Abstract 1153 Nuclear Localization of SARS-CoV-1 Nsp1 and Its Role in RNA Processing

The 9.2 kb unspliced viral RNA of Human Immunodeficiency Virus type 1 (HIV-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) 5 guanosines and a modified 5 cap.Despite 3G being the predominant species in the cell, 1G is selectively packaged as genomic RNA (gRNA).We have shown previously that the 5 UTR of 1G RNAs adopt distinct conformational ensembles that favor selective 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 5 UTR abolish selective 1G packaging.Using in vitro and cell-based assays, we recently demonstrated that 3G RNAs are translated more efficiently than 1G RNAs because of their distinct 5 UTR conformation.In vitro assays also 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 4F, the heterotrimeric complex responsible for mRNA activation, and load 43S ribosomes with similar efficiencies.Ongoing work is testing the hypotheses that distinct 5 UTR conformational ensembles result in altered host factor binding and ribosome scanning efficiencies.