We propose a broad-based program to characterize in detail the replication of human immunodeficiency virus (HIV). Understanding of retroviral replication is key to the design of agents which can effectively intervene to prevent the devastating in vivo effects of viral replication. In this application a group of molecular and cell biologists will utilize expertise gained in other systems, including studies of avian and murine retroviral replication, oncogenesis, and gene expression, in order to achieve this goal. Specific approaches of the individual projects will be: 1. Identification of sequences required for HIV genomic packaging and development of packaging lines to deliver sequences to HIV target cells; 2. Detailed characterization of the processing of the HIV gag gene product and the role of the virally encoded protease in viral assembly; 3. Analysis of transcriptional termination in the HIV genome and the role of the tat gene product in this process; 4. Development of vectors containing antisense viral constructs for the inhibition of viral replication and the elucidation of the mechanism of antisense inhibition; 5. Molecular analysis of the sequences in cytomegalovirus (CMV) which activate HIV gene expression and determination as to whether reciprocal activation occurs; 6. Mutagenesis of the HIV genome in order to identify trans-acting dominant-negative mutants of HIV which could inhibit replication of wild-type HIV; 7. Analysis of whether HIV replication requires specific phases of the host cell cycle and which phases of HIV replication are cell-cycle dependent. These projects interdigitate in their use of common reagents and the generation of reagents and information necessary for the completion of the specific goals. Collectively, the investigators provide all of the technical expertise required for the successful attainment of the goals including cloning and sequencing, vector construction, cell culture and virus growth, protein analysis, in vitro and in vivo assay of transcription products, and cell cycle analysis.
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