Poster abstracts
Poster number 15 submitted by Kevin A. Garayalde Batista
Probing the role of HIV-1 genomic RNA transcriptional start site heterogeneity in Gag binding
Kevin Garayalde (Department of Chemistry and Biochemistry, Center for Retrovirus Research and Center for RNA Biology, Ohio State University, Columbus OH), Shouhui Liu (Department of Chemistry and Biochemistry, Center for Retrovirus Research and Center for RNA Biology, Ohio State University, Columbus OH), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for Retrovirus Research and Center for RNA Biology, Ohio State University, Columbus OH)
Abstract:
Approximately 38 million people currently live with Human Immunodeficiency Virus type 1 (HIV-1). Current FDA approved antiviral treatments target several steps of the lifecycle, but do not target the virion assembly or genomic RNA (gRNA) packaging process. Two copies of viral gRNA are selected as dimers by the HIV-1 Gag protein through interactions with a packaging signal (Psi) within the highly conserved 5′ untranslated region (5′UTR). The 5′UTR exists in two mutually exclusive conformational states: the monomeric state where the palindromic dimerization initiation site (DIS) is sequestered through intramolecular base pairing, and the dimeric state where the DIS is exposed. A surprising recent finding suggests that sequence heterogeneity at the gRNA transcriptional start site (TSS) results in variable numbers of guanosines (1G, 2G or 3G) at the 5′ end of the gRNA, which affect the fate of the RNA. The 1G transcripts are preferentially dimeric and packaged into newly synthesized virions, while the monomeric 3G transcripts are enriched on polysomes and serve as mRNAs. We hypothesize that the 1G transcripts are preferentially bound to HIV-1 Gag. Unexpectedly, using direct fluorescence anisotropy (FA) binding and FA-based salt-titration assays and excess Gag protein, we failed to observe a Gag binding preference for the fluorophore-labeled 1G 5′UTR RNA over the 3G RNA. In this work, we aim to selectively label HIV-1 Gag through an engineered extra cysteine residue (A120C) in the Matrix-Capsid linker region for use in FA binding assays using low Gag concentrations. By more closely mimicking native conditions, incubating labeled A120C Gag with excess HIV-1 5′UTR, we expect to see a Gag binding preference for the dimer-stabilizing 1G RNA. This work will elucidate the role of gRNA transcriptional start site heterogeneity in HIV-1 Gag binding and packaging preference and could pave the way for new therapies aimed at the critical assembly step of the viral lifecycle.
Keywords: Human Immunodeficiency Virus type 1 (HIV-1) , Transcriptional Start Site (TSS), Fluorescence Anisotropy (FA)