Poster abstracts
Poster number 52 submitted by Yu-Ci Syu
Mechanism of initiation of reverse transcription in HTLV-1
Yu-Ci Syu (Molecular, Cellular, and Developmental Biology Program, Center for RNA Biology, and Center for Retrovirus Research), Joshua Hatterschide (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research), 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), Patrick Green (Molecular, Cellular, and Developmental Biology Program, Department of Veterinary Biosciences, and Center for Retrovirus Research), Karin Musier-Forsyth (Molecular, Cellular, and Developmental Biology 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 first oncogenic and infectious human retrovirus discovered and therapeutics targeting HTLV-1 replication could prevent cancer progression. In contrast to HIV-1, several critical details of HTLV-1 reverse transcription (RT), the first enzymatic step of the retroviral lifecycle, remain unclear. All retroviruses use a host cell tRNA to prime RT. The 3' end of the tRNA anneals to an 18-nucleotide (nt) region of the genome known as the primer binding site (PBS). In HTLV-1, this region is perfectly complementary to human tRNAPro. Structure-probing of the genomic RNA (gRNA) revealed that the PBS is embedded in a highly-structured hairpin.1 Previous studies also showed that tRNAPro and a fragment derived from the 3'-18 nt of tRNAPro (tRFPro) are present in HTLV-1 virions, and both RNAs are capable of priming RT in vitro.2 To determine the authentic RT primer used, we purified the primer-PBS binary complex from HTLV-1 virions and added radiolabeled nucleotides and reverse transcriptase to synthesize a short extension. Analysis of this reaction on a high-resolution sequencing gel indicated that full-length tRNAPro is likely to be the RT primer in HTLV-1. However, neither HTLV-1 nucleocapsid protein (NC), matrix (MA) nor the robust HIV-1 NC chaperone protein are capable of annealing tRNAPro to the highly-structured PBS. In contrast, tRFPro anneals to the PBS without the assistance of an RNA chaperone protein. Mutation of the gRNA to disrupt the base-pairing interactions of the PBS region significantly increased tRNAPro annealing. Current studies are focused on preparing full-length HTLV-1 Gag to establish if this viral protein is capable of forming the tRNAPro/PBS complex. Work aimed at elucidating the role of tRFPro in the HTLV-1 lifecycle is also currently underway. These studies will lead to new mechanistic insights that could be targeted for therapeutic development.
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 Ruggero, K. et al. 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, (2014).
Keywords: HTLV-1, tRNA, Reverse transcription