Talk abstracts
Talk on Friday 02:00-02:15pm submitted by Joe Kanlong
The role of transcriptional start site heterogeneity in the translation of HIV-1 genomic RNA
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), 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), Christina R. Budding, Heewon Seo, Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, Center for Retrovirus Research, 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. Full-length 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 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 adopts 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 transcription start site heterogeneity on HIV-1 RNA translation is still unknown. To investigate this open question, we adapted in vitro nano-luciferase reporter assays to compare translation efficiency (TE) of the 1G and 3G 5ʹ UTRs. Our data indicate that 3G RNAs are translated significantly more efficiently. Assays with mutant 5ʹ UTR reporters indicate that TE differences arise from 5ʹ UTR structural differences, rather than differences in 5ʹ cap recognition. Sucrose gradient fractionation of HIV-infected cells and analysis by next-generation sequencing-based 5ʹ RACE showed that both 1G and 3G co-sediment with monosomes and polysomes, but that 3G RNAs co-sediment predominantly with polysomes, which is consistent with 3G RNAs being translated more efficiently. Polysome analysis of 5ʹ UTR structural mutants that abolish selective 1G packaging support the conclusion that differences in 1G and 3G translation are a result of 5ʹ UTR structural differences. New insights into how RNA structure governs HIV-1 RNA translation suggest a complex host polymerase-dependent mechanism of retroviral replication.
Keywords: HIV-1, Translation, RNA structure