Substitutions of stem-loop subdomains in internal ribosome entry site of Senecavirus A:Impacts on rescue of sequence-modifying viruses  

作　　者：Qianqian Wang Jie Wang Lei Zhang Xiaoxiao Duan Lijie Zhu Youming Zhang Yan Li Fuxiao Liu 

机构地区：[1]College of Veterinary Medicine,Qingdao Agricultural University,Qingdao 266109,China [2]College of Veterinary Medicine,Inner Mongolia Agricultural University,Huhhot 010018,China [3]Shandong New Hope Liuhe Group Co.,Ltd.,Qingdao 266100,China [4]Qingdao Center for Animal Disease Control&Prevention,Qingdao 266199,China [5]State Key Laboratory of Microbial Technology,Shandong University,Qingdao 266237,China

出　　处：《Journal of Integrative Agriculture》2024年第7期2391-2406,共16页农业科学学报（英文版）

基　　金：This work was supported by the National Natural Science Found ation of China(32273000);the Qingdao Demonstration Project for People-benefit from Science and Techniques,China(23-2-8-xdny-14nsh and 24-2-8-xdny-4-nsh);the National Program of Undergraduate Innovation and Entrepreneurship,China(202310435039);the Open Project Fund of State Key Laboratory of Microbial Technology,China(M2023-03)。

摘　　要：Senecavirus A(SVA)has a positive-sense,single-stranded RNA genome.Its 5´untranslated region harbors an internal ribosome entry site(IRES),comprising 10 larger or smaller stem-loop structures(including a pseudoknot)that have been demonstrated to be well conserved.However,it is still unclear whether each stem-loop subdomain,such as a single stem or loop,is also highly conserved.To clarify this issue in the present study,a set of 29 SVA cDNA clones were constructed by site-directed mutagenesis(SDM)on the IRES.The SDM-modified scenarios included:(1)stem-formed complementary sequences exchanging with each other;(2)loop transversion;(3)loop transition;and(4)point mutations.All cDNA clones were separately transfected into cells for rescuing viable viruses,whereas only four SVAs of interest could be recovered,and were genetically stable during 20 passages.One progeny grew significantly slower than the other three did.The dual-luciferase reporter assay showed that none of the SDM-modified IRESes significantly inhibited the IRES activity.Our previous study indicated that a single motif from any of the ten stem structures,if completely mutated,would cause the failure of virus recovery.Interestingly,our present study revealed three stem structures,whose individual complementary sequences could exchange with each other to rescue sequence-modifying SVAs.Moreover,one apical loop was demonstrated to have the ability to tolerate its own full-length transition,also having no impact on the recovery of sequence-modifying SVA.The present study suggested that not every stem-loop structure was strictly conserved in its conformation,while the full-length IRES itself was well conserved.This provides a new research direction on interaction between the IRES and many factors.

关 键 词：SVA HCV IRES HCV-like IRES stem-loop structure cDNA clone virus rescue mutation 

分 类 号：S852.65[农业科学—基础兽医学]