The noncytolytic CD8+ T lymphocyte response to HIV infection involves inhibition of virus replication around the time of early gene expression in the host cell. The precise mechanism(s) and the target(s) of suppression are unknown. To better understand this cellular immune response to HIV infection, we propose to perform detailed mapping of how HIV-1 gene expression is inhibited and identify the viral genetic elements responsible for this process.
The specific aims are: 1.) Identify the stage of the HIV life cycle that is inhibited 2.) Identify which viral genetic elements are involved in the sensitivity to this activity. Our kinetic analyses of HIV suppression in primary CD4+ T lymphocytes by CD8+ T lymphocytes revealed that inhibition occurred after reverse transcription, but before (or during) the time of early viral gene expression. Here, we will more precisely define this suppression by examining the different stages of the HIV life cycle during this time frame including nuclear transport, integration, transcription, elongation, viral mRNA expression, and viral protein expression. To dovetail with these approaches, the genetic elements of the virus that are responsible for sensitivity to CD8+ T cell mediated suppression will be examined by deriving suppression-resistant viruses in vitro. The contribution of mutations in the resistant viruses will be confirmed using chimeric constructs and site directed mutagenesis to fully map and characterize the viral regions involved in sensitivity. These studies will further our understanding of this potent means of virologic control and promote the development of this host cellular immune response in future therapeutic and vaccination strategies.
