Simian immunodeficiency virus from rhesus macaques (SIVmac) is a close genetic and biological relative of HIV-1. This relationship makes SIV infection of monkeys one of the most significant animal models for the study of human AIDS. The broad goal of the experiments outlined in this proposal is to understand the role that TAR RNA splicing plays in the life cycle of SIVmac. These experiments are part of a long term effort to define and evaluate the contribution that different aspects of viral gene regulation make to the pathobiology of the AIDS viruses. Three lines of evidence indicate that TAR splicing is a pivotal regulatory event in the SIV life cycle. First, the proportion of viral MRNAS containing a spliced TAR decreases throughout the course of infection. Second, viruses containing mutations which prevent TAR splicing display significantly delayed kinetics of virus production. Third, MRNAS containing a spliced TAR leader sequence are more efficiently translated than similar MRNAS containing an unspliced TAR leader.
Specific Aim 1 is to determine the role of TAR splicing in the SIVmac life cycle. Viruses containing mutations that either inhibit or enhance TAR splicing will be introduced into cells in parallel with a wild-type virus. The levels and patterns of virus replication and gene expression will be monitored by examining virion production and the expression of viral RNA and protein. In addition, the influence of the physiological state of the cell on TAR splicing will be examined in cytokine and phorbol ester treated cells.
Specific Aim 2 is to determine whether the differences in translational competence between spliced and unspliced TAR-containing MRNAS is mediated by a cis and/or trans acting mechanism. Further studies will be conducted to determine whether TAR splicing also effects other levels of virus gene expression, including tat-mediated transcription, MRNA stability and nuclear export.
Specific Aim 3 is to determine whether the level of spliced TAR RNA is regulated by viral or cellular gene products. A reporter gene containing the TAR intron and all of the cis-acting sequences necessary for the regulated accumulation of the spliced MRNAS will be co-transfected into cells along with plasmids expressing various SIVmac proteins and the levels of spliced and unspliced MRNAS will be compared.
