Study of translation control by a RNA helicase A-responsive post-transcriptional control element in Retroviridae.

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). 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 capdependent 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 44 divergent retroviruses have evolved a convergent RNA-protein interaction to modulate translation of their highly structured mRNA. INTRODUCTION Retrovirus structural and enzymatic proteins are synthesized from mRNA transcript that contains a complex 5’ UTR that inhibits ribosome scanning [14-16,186]. The process used to circumvent this barrier to translation initiation is an issue that remains to be fully elucidated. Translation initiation is the rate-limiting step in protein synthesis and the primary target for translational control [187]. The majority of eukaryotic mRNAs use a cap-dependent mode of translation initiation in which the multicomponent eIF4F complex binds the 5’ 7mpppG cap of mRNA and is joined by the small ribosomal subunit [1,187]. Once assembled, the initiation complex scans the mRNA until an AUG or, in some cases CUG, in appropriate Kozak consensus is located. The large ribosomal subunit then joins the complex and the elongation phase begins. A 5’ UTR greater than 100 nt in length or with structural motifs with free energy of < -50 kcal/mol has been shown to block efficient ribosome scanning and significantly dampen the efficiency of translation initiation [186,188]. The UTR of retroviruses is typically greater than 200 nt in length and contain a collection of cis-acting replication motifs with free energy greater than -50 kcal/mol [20]. These motifs include the packaging signal and primer binding site, which are stem loop structures that perform essential functions during particle assembly and reverse transcription, respectively. In order to ensure translation of the viral transcript, retroviruses require a specialized translational control mechanism to overcome structural barriers to cap-dependent ribosome scanning. Two viral RNA elements that facilitate translation despite a long and highly structured 5’ UTR 45 are: 1) internal ribosome entry site (IRES); and 2) post-transcriptional control element (PCE). Both viral RNA elements operate by recruiting host effector proteins and have been shown to have a homolog in cellular genes. The IRES is an RNA structural motif originally identified in the picornaviruses encephalomyocarditis virus (EMCV) [25,189,190] and poliovirus [191], and more recently in selected cellular mRNAs including BIP [192], eIF4G [193], c-Myc [194,195], DAP5 [52], Apaf-1 [49], BAG-1 [44,45], c-IAP1 [196], FGF2 [41,43], and BCL-2 [50]. Picornaviruses lack a 5’ cap and contain a 5’ UTR ~750nt in length that is comprised of structural motifs, oligopyrimidine tracts, and cryptic AUG and CUG codons upstream of the authentic initiation codon. Features of this complex 5’ UTR serve as a recognition site for a modified pre-initiation complex that delivers the small ribosomal subunit to an appropriate initiation codon independently of cap-binding and ribosome scanning. Generally, picornaviruses encode proteins that modify the eIF4F complex and efficiently shut-down cap-dependent host translation initiation. Under such conditions, capindependent translation initiation at the viral IRES is favored [197]. Unlike picornaviruses, retroviral mRNAs are capped, polyadenylated, and in some cases lack upstream AUGs. They also generally contain AUGs in appropriate Kozak consensus [20]. These features are consistent with translation initiation by a capdependent mechanism. However, the structured replication motifs between the 5' cap and distal translation start codon of retroviral RNAs have been directly demonstrated to inhibit efficient cap-dependent ribosome scanning and impede translation initiation at the downstream open reading frame [14-16]. A growing collection of retroviruses have been identified to contain a 5’ terminal PCE that functions in conjunction with cellular protein