Poster abstracts
Poster number 76 submitted by Joe Kanlong
Translational control of HIV-1 unspliced RNA is regulated at the level of transcription through an RNA structure-guided mechanism
Joseph G Kanlong (Department of Chemistry and Biochemistry, Center for RNA Biology, Center for Retrovirus Research, The Ohio State University, Columbus, OH), Zetao Cheng, Saiful Islam, Olga A. Nikolaitchik, Wei-Shau Hu (Viral Recombination Section, HIV DRP, NCI, Frederick, MD), Crystal Stackhouse, Joseph D. Puglisi, Elisabetta Viani Puglisi, (Department of Structural Biology, Stanford University School of Medicine, Stanford, CA), Vinay K. Pathak (Viral Mutation Section, HIV DRP, NCI, Frederick, MD), Michael G. Kearse (Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University, Columbus OH), Madeline Sheppard, Heewon Seo, Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for Retrovirus Research, Center for RNA Biology, The Ohio State University, Columbus OH)
Abstract:
The 9.2 kb unspliced viral RNA of Human Immunodeficiency type 1 serves a dual purpose; it functions as the viral genome, packaged into new viral particles as a dimer, and as the mRNA template for the synthesis of the essential structural proteins Gag and Gag-Pol. During transcription of HIV-1 RNA by host cell RNA polymerase II, various transcription start sites are used resulting in heterogeneous RNAs containing either one (1G), two, or three (3G) 5ʹ guanosines and a modified 5ʹ cap. Despite 3G being the predominant species in the cell, 1G is selectively packaged as genomic RNA (gRNA). We have shown previously that the 5ʹ UTR of 1G and 3G RNAs adopt distinct conformational ensembles that favor selective 1G packaging by exposing structural elements required for efficient gRNA dimerization, Gag binding, and Gag multimerization. Additionally, mutations that eliminate structural differences between the 1G and 3G 5ʹ UTR abolish selective 1G packaging. Our recent in vitro and cell-based assays demonstrated that 3G RNAs are translated more efficiently than 1G RNAs because of their distinct 5’ UTR conformational ensembles. However, in vitro assays indicated that both the 1G and 3G 5’ UTR structures are generally inhibitory to translation when compared to unstructured controls, consistent with a requirement for additional host factors in cells. While we have shown that these two species of RNA are translated with varying efficiencies, the underlying mechanisms that govern HIV-1 translation initiation are not well understood. Electrophoretic mobility shift assays and single-molecule FRET experiments revealed that both 1G and 3G RNAs are bound and unwound similarly by eukaryotic initiation factor (eIF) 4F, the heterotrimeric complex responsible for mRNA activation, and load 43S ribosomes with similar efficiencies. Future work will test the hypothesis that distinct 5ʹ UTR conformational ensembles result in altered host factor binding and ribosome scanning efficiencies.
Keywords: HIV-1, Translation, RNA structure