Talk abstracts
Talk on Tuesday 09:45-10:00am submitted by Joe Kanlong
Role of transcriptional start site heterogeneity in HIV-1 genomic RNA translation
Joseph G. Kanlong (Ohio State Biochemistry Graduate Program), Zetao Cheng, Saiful Islam, Olga A. Nikolaitchik, Wei-Shau Hu (Viral Recombination Section, HIV DRP, NCI, Frederick, MD), Madeline Sheppard, Heewon Seo (Department of Chemistry and Biochemistry, Center for Retrovirus Research, Center for RNA Biology, The Ohio State University, Columbus OH), 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), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for Retrovirus Research, Center for RNA Biology, The Ohio State University, Columbus OH)
Abstract:
Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that encodes a 9.4 kb viral RNA genome. The RNA is reverse transcribed and integrated into the host genome during infection. Unspliced HIV-1 viral RNA has two primary functions: it serves as the mRNA template for the synthesis of HIV-1 Gag and Gag-Pol, and it is packaged as a dimer into new viral particles. During transcription of HIV-1 RNA by host cell RNA pol II, various transcription start sites (TSS) 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). The mechanism by which a two-nucleotide difference in a 9.4 kb RNA dictates viral RNA fate is an intriguing question. We have shown previously that the 5ʹ UTR of 1G and 3G RNAs adopt unique conformational ensembles that favor selective 1G packaging by exposing structural elements required for efficient gRNA dimerization and Gag binding. Additionally, mutations that eliminate structural differences between the 1G and 3G 5ʹ UTR, abolish selective 1G packaging. However, the effect of TSS heterogeneity on HIV-1 RNA translation is still unknown. To investigate this open question, we adapted nano-luciferase reporter assays to compare translation efficiency (TE) of RNAs encoding the 1G and 3G 5ʹ UTRs. Our data indicate that 3G RNAs are translated significantly more efficiently than 1G RNAs in vitro. Polysome profiling analysis of HIV-1 infected cell lysates showed that although both RNAs are competent for translation, 3G RNAs are translated more efficiently than 1G RNAs. In vitro and cell-based assays with mutant 5ʹ UTR reporters support the conclusion that TE differences arise from 5ʹ UTR structural differences. Thus, the host machinery translates two 99.9% identical HIV-1 RNAs with different efficiencies and the differential translation is regulated by 5′ UTR structure.
Keywords: HIV-1, Translation, RNA structure