Poster abstracts
Poster number 78 submitted by Yu-Ci Syu
Role of HTLV-1 Gag-host factor interactions in chaperoning tRNA-Pro primer annealing
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. In contrast to HIV-1, several critical details of HTLV-1 reverse transcription remain unclear. 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 domain 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 primer-annealing assays. Relative to NC and MA, HTLV-1 Gag is 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). Three significant AP-MS hits, DDX21, RPL7, and YBX1, were further validated by reciprocal co-IP studies in both HEK293T and MT-2 cells. Domain mapping studies revealed that the zinc fingers in the NC domain of HTLV-1 Gag interact with both the N-terminal basic leucine zipper (bZIP) and C-terminal domains (CTD) of RPL7. In addition, we showed that the interactions depend on the intact zinc finger structures but not on the presence of RNA, Gag myristoylation, or Gag oligomerization. RPL7 alone was more effective than either HTLV-1 MA or Gag at annealing tRNAPro to the PBS. Domain deletion studies showed that both bZIP and CTD of RPL7 are involved in facilitating primer annealing. Current studies are focused on investigating the annealing activity of another HTLV-1 Gag-interacting partner, the DDX21 helicase, and the influence of RPL7 and DDX21 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., Hatterschide, J., Syu, Y.C., Cantara, W.A., Blower, R.J., Hanson, H.M., Mansky, L.M. and Musier-Forsyth, K. (2018) 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.
Keywords: Gag, RPL7, DDX21