Translational control of HIV will be investigated in this project. HIV-1 leader RNA contains a double stem-loop structure which activates the interferon-inducible antiviral proteins, dsRNA-dependent protein kinase and 2-5A synthetase. HIV Tat Protein inhibits activation of the dsRNA- dependent protein kinase. The activity of the 2-5A and protein kinase pathways will be determined in HIV-1 infected, control and interferon-tested cells. Phosphorylation of protein synthesis factor eIF2-alpha by the dsRNA-dependent protein kinase will be determined using an immunoblotting method. Activation of the 2-5A per se and from 2-5A-mediated rRNA cleavages. The specific sequences and structures in HIV-1 leader RNA which are responsible for translational control will be determined. Natural and mutated HIV-1 leader RNA sequences will be linked to mRNA for an indicator protein. The relative contribution of HIV-1 leader sequences to cis and trans inhibition systems deficient or inhibited in one or both of the dsRNA-dependent pathways. We will establish whether the double-stem loop structure in HIV-1 leader RNA can exist in intact cells. The effects of HIV-1 leader RNA on both protein synthesis and eIF-2-alpha phosphorylation will be determined in cells which contain or lack dsRNA-dependent protein kinase. This project will extend our knowledge about the mechanisms by which HIV-1 leader RNA, Tat, and dsRNA-dependent enzymes interact to control translation during HIV-1 replication.
