The disease associated with HIV-1 infection of the CNS, HIV-1 associated dementia complex (HAD), occurs in approximately 15% to 25% of infected individuals. The pathogenesis associated with HAD is due to infection of macrophage and microglial cells, and the subsequent release of cytokines and neurotoxins that serve to amplify the pathological and clinical manifestation of the disease. Although many of the features of HIV-1 infection of monocytes, macrophages and microglia are known, there are still important gaps in our knowledge. One of the most important areas that has been neglected is the process by which HIV-1 selects the tRNA primer required for initiation of reverse transcription. All HIV-1 exclusively use tRNA Lys,3 to initiate reverse transcription. The mechanism for the selection of tRNA Lys,3 by HIV-1 is not completely understood. A genetic element consisting of a RNA stem loop has been found upstream of the primer binding site (PBS) in the U5 region that can dramatically influence the selection of the tRNA and virus replication in continuous and primary T-cells. The experiments in this proposal will focus on one of the key steps for the production of infectious HIV-1, the selection process for tRNA Lys,3. For these studies, we will compare the replication in monocytes, macrophages and microglia of HIV-1 with mutated U5-PBS, and determine whether the production of virus from these cell types correlates with the elaboration of known neuroimmunomodulatory molecules (TNF-alpha, IL-1beta and oncostatin-M (OSM)) and eicosanoids (PGE2) that have been shown to be neurotoxic. The following Specific Aims are proposed:
Specific Aim 1 : To determine the importance of the U5-PBS for HIV-1 replication and elaboration of neurotoxic proteins from monocytes, macrophages and microglia. Viruses will be constructed that contain mutations in the U5-PBS and will be assayed for replication in monocytes, macrophages and microglia. The capacity of the wild type virus and mutants to elicit known cytokines and molecules that are neurotoxic will be compared.
Specific Aim 2 : To determine if intracellular availability of tRNA Lys,3 in monocytes, macrophages and microglia correlates with HIV-1 replication. Although HIV-1 is dependent upon tRNA Lys,3 as a primer for reverse transcription, HIV-1 and cellular translation require tRNA Lys,3 for protein synthesis. Understanding how HIV-1 has evolved to balance these dual needs for tRNA Lys,3 will provide insights in the control of replication and translation. We will determine the levels of free and aminoacylated tRNA in monocytes, macrophages and microglia pre and post infection with HIV-1. We will also assess whether HIV-1 proteins Tat and Vpr can modulate the intracellular expression and location of tRNA Lys,3 that will have impact on the production of infectious virus. The results of our studies will provide us with a foundation to understand how genetic elements of HIV-1 (U5- PBS) have evolved to effectively select tRNA Lys,3 in monocytes, macrophages and microglia, and how this correlates with the elaboration of neurotoxic molecules from these cells that are an important indication for the severity of HAD.
