Spontaneous and activation-induced T-cell apoptosis have been well-documented in PBMC cultures of HIV-infected individuals and have been proposed to be involved in both the functional T cell defects observed even at early stages of the disease and the later depletion of T lymphocytes. However, the biochemical mechanisms involved in these cellular defects have not been fully elucidated. Growth factor bound protein 2 (Grb2) is an adaptor protein that is known to participate in a variety of signal transduction pathways, including the MAPK pathway and T-cell activation. We have recently demonstrated that Grb3-3, an isoform of Grb2 that is normally not expressed in healthy lymphocytes, is upregulated in CD4+ T-cells infected with HIV-1. Importantly, an analysis of the HIV-1 gene products responsible for Grb3-3 up-regulation have indicated that Tat and Nef, both of which have been implicated in modulating T-cell function and causing apoptosis, can independently induce expression of Grb3-3. We found that Grb3-3 activates NFATc activity and in doing so potentiates HIV-1 LTR promoter activity following T-cell receptor stimulation. This in turn aids in establishing an intracellular environment that can optimally support HIV-1 replication. We also found that Grb3-3 expression is coincident with HIV-1-infected CD4+ T-cells undergoing apoptosis and that Grb3-3 can induce apoptosis in T-cells. In this proposal, we will use the massively parallel signature sequencing (MPSS) technology to perform an analysis of differentially expressed mRNAs from different populations of T-cells. Specifically, we will perform global transcriptional analysis of in vitro infected human peripheral blood CD4+ T-cells at sequential steps after acute HIV-1 infection and prior to apoptosis to identify gene(s) that may be turned on or turned off during these processes. We will also analyze the gene expression profiles that result from expression of the individual HIV-1 Tat, Nef and Vpr genes and the cellular Grb3-3 gene that are known to be involved in the pathogenesis of AIDS. We will use our autoregulated tetracycline-inducible self-inactivating lentiviral vectors to express these individual genes in transduced CD4+ T-cells and to analyze the time course of their effects on cellular gene expression. The information obtained from these studies can then be used in model screening systems for the development of drugs to treat the cellular defects seen in HIV-1 infection.
| Bal, Harshawardhan P; Cheng, Jihua; Murakami, Akikazu et al. (2005) GITR overexpression on CD4+CD25+ HTLV-1 transformed cells: detection by massively parallel signature sequencing. Biochem Biophys Res Commun 332:569-84 |
