Poster abstracts

Poster number 45 submitted by Yu-Ci Syu

HTLV-1 Gag purification, cellular interactome, and in vitro chaperone activity

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

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 reverse transcription. In HTLV-1, the primer binding site (PBS) in the genomic RNA (gRNA) is complementary to the 3'-18-nucleotides of human tRNAPro. Structure-probing of the gRNA revealed that the PBS is embedded in a highly-structured hairpin.1 Neither HTLV-1 nucleocapsid (NC) nor matrix (MA) proteins are capable of annealing tRNAPro to the stable PBS in vitro. We hypothesize that HTLV-1 Gag, the polyprotein made up of MA, capsid, and NC domains, may have more robust chaperone activity than mature NC or MA 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 that HTLV-1 Gag displayed stronger RNA-binding affinity than HTLV-1 NC or MA. Relative to NC and MA, HTLV-1 Gag was only slightly more effective at chaperoning the annealing of tRNAPro to the PBS. To identify potential HTLV-1 Gag interacting partners and co-chaperones of tRNA annealing in cells, we performed affinity tagging/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. Domain mapping studies revealed the zinc fingers in the NC domain of HTLV-1 Gag are important for interaction with both binding partners. In addition, we showed the interactions are RNA-independent, Gag myristoylation-independent, and Gag oligomerization-independent. RPL7 is a ribosomal protein that has been reported to be packaged into HIV-1 virions and interact with the NC domain of HIV-1 Gag to increase chaperone activity.2 RPL7 alone was more effective than either HTLV-1 MA or Gag at annealing tRNAPro to the PBS. Current studies are focused on investigating the synergistic effects of RPL7 on HTLV-1 Gag chaperone activity and the influence of RPL7 knockdown on viral infectivity. 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: Gag, tRNA, RPL7