Poster abstracts
Poster number 141 submitted by Yu-Ci Syu
Identification of specific tRNA-Pro isodecoder used by HTLV-1 to prime reverse transcription and role of host factors 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), Christina R. Budding, Zixi Long (Molecular, Cellular, and Developmental Biology Graduate Program, 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, 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:
All retroviruses are believed to use a host cell tRNA to prime reverse transcription (RT). In oncogenic human T-cell leukemia virus type 1 (HTLV-1), the primer binding site (PBS) in the genomic RNA (gRNA) is complementary to the 3'-18 nucleotides (nt) of human tRNAPro. The human genome encodes 20 tRNAPro genes representing 7 isodecoders; all of them share the same 3' 18-nt sequence but vary at other locations. Moreover, a previous study showed that a 3' 18-nt tRNAPro-derived fragment is packaged into HTLV-1 particles and can prime RT in vitro1. To determine the identity of the authentic RT primer, we sequenced the minus-strand strong-stop RT product containing the intact primer and established HTLV-1 primes RT using full-length tRNAPro3UGG. Structure-probing of the gRNA revealed the PBS is embedded in a highly-structured hairpin2. We successfully purified recombinant HTLV-1 Gag and performed primer-annealing assays. Similar to nucleocapsid (NC) and matrix, HTLV-1 Gag is not 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. Moreover, both were shown to be packaged into HTLV-1 virions. Domain mapping studies revealed that HTLV-1 Gag interacts with RPL7 and DDX21 through the zinc fingers (ZF) in NC 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 ZF 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. Current studies are focused on knocking down RPL7 and/or DDX21 and investigating the impact on tRNAPro primer occupancy at the PBS in virions. Taken together, the mechanistic insights gained from these studies can be exploited for the development of new therapeutic strategies aimed at targeting HTLV-1 RT.
References:
1. Ruggero K, Guffanti A, Corradin A, Sharma VK, De Bellis G, Corti G, Grassi A, Zanovello P, Bronte V, Ciminale V et al. 2014. Small noncoding RNAs in cells transformed by human T-cell leukemia virus type 1: a role for a tRNA fragment as a primer for reverse transcriptase. J Virol 88: 3612-3622.
2. Wu W, Hatterschide J, Syu YC, Cantara WA, Blower RJ, Hanson HM, Mansky LM, 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: tRNA, DDX21, RPL7