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

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 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.

Features of Double-stranded RNA-binding Domains of RNA Helicase A Are Necessary for Selective Recognition and Translation of Complex mRNAs

“…Conversely, mutations of its conserved lysine residues eliminated interaction with PCE RNA in cells and did not block translation of PCE RNA. Our results indicate that N-term basic residues of RHA are necessary to engage the RNA structure and to deliver the ATP-dependent helicase activity that is necessary for translation of PCE mRNA (3). This N-term interaction is selective for functional PCE because the binding affinity for nonfunctional PCE (mutAC) is reduced by a factor of 2-3, and binding to negative control dsRNAs is not detectable.…”

“…PCE activity is attributable to two functionally redundant stem-loop structures (A and C) (2,21). Mutations that disrupt structural features of structures A and C (referred to as mutAC) in spleen necrosis virus (SNV), an avian retrovirus, eliminate translation activity and detectable interaction with epitope-tagged RHA (FLAG-RHA) in cells, whereas compensatory mutations restore translation activity and interaction with FLAG-RHA (1)(2)(3)24). These observations provide a framework to determine the residues of RHA necessary for functionally relevant interaction with PCE.…”

“…RHA is necessary for the translation of the junD proto-oncogene and the retroviruses that infect a variety of metazoan host cells (1-3, 21, 22). RHA interacts with structural features of their complex 5Ј-UTR and facilitates polyribosome loading and productive translation (1)(2)(3). JunD is an AP1 transcription factor that is necessary for healthy cell growth and the expression of which is stringently controlled at the transcriptional and post-transcriptional levels (23).…”

“…Site-directed mutagenesis and reporter assays have determined structural features of the complex 5Ј-UTR that are necessary for RHA interaction and translation activity (1)(2)(3)24). RHA-dependent translation activity is conferred by orientation-dependent activity of the ϳ150-nucleotide 5Ј terminus, which is designated the post-transcriptional control element (PCE) (13,14).…”

Translation initiation of the HIV-1 mRNA

“…55,[58][59][60] In this process, these enzymes are delivered to the 5 0 end of the mRNA either by binding to a specific post-transcriptional element (PCE) in the case of RHA, as part of the eIF4F complex for Ded1, or together with the 40S ribosomal subunit for DHX29. [60][61][62] In the case of HIV-1, BorisLawrie and colleagues have addressed the role of DHX9/RHA in viral translation and they showed that the latter was required to promote RNP remodeling through the 5 0 UTR to ensure ribosome progression. 61 This occurs by the initial binding of RHA to the PCE element found in the HIV-1 5…”