Poster abstracts

Poster number 35 submitted by Alice Duchon

Localization of the LysRS/tRNALys/Gag Packaging Complex During HIV-1 Assembly

Alice Duchon (Department of Chemistry and Biochemistry, Center for RNA Biology and Center for Retroviral Research, The Ohio State University, Columbus, OH 43210), Corine St. Gelais (Center for RNA Biology, Center for Retroviral Research and Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210), Christopher P. Jones (Department of Chemistry and Biochemistry, Center for RNA Biology and Center for Retroviral Research, The Ohio State University, Columbus, OH 43210), Jaisri Lingappa (Department of Global Health and Department of Medicine, University of Washington, Seattle, Washington 98102), Li Wu (Center for RNA Biology, Center for Retroviral Research and Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210), Karin Musier-Forsyth1 (Department of Chemistry and Biochemistry, Center for RNA Biology and Center for Retroviral Research, The Ohio State University, Columbus, OH 43210)

Abstract:
Aminoacyl-tRNA synthetases (aaRS) are enzymes primarily responsible for charging tRNAs with their cognate amino acid, however, most have alternative functions. A subset of higher eukaryotic aaRSs are associated with a multi-synthetase complex (MSC). Human lysyl-tRNA synthetase (LysRS) is normally a core member of the MSC, but is packaged into HIV-1 particles through interactions with the Gag polyprotein. This interaction brings host cell tRNA^Lys3, which serves as the replication primer, into virions. Disruption of this interaction, and subsequent packaging, inhibits infectivity of HIV-1, underscoring the importance of this protein in the viral lifecycle. The mechanism by which LysRS associates with HIV-1 components is unclear. During HIV-1 assembly, Gag forms a series of transient assembly intermediates, some of which contain an ATP-binding protein, ABCE1. These complexes were isolated by sucrose gradient with sedimentation coefficients ranging from 10S to 500S, and immunoprecipitated to extract ABCE1 assembly complexes. We have shown that both LysRS and tRNA^Lys3 are present in the 80S, 150S and 500S fractions. Reverse transcriptase assays are underway using samples containing tRNA^Lys3, to determine if the tRNA is annealed to genomic RNA prior to viral budding. To further examine LysRS trafficking, immunofluorescence is being used to co-localize endogenous LysRS and GFP-reporter HIV-1 in HEK293T and CD4+ HuT/CCR5 T-cells.

Keywords: LysRS, HIV, tRNA