Poster abstracts
Poster number 134 submitted by Nathan Titkemeier
Determination 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, The Ohio State University), Michael E Hoover (Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University Medical School), Corine St.-Gelais (Center for Retroviral Research, College of Veterinary Medicine, The Ohio State University), Li Wu (Center for Retroviral Research, College of Veterinary Medicine, The Ohio State University), Michael A Freitas (Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University Medical School), Karin Musier Forsyth (Department of Chemistry and Biochemistry, 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 tRNALys3, the primer for HIV-1 reverse transcription, into HIV-1 virions. Like most 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. Preliminary data suggests the presence of cytoplasmic LysRS within the virions, and we are employing a multiple reaction monitoring MS/MS approach to definitively identify the packaged isoform. Preliminary data also suggest an increase in phosphoserine on LysRS following HIV-1 infection, but changes in the PTM profile remain to be elucidated through LC-MS and LC-MS/MS. LysRS proteolysis has been optimized to provide better sequence coverage and SILAC studies are planned to confirm changes in LysRS modifications in HIV-1 infected cells.
Keywords: HIV-1, aminoacyl-tRNA synthetase