Poster abstracts

Poster number 32 submitted by Andrew Goodrich

Probing the role of HIV Gag in regulation of reverse transcription

Andrew C. Goodrich (Departments of Chemistry and Biochemistry, Center for Retroviral Research, and Center for RNA Biology, The Ohio State University), Christopher P. Jones (Departments of Chemistry and Biochemistry, Center for Retroviral Research, and Center for RNA Biology, The Ohio State University), Karin Musier-Forsyth (Departments of Chemistry and Biochemistry, Center for Retroviral Research, and Center for RNA Biology, The Ohio State University)

Abstract:
Human immunodeficiency virus (HIV) is a retrovirus that relies on nucleic acid (NA) chaperone proteins, which function to remodel NAs at critical steps throughout the viral lifecycle. Upon entering a host cell, HIV reverse transcribes its RNA genome into DNA, which is integrated into the host DNA. Following the production of new viral RNAs and proteins, the assembly of new virus particles takes place at the plasma membrane. The polyprotein Gag, which consists of the matrix, capsid, nucleocapsid (NC), and p6 domains, directs virus assembly and packages the viral RNA genome into new virions. Gag is a chaperone protein that is also responsible for annealing of a host cell tRNA^Lys primer to the HIV genome, the first step of reverse transcription. After virus budding, Gag is proteolyzed, and freestanding NC assumes the role as the chaperone for the remaining steps of reverse transcription. Although both Gag and NC promote tRNA^Lys annealing to the genome in vitro, we have found that extension of the primer by reverse transcriptase is inhibited by Gag. Interestingly, in vivo studies have found that virus variants with specific mutations in the NC domain of Gag undergo premature reverse transcription, which causes the packaging of DNA and abolishes infectivity. Thus, we hypothesize that Gag’s role in inhibiting reverse transcription depends upon critical structures within the NC domain of Gag, which remodel the primer/genome complex in such as way as to prevent premature reverse transcription. In this work, we begin to test this hypothesis by preparing Gag variants with mutations in the NC domain and testing their capability to aggregate and bind NA, destabilize NA secondary structure, and facilitate tRNA annealing and reverse transcription in vitro. We show that these mutants bind to NAs with reduced affinity relative to wild-type (WT) Gag. However, they aggregate and anneal NA as efficiently as WT Gag. Surprisingly, these mutants are better than WT Gag at destabilizing NA secondary structure. Studies to examine the effect of NC domain mutants on Gag’s inhibition of reverse transcription are underway.

Keywords: HIV, reverse transcription