Poster abstracts

Poster number 128 submitted by Nathan Titkemeier

Characterization of the isoform and post-translational modification state of lysyl-tRNA synthetase packaged into HIV-1 particles by mass spectrometry

Nathan P. Titkemeier (Department of Chemistry and Biochemistry; Center for Retroviral Research; Center for RNA Biology; Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University), Michael E. Hoover (Department of Molecular Virology, Immunology, and Medical Genetics; Arthur G James Comprehensive Cancer Center, The Ohio State University), Corine St. Gelais, Li Wu (Center for Retroviral Research; Center for RNA Biology; Department of Veterinary Biosciences, The Ohio State University), Xiaoyan Guan (Department of Chemistry and Biochemistry, The Ohio State University), Michael A. Freitas (Department of Molecular Virology, Immunology, and Medical Genetics; Arthur G James Comprehensive Cancer Center, The Ohio State University), Karin Musier-Forsyth (Department of Chemistry and Biochemistry; Center for Retroviral Research; Center for RNA Biology, The Ohio State University)

Abstract:
Human lysyl-tRNA synthetase (LysRS) is critical for the replication of Human Immunodeficiency Virus 1 (HIV-1), as it directs the selective packaging of host tRNA^Lys3, the primer for HIV-1 reverse transcription, into HIV-1 virions. Like several other aminoacyl-tRNA synthetases (aaRSs), LysRS has been demonstrated to perform a number of non-canonical functions beyond its role in aminoacylating or “charging” amino acids onto their cognate tRNAs. In most of these cases, LysRS becomes activated to perform non-canonical functions through post-translation modifications (PTMs), which cause it to dissociate from the high molecular weight multi-synthetase complex (MSC), where it exists as a homotetramer in complex with 8 other aaRSs and 3 scaffold proteins. In addition, three forms of LysRS are produced from the same gene: the cytoplasmic isoform, an immature pre-mitochondrial isoform, and a processed mitochondrial isoform. The exact isoform(s) packaged into HIV-1 virions has been a point of debate. We are using liquid chromatography-coupled tandem mass spectrometry to identify whether the PTM profile of human LysRS is altered upon HIV-1 infection, as well as to establish the specific isoform(s) of LysRS that is/are packaged into HIV-1 virions. To date, we have identified both cytoplasmic and pre-mitochondrial isoforms within HIV virions, and we will employ a targeted MS approach to identify the presence or absence of the mature mitochondrial species. While preliminary data suggest that LysRS is post-translationally modified, changes in the PTM profile remain to be elucidated. LysRS proteolysis is being optimized to provide better sequence coverage and SILAC studies are planned to probe changes in LysRS modifications in HIV-1 infected cells.

Keywords: HIV, Aminoacyl-tRNA Synthetase, Mass Spectrometry