The goal of the research in this project is to construct and analyze replication competent strains of rhesus monkey rhadinovirus (RRV) that express SIV antigens at high levels. RRV is a naturally-occurring gamma herpesvirus of rhesus monkeys that can be used for experimental infection of RRV-negative animals. RRV persists in a non-pathogenic fashion for the lifespan of infected monkeys and its growth properties in culture will allow ready manipulation of the viral genome. Our discovery of this virus provides an ideal system to investigate the ability of herpesvirus recombinants to provide vaccine protection against pathogenic SIV. In addition, our recent determination of the RRV genomic DNA sequence will greatly facilitate the genetic manipulations needed for the recombinant constructions. Initial research will focus on the development of procedures for construction of deletion mutant and recombinant RRV. RRV with deletions in targeted genes will be analyzed for growth properties in permissive cells and for persistence in B cells in culture. Replication- competent strains of RRV will be constructed in which RRV genes are replaced by SIV env or gag-pol expressing cassettes. We will compare constitutive SV40 promoter versus endogenous herpesvirus promoter, rev-dependent versus rev-independent modes of expression, and different insertion sites in the RRV genome for level of expression of SIV antigens during permissive infection of rhesus monkey fibroblasts and during persistent infection of rhesus monkey B cells. Well-characterized, recombinant RRV strains expressing SIV antigens derived from these studies will be sued for vaccine testing in Project III.
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