Abstract
BackgroundConventional 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.ResultsshRNA-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.ConclusionsThis 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.
Highlights
Conventional anti-HIV drug regimens targeting viral enzymes are plagued by the emergence of drug resistance
Tat-SF1 has previously been shown to function as an HIV-dependency factors (HDFs) in TZM-bl cells [18], we further demonstrated that the inhibitory effect on HIV-1 following RNA interference (RNAi)-mediated Tat-SF1 suppression was not a result of cellular toxicity or induction of an immune response (Figure 1E and F) and includes inhibition of infectous particle production (Figure 2C)
Focusing on the HIV-1 cofactor Tat-SF1, this study highlights the limitations associated with HDF validation in the TZM-bl reporter cell line
Summary
Conventional anti-HIV drug regimens targeting viral enzymes are plagued by the emergence of drug resistance. Host factors required by the virus for replication, socalled HIV-dependency factors (HDFs), represent attractive therapeutic targets since their coding sequences remain constant relative to the sequence variability of viral targets within a patient and across the pandemic. There have been several clinical trials showing the positive impact CCR5 deletion from CD4+ T cells has on T cell longevity, viral suppression and patient health (reviewed in [4]). This was most emphatically demonstrated by the apparent cure of the ‘Berlin patient’ [5,6,7]
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