RNA helicase A modulates translation of HIV-1 and infectivity of progeny virions

Retroviruses rely on host RNA-binding proteins to modulate various steps in their replication. Previously several animal retroviruses were determined to mediate Dhx9/RNA helicase A (RHA) interaction with a 5′ terminal post-transcriptional control element (PCE) for efficient translation. Herein PCE reporter assays determined HTLV-1 and HIV-1 RU5 confer orientation-dependent PCE activity. The effect of Dhx9/RHA down-regulation and rescue with siRNA-resistant RHA on expression of HIV-1NL4–3 provirus determined that RHA is necessary for efficient HIV-1 RNA translation and requires ATPase-dependent helicase function. Quantitative analysis determined HIV-1 RNA steady-state and cytoplasmic accumulation were not reduced; rather the translational activity of viral RNA was reduced. Western blotting determined that RHA-deficient virions assemble with Lys-tRNA synthetase, exhibit processed reverse transcriptase and contain similar level of viral RNA, but they are poorly infectious on primary lymphocytes and HeLa cells. The results demonstrate RHA is an important host factor within the virus-producer cell and within the viral particle. The identification of RHA-dependent PCE activity in cellular junD RNA and in six of seven genera of Retroviridae suggests conservation of this translational control mechanism among vertebrates, and convergent evolution of Retroviridae to utilize this host mechanism

RNA helicase A interacts with divergent lymphotropic retroviruses and promotes translation of human T-cell leukemia virus type 1

The 5′ untranslated region (UTR) of retroviruses contain structured replication motifs that impose barriers to efficient ribosome scanning. Two RNA structural motifs that facilitate efficient translation initiation despite a complex 5′ UTR are internal ribosome entry site (IRES) and 5′ proximal post-transcriptional control element (PCE). Here, stringent RNA and protein analyses determined the 5′ UTR of spleen necrosis virus (SNV), reticuloendotheliosis virus A (REV-A) and human T-cell leukemia virus type 1 (HTLV-1) exhibit PCE activity, but not IRES activity. Assessment of SNV translation initiation in the natural context of the provirus determined that SNV is reliant on a cap-dependent initiation mechanism. Experiments with siRNAs identified that REV-A and HTLV-1 PCE modulate post-transcriptional gene expression through interaction with host RNA helicase A (RHA). Analysis of hybrid SNV/HTLV-1 proviruses determined SNV PCE facilitates Rex/Rex responsive element-independent Gag production and interaction with RHA is necessary. Ribosomal profile analyses determined that RHA is necessary for polysome association of HTLV-1 gag and provide direct evidence that RHA is necessary for efficient HTLV-1 replication. We conclude that PCE/RHA is an important translation regulatory axis of multiple lymphotropic retroviruses. We speculate divergent retroviruses have evolved a convergent RNA–protein interaction to modulate translation of their highly structured mRNA

RNA transfection assays indicate that HTLV-1 and REV-A 5′ UTR sequences lack IRES activity

<p><b>Copyright information:</b></p><p>Taken from "RNA helicase A interacts with divergent lymphotropic retroviruses and promotes translation of human T-cell leukemia virus type 1"</p><p></p><p>Nucleic Acids Research 2007;35(8):2629-2642.</p><p>Published online 10 Apr 2007</p><p>PMCID:PMC1885656.</p><p>© 2007 The Author(s)</p> Diagram of the synthetic bicistronic transcripts that were transcribed , capped, polyadenylated and transfected into HeLa cells. Labels are as described in . Northern blot with F-luc probe verified the homogeneous transcript populations. Dual Luciferase assays were performed on total cellular protein at 20 h post-transfection. Box plot analysis of the five independent experiments summarizes range of F-Luc activity with mean indicated by the internal line and extending lines connect box to extreme values. Tukey method was used to measure the significance level of each group relative to pNoIRESfs. Asterisk indicates -value that is statistically different from pNoIRESfs group

RNA helicase A is necessary for robust REV-A PCE activity and HTLV-1 Gag production

<p><b>Copyright information:</b></p><p>Taken from "RNA helicase A interacts with divergent lymphotropic retroviruses and promotes translation of human T-cell leukemia virus type 1"</p><p></p><p>Nucleic Acids Research 2007;35(8):2629-2642.</p><p>Published online 10 Apr 2007</p><p>PMCID:PMC1885656.</p><p>© 2007 The Author(s)</p> COS cells were transfected with siRNAs targeting RHA or with scrambled sequences (Sc), incubated for 72 h and co-transfected with the siRNAs and indicated PCE reporter plasmid or HTLV-1 provirus. After 48 h, total cellular protein was harvested and subjected to immunoblot with antiserum to RHA to assess downregulation, Gapdh antiserum to control for sample loading, and F-luc to control for non-specific effects of siRNAs. () Measurements of PCE activity by Gag ELISA on the total cellular protein are summarized below immunoblot. The detection limit of the assay was (25 pg/ml). Results are representative of three independent experiments. () Lysates from three independent transfection assays were used for immunoblot for RHA and Gapdh. Results of HTLV Gag ELISA on cell-associated or cell-free protein preparations are summarized below immunoblot

Enhanced expression of reporter RNA by REV-A RU5 is not due to altered cytoplasmic accumulation

<p><b>Copyright information:</b></p><p>Taken from "RNA helicase A interacts with divergent lymphotropic retroviruses and promotes translation of human T-cell leukemia virus type 1"</p><p></p><p>Nucleic Acids Research 2007;35(8):2629-2642.</p><p>Published online 10 Apr 2007</p><p>PMCID:PMC1885656.</p><p>© 2007 The Author(s)</p> () Nucleoplasm and cytoplasm were effectively isolated for analysis of mRNA cytoplasmic accumulation. Here, 293 cells were transiently transfected with indicated REV-A or SNV PCE reporter plasmids for 48 h. Nucleoplasm and cytoplasm were isolated and aliquots were subjected to immunoblot with antiserum to nuclear protein histone H1 and cytoplasmic protein β-tubulin, and β-actin loading control. () Summary table of results of Gag ELISA and RNA quantification. 293 cells transfected with indicated reporter plasmids were seperated into nuculear and cytoplasmic fractions. p(A) + RNA was isolated from each fractions, subjected to reverse transcption and real-time PCR with gag and β-actin primers. Values represent gag copy number per nanogram standarized to β-actin copy number. Ratio of Gag protein production determined by ELISA relative to cytoplasmic gag RNA level