Poster abstracts

Poster number 47 submitted by Yu-Ci Syu

Analysis of HTLV-1 Gag interactome and characterization of its chaperone activity

Yu-Ci Syu (Molecular, Cellular, and Developmental Biology Graduate Program, Center for RNA Biology, and Center for Retrovirus Research), Joshua Hatterschide, Yingke Tang (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research), Jacob Al-Saleem (Department of Veterinary Biosciences, and Center for Retrovirus Research), Amanda R. Panfil (Molecular, Cellular, and Developmental Biology Graduate Program, Department of Veterinary Biosciences, and Center for Retrovirus Research), Patrick L. Green (Molecular, Cellular, and Developmental Biology Graduate Program, Department of Veterinary Biosciences, and Center for Retrovirus Research), Karin Musier-Forsyth (Molecular, Cellular, and Developmental Biology Graduate Program, Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research)

Abstract:
Human T-cell leukemia virus type 1 (HTLV-1) is the only oncogenic human retrovirus discovered to date. All retroviruses use a host cell tRNA to prime RT. The 3' end of the tRNA anneals to an 18-nucleotide region of the genome known as the primer binding site (PBS). In HTLV-1, the PBS is complementary to human tRNAPro. Structure-probing of the genomic RNA revealed the PBS is embedded in a highly-structured hairpin.1 HTLV-1 nucleocapsid (NC), matrix (MA), and even the HIV-1 NC protein are incapable of annealing tRNAPro to the stable PBS domain in vitro. We hypothesize that HTLV-1 Gag may have more robust chaperone activity and that a cellular co-factor may be required to facilitate primer tRNA annealing. We successfully purified recombinant HTLV-1 Gag for the first time and performed fluorescence anisotropy RNA-binding assays, as well as primer-annealing assays. In direct-binding assays to HTLV-1 5' UTR-derived RNAs, we found HTLV-1 Gag displayed stronger RNA-binding affinity (Kd ~0.5-5 uM) than HTLV-1 NC (~3 uM) or MA (~1-8 uM). As expected, mutant HTLV-1 Gag with deletion of the highly acidic 29 residues at the C-terminus displayed significantly better RNA-binding activity than WT HTLV-1 Gag (Kd ~0.2-1.5 uM). In preliminary annealing assays, HTLV-1 Gag chaperoned the annealing of tRNAPro to the PBS to a comparable degree as a heat-annealed primer/template complex (positive control). To identify possible interacting partners of HTLV-1 Gag in cells, we performed affinity purification mass spectrometry (AP-MS). Two significant AP-MS hits, RPL7 and YBX1, were further validated by reciprocal co-IP in both HEK293T and MT-2 cells. RPL7 has been reported to interact with the NC domain of HIV-1 Gag to increase chaperone activity.2 Current studies are focused on preparing recombinant RPL7 and investigating whether it enhances HTLV-1 Gag’s chaperone activity. Taken together, these studies will lead to mechanistic insights that could be exploited for new therapeutic strategies.

References:
1. Wu, W. et al. Human T-cell leukemia virus type 1 Gag domains have distinct RNA-binding specificities with implications for RNA packaging and dimerization. J Biol Chem 293, 16261-16276, (2018).
2. Mekdad, H. E. et al. Characterization of the interaction between the HIV-1 Gag structural polyprotein and the cellular ribosomal protein L7 and its implication in viral nucleic acid remodeling. Retrovirology 13, 54, (2016).

Keywords: HTLV-1, tRNA, Reverse transcription