title: Impact of sustained RNAi-mediated suppression of cellular cofactor Tat-SF1 on HIV-1 replication in CD4+ T cells.
abstract: 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.
authors.affiliation: Antiviral Gene Therapy Research Unit, Health Sciences Faculty, University of the Witwatersrand, Johannesburg, South Africa.
authors.forename: Victoria A
authors.initials: VA
authors.lastname: Green
authors.forename: Patrick
authors.initials: P
authors.lastname: Arbuthnot
authors.forename: Marc S
authors.initials: MS
authors.lastname: Weinberg
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism* / Q000821:virology*; D002460:Cell Line; D015870:Gene Expression; D005786:Gene Expression Regulation; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics*; D014779:Virus Replication
chemical_list: C101819:HTATSF1 protein, human; D034741:RNA, Small Interfering; D015534:Trans-Activators