1686–1696 Nucleic Acids Research, 2010, Vol. 38, No. 5 doi:10.1093/nar/gkp1075 Published online 9 December 2009 RNA helicase A modulates translation of HIV-1 and infectivity of progeny virions Cheryl Bolinger1, Amit Sharma1,2, Deepali Singh1, Lianbo Yu3 and Kathleen Boris-Lawrie1,2,4,* 1Department of Veterinary Biosciences, Center for Retrovirus Research and Center for RNA Biology, 2Graduate Program in Molecular Genetics and 3Center for Biostatistics and 4Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210-1093, USA Received September 2, 2009; Revised November 2, 2009; Accepted November 3, 2009 ABSTRACT 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 50 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 expres- sion 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 deter- mined 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 demon- strate 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 conserva- tion of this translational control mechanism among vertebrates, and convergent evolution of Retroviridae to utilize this host mechanism. INTRODUCTION Retroviruses rely on host RNA-binding proteins to modulate post-transcriptional control of viral gene expres- sion. Several animal retroviruses contain a RNA struc- tural element within their 50 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 inter- action with host Dhx9/RNA helicase A (RHA) (2). PCE was originally identiﬁed in the 50 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, delta- retrovirus, gammaretrovirus and spumavirus). The possi- bility 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 (2,7). 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 suﬃ- cient for PCE activity although a suﬃcient role for the RU5 regions remains to be evaluated. Subsequent to identiﬁca- tion in Retroviridae, PCE activity was identiﬁed in the complex 50 UTR of rat and human junD (2). RHA is nec- essary 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 *To whom correspondence should be addressed. Tel: +1 614 292 1392; Fax: +1 614 292 6473; Email: boris-lawrie.1@osu.edu The authors wish it to be known that, in their opinion, the ﬁrst two authors should be regarded as joint First Authors. The Author(s) 2009. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.