Poster abstracts

Poster number 98 submitted by Alice Duchon

Mechanism of Human Lysyl-tRNA Synthetase/tRNALys Primer Recruitment and Packaging into HIV-1

Alice Duchon (Department of Chemistry and Biochemistry, Center for RNA Biology, Center for Retroviral Research, The Ohio State University), Nathan Titekemeier (Department of Chemistry and Biochemistry, Center for RNA Biology, Center for Retroviral Research, The Ohio State University), Corine St. Gelais (Center for RNA Biology, Center for Retroviral Research, Department of Veterinary Biosciences, The Ohio State University), Michael Freitas (Department of Molecular Virology Immunology and Medical Genetics, The Ohio State University), Li Wu (Center for RNA Biology, Center for Retroviral Research, Department of Veterinary Biosciences, The Ohio State University), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, Center for Retroviral Research, The Ohio State University)

Abstract:
The primer for reverse transcription in HIV-1, human tRNA^Lys3, is selectively packaged into virions along with tRNA^Lys1,2. Human lysyl-tRNA synthetase (LysRS), the only cellular factor known to interact specifically with all three tRNA^Lys isoacceptors, is also packaged into HIV-1. Selective packaging of tRNA^Lys depends on the ability of the tRNA to bind to LysRS and the presence of both host cell factors within virions is required for optimal viral infectivity. LysRS is normally part of a dynamic mammalian multisynthetase complex (MSC). Recent studies show that extra-cellular stimuli (e.g., interferon-γ, TNF-α) can trigger phosphorylation of LysRS, releasing it from the complex to act in non-translational pathways. Elucidating the detailed molecular mechanism for the alternative function of LysRS in HIV-1 infectivity is needed in order to develop effective therapeutics aimed at targeting this essential host cell factor. The mechanism by which the LysRS/tRNA complex is diverted from its normal function in translation and recruited into particles is unclear. Here, we show that the expression of LysRS is unaltered upon HIV-1 infection of HEK293T and CD4⁺ HuT/CCR5⁺ T cells, suggesting that the LysRS species packaged is recruited from an existing pool of LysRS. Interestingly, using immunofluorescence and confocal microscopy, we find that LysRS trafficking is altered upon HIV-1 infection. Confocal analysis and cellular fractionation studies indicate that LysRS localizes to the nucleus during HIV-1 infection. Studies with a LysRS mutant lacking a nuclear localization signal are planned, and LysRS-Gag and LysRS-genomic RNA co-localization studies are currently underway. Our studies also indicate that phosphorylation of LysRS on Ser and/or Thr residues occurs after HIV-1 infection. We hypothesize that phosphorylation results in release from the MSC, which may re-direct LysRS to assembling virions. Studies to understand the significance of these findings for HIV infectivity are in progress.

Keywords: LysRS, HIV-1, Assembly