For most, if not all, retroviruses, successful completion of the life cycle requires that the infected host cell actively traverse the cell cycle. Cells blocked in GO don not synthesize viral proteins or produce high levels of extracellular virus until the cell cycle is reinitiated. The precise events in the retroviral life cycle which are sensitive to the growth state of the host are not yet clear. Most available evidence for avian sarcoma virus (ASV) and other classic retrovirus systems indicates that at least synthesis of the DNA provirus and integration of proviral DNA into the cell genome are cell cycle dependent. In contrast, one lentivirus, visna, may not require proviral integration for viral RNA synthesis and productive infection. For the human immunodeficiency virus (HIV), cellular tropism is determined by the affinity of the major virus envelope glycoprotein, gp120, for the CD4 cell surface molecule. However, HIV replicates selectively in activated, proliferating T helper cells. In this case, host cell activation and DNA synthesis may be required to establish infection or, alternatively, they may serve to amplify production of progeny virus. In either case the operant host cell co-factors have yet to be defined. The objectives of this proposal are a) to determine which steps, if any, of HIV infection/replication are restricted to a particular phase of cell cycle and b) to identify the specific host elements involved. Normal CD4+ T cells synchronized in early stages of activation will be compared to GO and S phase cells for their ability to support proviral DNA synthesis, integration, and productive infection. In parallel we will establish a cell-free in vitro system for monitoring HIV integration into a standard target sequence. We will use this assay to identify host cell trans-acting factors which foster integration and to ask whether the replicative state of the target sequence determines integration efficiency. Proviral integration is one step in the virus life cycle which has no obvious counterpart in host cell biology. Consequently, it may be an ideal target for anti-viral drug therapy. Our experiments are designed to identify the integration requirements of HIV.
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