Poster abstracts
Poster number 161 submitted by Weixin Wu
Elucidation of the protein-RNA interactions that dictate human T-lymphotropic virus type-1 viral genomic RNA packaging
Weixin Wu (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH 43210), William A. Cantara (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH 43210), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH 43210)
Abstract:
In most retroviruses, viral genomic RNA (vRNA) packaging is mediated by a specific interaction between the nucleocapsid (NC) domain of the viral Gag polyprotein and vRNA psi (Ψ) packaging signals. Recent studies have shown that the matrix (MA) domain of Gag also plays a role in vRNA packaging (1). In HIV-1, NC is critical for selective packaging of vRNA over various other cellular RNAs. By comparison, the mechanisms of vRNA packaging in deltaretroviruses, which include bovine leukemia virus (BLV), human T-lymphotropic virus type-1 and -2 (HTLV-1 and -2), are less clear. Previous studies showed that both BLV Gag NC and MA domains are involved in vRNA packaging (2). Our recent results support an important role of HTLV-2 MA in nucleic acid binding (3). Therefore, we have proposed that deltaretroviruses may use a different vRNA packaging mechanism from that of HIV-1 in which MA plays a larger role in vRNA selection at the initial stage. This project aims to understand the HTLV-1 MA-vRNA interactions that are responsible for selective vRNA interaction and packaging. We have prepared and optimized the purification of recombinant HTLV-1 MA. Fluorescence anisotropy binding assays reveal that HTLV-1 NC bound to a non-specific 20-mer DNA oligonucleotide (Kd = 230 nM), but failed to bind to RNA stem loops SL1 or SL2 derived from the putative vRNA packaging signal. In contrast, HTLV-1 MA binds with moderate affinity to ssDNA, SL2 and SL1-SL2 (Kd = 1.1 μM, 1.9 μM and 1.7 μM, respectively); no binding was detected to SL1 alone. These results suggest that HTLV-1 MA has moderate nucleic acid binding ability and may play a role in specific vRNA interaction and selection. Salt-titration and competition-binding studies are underway to examine binding specificity. We are also currently mapping the interactions between HTLV-1 proteins and larger vRNA fragments encompassing the full-length 5’-UTR (nt 1-448) and upstream gag (nt 449-624), which are the likely locations of Ψ.
References:
1. Parent, L. J.; Gudleski, N., Beyond plasma membrane targeting: role of the MA domain of Gag in retroviral genome encapsidation. J Mol Biol 2011, 410 (4), 553-64.
2. Wang, H.; Norris, K. M.; Mansky, L. M., Involvement of the matrix and nucleocapsid domains of the bovine leukemia virus Gag polyprotein precursor in viral RNA packaging. J Virol 2003, 77 (17), 9431-8.
3. 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 2014, 88 (2), 1271-80.
Keywords: Deltaretrovirus, HTLV-1 , MA