Regulation Mechanism Of Cap-independent Translation In Bean Common Mosaic Virus

Bean common mosaic virus was a member of the genus Potyvirus in the famlily Potyviridae,viral genome was a single-stranded sense RNA,approximately 10 kb in total length,genome 5’end with viral protein genome-linked,3’end with poly（A）tail.BCMV encoded a polymeric protein of approximately 358 k Da,which was subsequently cleaved into10 mature functional proteins by protease.In addition,a novel protein,PIPO,was formed at the N terminal of P3 protein by transcriptional frameshift strategy.In this study,the regulatory effects of BCMV 5’UTR and 3’UTR on cap-independent translation were analyzed,as well as the RNA structural characteristics of the core regulatory elements,and finally the regulatory mechanism of BCMV cap-independent translation was analyzed.By using firefly luciferase（Fluc）reporter gene vector and in vitro translation system,it was found that the 5’UTR of BCMV Taian isolate and Jinan isolate could positively regulate cap-independent translation.The presence of 3’UTR alone did not regulate cap-independent translation,but synergically enhanced the regulation of 5’UTR,suggesting that there might be potential long-distance RNA-RNA interactions between 5’UTR and 3’UTR.Since the two isolates had little difference in the regulation of cap-independent translation,the Taian isolate was selected as the research object to conduct an in-depth study on the regulation mechanism of cap-independent translation in BCMV.By inserting a hairpin upstream of BCMV 5’UTR,it showed internal ribosomal entry site（IRES）activity.Moreover,the 5’UTR still showed positive regulation in the presence of cap structure.A series of 5’UTR deletions showed that the core active region in 5’UTR,which regulates cap-independent translation,is mainly located in 20-101 nt.The RNA structures of BCMV 5’UTR and 3’UTR were analyzed by in-line probing technique.The core region of the 5’UTR regulation cap-independent translation contained 3hairpins and the 3’UTR contained 4 hairpins.In the presence of 3’UTR,there were two regions with reduced cleavage in 5’UTR,and potential long-distance RNA-RNA interactions sites between 5’UTR and 3’UTR were tentatively located.Then the long-distance RNA-RNA interaction between 5’UTR and 3’UTR was verified by EMSA experiment.In combination with RNA structural information and mutation analysis,possible remote RNA-RNA interaction sites were located in both 5’UTR and 3’UTR,showing a"1 vs 3"RNA-RNA interaction feature,one site in the 5’UTR(³⁶AAAC)was involved in RNA-RNA interaction,while three sites in the 3’UTR(⁹⁸⁸⁰ GUUU、⁹⁸⁹¹ GUUU、⁹⁹⁵⁹GUUU)could interact with the 5’UTR.In this study,we analyzed the core regulatory elements of cap-independent translation of BCMV,and enriched the data of cap-independent translation.We found that there may be"1vs 3"type long-distance RNA-RNA interactions in cap-independent regulation of plant RNA viruses.It provides a new target for the prevention and control of BCMV virus disease.