Impact of sustained RNAi-mediated suppression of cellular cofactor Tat-SF1 on HIV-1 replication in CD4+ T cells
Antiviral Gene Therapy Research Unit, Health Sciences Faculty, University of the Witwatersrand, Johannesburg, South Africa; Department of Molecular and Experimental Medicine, Scripps Research Institute, San Diego, CA, United States
Background: Conventional anti-HIV drug regimens targeting viral enzymes are plagued by the emergence of drug resistance. There is interest in targeting HIV-dependency factors (HDFs), host proteins that the virus requires for replication, as drugs targeting their function may prove protective. Reporter cell lines provide a rapid and convenient method of identifying putative HDFs, but this approach may lead to misleading results and a failure to detect subtle detrimental effects on cells that result from HDF suppression. Thus, alternative methods for HDF validation are required. Cellular Tat-SF1 has long been ascribed a cofactor role in Tat-dependent transactivation of viral transcription elongation. Here we employ sustained RNAi-mediated suppression of Tat-SF1 to validate its requirement for HIV-1 replication in a CD4+ T cell-derived line and its potential as a therapeutic target. Results: shRNA-mediated suppression of Tat-SF1 reduced HIV-1 replication and infectious particle production from TZM-bl reporter cells. This effect was not a result of increased apoptosis, loss of cell viability or an immune response. To validate its requirement for HIV-1 replication in a more relevant cell line, CD4+ SupT1 cell populations were generated that stably expressed shRNAs. HIV-1 replication was significantly reduced for two weeks (∼65%) in cells with depleted Tat-SF1, although the inhibition of viral replication was moderate when compared to SupT1 cells expressing a shRNA targeting the integration cofactor LEDGF/p75. Tat-SF1 suppression was attenuated over time, resulting from decreased shRNA guide strand expression, suggesting that there is a selective pressure to restore Tat-SF1 levels. Conclusions: This study validates Tat-SF1 as an HDF in CD4+ T cell-derived SupT1 cells. However, our findings also suggest that Tat-SF1 is not a critical cofactor required for virus replication and its suppression may affect cell growth. Therefore, this study demonstrates the importance of examining HIV-1 replication kinetics and cytotoxicity in cells with sustained HDF suppression to validate their therapeutic potential as targets. © 2012 Green et al.; licensee BioMed Central Ltd.
lens epithelium derived growth factor; protein; protein p75; SF1 protein; short hairpin RNA; transactivator protein; unclassified drug; article; CD4+ T lymphocyte; cell growth; cell line; human; human cell; Human immunodeficiency virus 1; nucleotide sequence; protein depletion; protein expression; protein function; RNA interference; T lymphocyte subpopulation; virus inhibition; virus replication; CD4-Positive T-Lymphocytes; Cell Line; Gene Expression; Gene Expression Regulation; HIV-1; Humans; RNA Interference; RNA, Small Interfering; Trans-Activators; Virus Replication; Human immunodeficiency virus 1