The aims of this project are to study the macromolecular regulatory steps during the replication of vesicular stomatitis virus (VSV) and to discover underlying mechanisms by which viruses cause disease, with the hope that novel counter measures will be discovered for the better control of viral diseases. Molecular biochemical techniques will be used throughout. This will be coupled with genetic and biological approaches. The glycoprotein G of VSV will be studied in relation to its natural cleavage products Gs and anchor. Their functions in vitro and as immunogens in mice will be delineated. Virions, containing only anchors, will be tested for immune enhancement using non- neutralizing antibodies and macrophages. The point of cleavage and the overall structure of Gs will be determined by terminal amino acid analysis and, eventually, X-ray crystallography. Also, how efficiently VSV donates its G to HIV during coinfection of H9 cells with RF-HIV will be assessed. Pseudotypes of HIV (VSV) will be characterized by their susceptibility to specific neutralizing antibodies and by their expanded host range. The role of defective interfering (DI) particles in eliciting or modulating immunogenic responses will be studied in mice and in reconstituted immune cell in vitro. Preliminary data show that DI particles differ from standard infectious VSV in their interactions with T lymphocytes. VSV mRNA's will be utilized to detect cytoplasmic regulatory controls. Cloned cDNA's will be engineered to contain mutations, deletions or extra sequences. mRNA made from these cDNA's will be tested in vitro or in vivo for translational efficiency, ribosome binding and dissociation, mRNA stability, and targeting to membrane organelles. Initially, engineered sequences will be made in the 5' and 3' non-coding leader regions Then nonessential coding regions and regions containing other open reading frames will be altered. If sequences in the mRNA are found to respond to cytoplasmic regulation then those sequences will be used in binding assays to detect the proteins or ribonucleoproteins that may be responsible. Also, peptides made from these minor open reading frames will be used to make antibody for the purpose of determining whether these products exist during vsv infections.
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