Poster abstracts
Poster number 47 submitted by Yu-Ci Syu
Identification of the authentic HTLV-1 reverse transcription primer and role of Gag-host factor interactions in chaperoning 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 HTLV-1, the primer binding site (PBS) for reverse transcription (RT) in the genomic RNA (gRNA) is complementary to the 3'-18 nucleotides (nt) of human tRNAPro. Humans encode 22 tRNAPro isodecoders, the majority of which share the same 3'-18 nt sequence but vary at other locations. To establish the identity of the authentic RT primer, we sequenced the minus-strand strong-stop RT product containing the intact primer and established that HTLV-1 primes RT using full-length tRNAPro,3UGG. Structure-probing of the gRNA revealed that the PBS is embedded in a highly structured hairpin. We showed that 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 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. Two significant hits from this screen, ribosomal protein RPL7 and RNA helicase DDX21, were further validated by reciprocal co-IP studies in both HEK293T and MT-2 cells. Domain mapping studies revealed that HTLV-1 Gag interacts with RPL7 and DDX21 through the zinc fingers in the NC domain and that RPL7 interacts with HTLV-1 Gag and DDX21 through both the N-terminal basic leucine zipper and C-terminal domains. The interactions depend on the intact zinc finger structures but not on the presence of RNA, Gag myristoylation, or Gag oligomerization. RPL7 or DDX21 alone were significantly more effective than HTLV-1 Gag at annealing tRNAPro to the PBS. Taken together, the mechanistic insights gained from these studies could be exploited for the development of new therapeutic strategies aimed at targeting HTLV-1 RT.
Keywords: tRNA, RPL7, DDX21