Translational Control of Retroviral Unspliced mRNA

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Retroviruses vary widely in their ability to cause neoplastic transformation or immunodeficiency. In addition, retroviruses are used as a backbone for constructing nonpathogenic vectors for gene transfer applications. However, in all cases, retroviruses recruit host cell proteins to achieve cytoplasmic expression of their unspliced genome-length RNA. We have identified sequences adjacent to the 5' cap of spleen necrosis virus (SNV) that facilitate Rev/Rev responsive element (RRE)-independent expression of HIV-1 unspliced reporter RNA. The RU5 region of the SNV long terminal repeat functions as a distinct position- and orientation-dependent cap-dependent translational enhancer of intron-containing HIV-1 gag RNA as well as nonviral luciferase (luc) RNA. Designated the SNV post-transcriptional control element (PCE). polysome analyses indicate that its functional mechanism is to stimulate translation initiation, although the PCE is not an internal ribosome entry sequence. Recently, we identified PCE activity in the 5' RNA terminus of two divergent retroviruses and in a cellular protooncogene mRNA. Our novel hypothesis is that 5' PCEs are a feature shared among divergent retroviruses and selected cellular mRNAs to achieve efficient translation in the face of multiple barriers to efficient cytoplasmic expression. Combined results of site-directed mutagenesis, RNA affinity chromatography and MALDI-TOF mass spectroscopy have determined redundant stem-loop motifs are necessary for PCE activity and that the structural features of the PCE present unpaired nucleotides for interaction with RNA helicase A (RHA). Knockdown of endogenous RHA by RNA silencing eliminates PCE translation stimulation and demonstrates that RHA is necessary for PCE activity. Our findings have generated the following essential questions: i) What conserved motifs necessaryfor translation stimulation are shared among retroviral PCEs? ii) What residues in RHA are necessary for interaction with the PCE and with translation factors or auxiliary proteins that mediate PCE activity? iii)What step of translation is stimulated? The overall goal of this proposal is to characterize the biochemical mechanism of PCE-RHA translational enhancement. Three integrated Specific Aims for this proposal are:1) to define conserved features in PCEs among divergent retroviruses; 2) to define the domains of RNA helicase A necessary for PCE translational enhancement; and 3) to evaluate the function of the PCE-RHA interaction in translation initiation. Our results will illuminate a unique control mechanism of eukaryotic post-transcriptional gene expression and define virus-host interactions that are important for viral replication and progression to disease. Our new fundamental knowledge of translational control will define the process by which cellular mRNAs become committed to cytoplasmic expression and produce new strategies to optimize vector systems for diverse gene transfer applications.