Poster abstracts
Poster number 115 submitted by Yu-Ci Syu
Specificities of HTLV-1 Gag RNA-binding domains: implications for genomic RNA dimerization and primer annealing
Yu-Ci Syu (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210), Weixin Wu (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210), Joshua Hatterschide (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210), Alice Duchon, Yingke Tang (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210), William Cantara (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210)
Abstract:
During assembly of retrovirus particles, the Gag polyprotein selectively packages two copies of full-length viral genomic RNA (gRNA) in a process mediated by a specific interaction between Gag and the gRNA packaging signal. In HIV-1, the nucleocapsid (NC) domain of Gag is critical for specific gRNA selection and tRNA primer annealing to the primer binding site (PBS). In contrast, the mechanism of gRNA packaging and primer annealing in deltaretroviruses such as human T-cell lymphotropic virus type 1 (HTLV-1) are less clear. Previous studies suggested that deltaretroviral matrix (MA) plays a more important role than NC in the initial gRNA selection process (Sun, M., et al, J. Virol 2014). To test this hypothesis, UV cross-linking and selective 2'-hydroxyl acylation analyzed by primer extension (XL-SHAPE) were used to determine the secondary structure of the HTLV-1 5' leader for the first time, and to characterize HTLV-1 MA- and NC-gRNA interactions. SHAPE probing of gRNA alone did not support the formation of the previously proposed stem-loop 1 (SL1) and revealed that the PBS is embedded in a highly-structured hairpin. XL-SHAPE identified sites of both MA and NC interaction with the gRNA, as well as conformational changes upon protein binding. Moreover, a new primary dimerization initiation site (DIS) located in the U5 region was identified and confirmed by in vitro dimerization assays. We also demonstrated that the putative primer for HTLV-1 reverse transcriptase (RT), tRNAPro, is not efficiently annealed to the complementary PBS by either HTLV-1 NC or MA. Deletion of the gRNA sequence that is base paired to the PBS significantly increases full-length tRNAPro annealing, which suggests this sequence may play a role in regulating primer annealing in HTLV-1. Interestingly, a fragment derived from the 3'-18 nt of tRNAPro is present in HTLV-1 particles (Ruggero, K., et al, J. Virol 2014) and can be fully annealed to the PBS even in the absence of a chaperone protein. Studies to identify the RT primer in HTLV-1 virions are currently underway.
References:
Ruggero, K., Guffanti, A., Corradin, A., Sharma, V.K., De Bellis, G., Corti, G., Grassi, A., Zanovello, P., Bronte, V., Ciminale, V., & D'Agostino, D.M. 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).
Sun, M., Grigsby, I.F., Gorelick, R.J., Mansky, L.M., & Musier-Forsyth, K. Retrovirus-specific differences in matrix and nucleocapsid protein-nucleic acid interactions: implications for genomic RNA packaging. J. Virol.
88, 1271-1280 (2014).
Keywords: HTLV-1, Genome dimerization, Primer annealing