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 pseudokno...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.展开更多
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)+1 种基金the National Program of Undergraduate Innovation and Entrepreneurship,China(202310435039)the Open Project Fund of State Key Laboratory of Microbial Technology,China(M2023-03)。
文摘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.
