistence of lytic RNA viruses such as TMEV requires continual cell-to-cell spread to maintain the infection. Low-neurovirulence TMEV use sialic acid as a co-receptor for cell binding before establishing infection. Experiments in which the low-neurovirulence DA virus was adapted to growth in sialic acid-deficient cells, three amino acid substitutions in the capsid arose, and the virus no longer used sialic acid as co-receptor. The adapted virus retained acute CNS virulence, but persistence in the CNS and pathological changes of white matter inflammation and demyelination were largely abrogated. Infection of murine macrophage but not oligodendrocyte cultures with the adapted virus was also significantly reduced. These data indicate a direct role for sialic acid binding and infection of macrophages in TMEV persistence and demyelination. We hypothesize that: TMEV persists in macrophages recruited into the CNS, which in turn drives a virus- specific CD4+ Th1 T cell response and delayed-type hypersensivity (DTH), leading to recruitment of macrophages;macrophage infiltration provides a continuous supply of susceptible cells to perpetuate the infection. TMEV requires sialic acid binding to persist in the CNS, whereas TMEV high-neurovirulence GDVII virus, which does not persist, infects macrophages less efficiently because it does not bind sialic acid and macrophages lack an essential heparan sulfate (HS) co-receptor epitope. To test this hypothesis, we will: 1) Identify the TMEV protein entry receptor, using biochemical, immunological and molecular biological approaches;2) Investigate potential mechanisms by which low-neurovirulence TMEV binding to sialic acid co-receptors leads to CNS persistence and demyelinating disease;and 3) Determine whether a relative block of GDVII virus attachment and entry into murine macrophages may further hinder the ability to cause persistent infection.
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