The human imunodeficiency virus (HIV) is a member of the lentivirus subfamily, a taxonomic group of nononcogenic retroviruses that persistently infect cells of the host immune system. These are enveloped viruses that have a highly glycosylated envelope glycoprotein (env gp) that stimulates strong immune responses in the infected host. However, immune responses elicited during natural infection fail to control virus replication resulting in a persistent infection that eventually leads to chronic multisystemic disease and death. Many of the functional domains that mediate cell tropism, cytopathicity, and neutralization of virus by antibody have been mapped to the env gp of HIV. However, the properties of the lentivirus envelope that underlie ineffective anti-lentivirus immune responses are poorly understood and difficult to evaluate with HIV itself. Insight into these areas are essential to developing effective immune based strategies against HIV. Caprine arthritis encephalitis virus (CAEV) of goats and visna virus of sheep are two closely related, well-characterized lentiviruses that mirror HIV in many of their biological and structural characteristics. Infectious molecular clones of both CAEV and visna that have been completely sequenced are available for study and provide a system in which to address questions of lentivirus immunogenicity and pathogenesis in the natural host. This proposal focuses on mapping and characterizing the functional domains of the ovine/caprine lentivirus env gp. A set of truncated and mutated env gp genes will be constructed using the polymerase chain reaction with synthetic oligonucleotides as primers and the subcloned into a baculovirus expression vector. Recombinant baculovirus expressing the various forms of the env gp will be used to infect spinner flasks of insect cells to obtain milligram quantities of purified env gp. The various purified glycoprotein constructs will be compared for their ability to block in various functional assays. In years 4-5 these mapping studies will be extended by using PCR to construct infectious molecular clones that are envelope chimeras of CAEV and visna. Switching specific domains between the env genes of the two viruses will map determinants of cell tropism, antibody neutralization, immunogenicity and pathogenicity for the ovine/caprine lentiviruses in their natural host. These biological properties are mediated at least in part by env gp and differ markedly between the well characterized parental viruses, CAEV and visna virus. The candidate has limited experience in applying techniques of molecular biology to the study of disease pathogenesis. The supervisor is a recognized expert in the field of lentivirus molecular biology with a large active lab. The candidate has been provided with independent laboratory space at the Bayview Research Campus of the Johns Hopkins University, School of Medicine along with equipment and support from the Division of Comparative Medicine and the university. This combination of factors should provide an excellent environment for completing the proposed studies and greatly enhance the candidate's development as an independent researcher.
