Retroviruses rely on host RNA-binding proteins to modulate post-transcriptional control of viral gene expression. Several animal retroviruses contain a RNA structural element within their 5′ untranslated region (UTR) that is necessary for efficient viral protein synthesis (1
). Viral proteins are not required. Instead this retroviral post-transcriptional control element (PCE) requires interaction with host Dhx9/RNA helicase A (RHA) (2
). PCE was originally identified in the 5′ long terminal repeat (LTR) of avian spleen necrosis virus (3
) and subsequently in retroviruses that infect feline, bovine, catarrhine, and human hosts (4–6
). These viruses represent five genera of the Retroviridae
(alpharetrovirus, betaretrovirus, deltaretrovirus, gammaretrovirus and spumavirus). The possibility of PCE activity in the lentivirus genus was addressed in this study.
RHA recognizes functionally redundant structural features encoded by the RU5 regions of the SNV LTR (2
). Experiments with human T-cell leukemia type 1 (HTLV-1) provirus determined that RHA down-regulation reduces the polysome association of HTLV-1 gag RNA and severely attenuates virion production, indicating that RHA is an important host factor for HTLV-1 replication (5
). Reporter assays determined the HTLV-1 LTR is sufficient for PCE activity although a sufficient role for the RU5 regions remains to be evaluated. Subsequent to identification in Retroviridae
, PCE activity was identified in the complex 5′ UTR of rat and human junD (2
). RHA is necessary for efficient translation of endogenous junD and results in the reporter assay demonstrated that RHA down-regulation eliminates junD PCE activity (2
Recently RHA/Dhx9 was identified as an important host factor in HIV-1 replication in meta-analysis of genome-wide studies (8
). Previous RHA overexpression studies detected increased HIV-1 gene expression as measured by HIV-1 LTR-luciferase
reporter gene activity (9
); an effect on balanced expression of the unspliced HIV-1 gag RNA; and increased virion protein production (10
). The results suggested RHA affects HIV-1 transcription and/or post-transcriptional expression. Biochemical analysis has revealed that RHA can act as a scaffold to bridge the association of CREB-binding protein and RNA polymerase II (11
). And a role for interaction with HIV-1 RNA was invoked from Northwestern analysis detecting RHA interaction with the HIV-1 trans
-activation response element (TAR) within the R region of the HIV-1 5′ LTR (9
); TAR is the target for the essential HIV-1 Tat transcriptional trans-activator. Recently, Liang and colleagues determined that RHA interacts with HIV-1 Gag and is packaged into HIV-1 particles in an RNA-dependent manner. RHA down-regulation by siRNA decreased HIV-1 infectivity that was attributed in part to lower reverse transcriptase (RT) activity (12
). In summary, RHA appears to influence HIV-1 gene expression and possibly the process of virus assembly. A possible role for RHA in translation of the virus and possible involvement of the ATP-dependent helicase activity remains an open issue.
Given the identification of RHA-dependent PCE activity in the RU5 of several animal retroviruses, we evaluated the RU5 regions of human retroviruses, HTLV-1 and HIV-1, for PCE reporter activity. We also examined the effect of RHA down-regulation and rescue with siRNA-resistant RHA on expression of HIV-1 provirus, virion production, content and infectivity in PBMC. The results demonstrated that RU5 of HIV-1 and HTLV-1 confer orientation-dependent PCE reporter activity, and that RHA affects two steps in replication of HIV-1: (i) translation of the viral RNA and (ii) infectivity of progeny virions.