Inhibiting HIV-1 using RNA interference (RNAi) to target novel HIV dependency factors (HDFs)

Blondeel, Mishka Dominique
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Three separate recent publications used genome-wide RNA interference (RNAi) to screen for novel host factors that are required for HIV-1 infection and replication. This was achieved using small interfering RNAs (siRNAs) to silence the expression of ~21 000 human genes and determining the effect of each gene’s loss of function on HIV-1 replication. Collectively, several hundred genes have now been implicated as novel HIV-1 host factors (termed HIV-1 Dependency Factors, HDFs). However, differences in study design resulted in little overlap and limited interpretive value from the three published datasets. To identify novel HDFs that are potential targets for anti-HIV therapy, five putative HDFs (SPTBN1, TMED2, KIAA1012, PRDM14 and SP110) were chosen for validation. RNAi effecters (both siRNAs and expressed short hairpin RNAs) were used to silence the selected genes. Gene suppression was measured by quantitative RT-PCR assay and two candidate genes were studied further (SPTBN1 and SP110) based on efficient mRNA inhibition (over 90%). As efforts to deliver the RNAi effecters to a T-cell line were unsuccessful, the effect of this knockdown on HIV-1 replication (both early- and late-stage) was assessed in cultured TZM-bl cells, a HeLa-derived cell line that expresses HIV-1 entry receptors and an integrated luciferase reporter for HIV-1 transcriptional activity (also used in the first genome-wide RNAi screen). An initial viral challenge assay with Subtype C-enveloped pseudovirus showed a 60% decrease in TZM-bl luciferase reporter activity in cells with suppressed SPTBN1 function, while knockdown of SP110 showed no effect on reporter activity. The final experiment, using fully-replicating Subtype B virus, showed a 75% decrease in late-stage viral replication when SPTBN1 expression was suppressed. In addition, SP110 suppression was confirmed to have no effect on TZM-bl reporter activity during any stage of HIV-1 replication. In conclusion, SPTBN1, but not SP110, is required for late-stage HIV-1 replication, though these results need to be confirmed in CD4+ T-cells. The absence of several important viral accessory factors from vi the virus used in the genome-wide screen may explain these findings and emphasises the need for using physiologically representative viral and cellular models to study the viral/cellular interactome.
MSc (Med), Molecular Medicine and Haematology, Faculty of Health Sciences, University of the Witwatersrand
gene suppression, RNA, HIV-1, AIDS, HIV dependency factors