Poster abstracts

Poster number 19 submitted by Varun Dewan

Identifying Peptide and RNA Inhibitors against HIV-1 Capsid

Varun Dewan (Department of Biochemistry , Center for Retroviral Research and Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Tao Liu, Dehua Pei (Departments of Biochemistry and Chemistry, The Ohio State University, Columbus, OH 43210), Wei Wang (Department of Chemistry , Center for Retroviral Research and Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Hiroshi Matsuo (Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455), Lawrence Kleiman (Lady Davis Institute for Medical Research and McGill AIDS Centre, Jewish General Hospital, Montreal, Quebec, Canada), Karin Musier Forsyth (Departments of Biochemistry and Chemistry, Center for Retroviral Research and Center for RNA Biology, The Ohio State University, Columbus, OH 43210)

Abstract:
Upon HIV-1 budding from the host cell membrane during the late phase of the viral lifecycle, the proteolytic cleavage of the Gag polyprotein leads to the formation of the characteristic conical core formed by the viral capsid (CA) protein in the mature virus. In the cytoplasm prior to viral assembly, CA interacts with several host cell factors, including human lysyl-tRNA synthetase (LysRS). Binding to LysRS facilitates selection and packaging of host cell tRNALys,3, which is the primer for reverse transcription. The numerous functions of CA in the viral lifecycle make it an important antiviral target. Small-molecule inhibitors against HIV-1 CA, such as a recently identified peptide-based CA assembly inhibitor (CAI), have potential clinical applications. In this work, a robust method has been employed for the high-throughput synthesis, screening and identification of cyclic peptidyl ligands against HIV-1 CA. Support-bound cyclic peptide libraries containing randomized amino acid sequences and different ring sizes were synthesized and screened for binding to Texas Red-labeled full-length CA and a monomeric form of the CA C-terminal domain (WM-CA CTD). Selective inhibitors identified from the library showed strong specific binding (Kd ~ 500 nM range) to both CA and WM-CA CTD in vitro. Two of the selected peptides also inhibited LysRS and CA interaction in vitro with an IC50 value of ~ 1 µM. To demonstrate the in vivo efficacy, we have designed cell permeable peptides and these studies are currently underway. Ongoing work is also aimed at studying the cyclic peptide binding site on CA by NMR. As an additional approach to discover small molecule inhibitors, we have also employed SELEX to select for high-affinity RNA aptamers that bind to HIV-1 CA. Briefly, a 40-nt randomized RNA with 20-nt flanking primer binding site regions is being used to select for RNAs that bind to His6-CA immobilized on a Ni-NTA resin. Results of preliminary rounds of selection will be presented.

Keywords: CA, LysRS