Mapping interactions between the HIV-1 genome and human lysyl-tRNA synthetase
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Date
2015-03-25
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Abstract
Human immunodeficiency virus type-1 (HIV-1) is a retrovirus with a complex life cycle that is the known cause of acquired immune deficiency syndrome (AIDS). As of 2013, approximately 35 million people were living with HIV-1. Although combination drug therapy has been successful in prolonging the life of AIDS patients, HIV-1 is a highly mutagenic virus and readily develops drug resistance. Thus, there is a continued need to develop new therapeutics. The HIV-1 RNA genome must be reverse transcribed into DNA prior to integration into the host genome. Our efforts are focused on the highly-conserved 5’ untranslated region (5’ UTR) of the HIV-1 genome: a highly-structured RNA segment that regulates much of the viral lifecycle. This region includes the primer binding site, which is the site of reverse transcription initiation. HIV-1 uses a host cell tRNALys to prime reverse transcription and our lab has previously shown that human lysyl-tRNA synthetase (LysRS) binds to a tRNA-like element of the 5’ UTR to facilitate this process. The purpose of this study is to use RNA structure-probing techniques to map the LysRS-viral genome interaction. Ribonucleases (RNases) are used to cleave the RNA with and without LysRS bound. The RNA fragments are then analyzed using capillary electrophoresis to find the RNA residues that are protected from cleavage by the bound protein. Three 356 nucleotide variants of the HIV-1 5’ UTR have been prepared and optimized for folding homogeneity and probing conditions. Preliminary RNase digestion experiments in the absence of bound protein are in agreement with the predicted secondary structure of the HIV-1 5’ UTR. We are presently mapping sites where LysRS binds to the viral genome. These experiments will help better understand this key viral regulatory mechanism, which may contribute to the future design of new therapeutics.
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Biological Sciences: 2nd Place (The Ohio State University Denman Undergraduate Research Forum)
Keywords
HIV-1, RNA, lysyl-tRNA synthetase, structure probing