| Senecavirus A(SVA)is an emerging virus that causes vesicular disease in pigs.This virus is classified into the genus Senecavirus in the family Picornaviridae.Its genome is a positive-sense,single-stranded and nonsegmented RNA,approximately 7300 nucleotides(nt)in length.A picornaviral genome is essentially an m RNA,which,albeit unmodified with 5’ cap structure,can still initiate protein expression by the internal ribosome entry site(IRES).The SVA CH-LX-01-2016(Gen Bank: KX751945.1)was a representative isolate from China.It was determined to have a 668-nt 5’UTR,and a 68-nt 3’UTR upstream of the3 ’poly(A)tail.We established a reverse genetics system based on this strain to rescue enhanced green fluorescent protein(e GFP)-tagged SVA for facilitating studies on viral molecular mechanism.The 5 ’UTR of the SVA genome contains a hepatitis C virus(HCV)-like IRES,which includes an RNA pseudoknot upstream of the initiation codon.It has been predicted that the 3’ UTR contains a kissing-loop structure.Pseudoknot structure plays an important role in initiating protein expressionTest 1: In this study,we constructed the whole set of SVA(CH-LX-01-2016 strain)minigenomes with all combinations of point mutations in its pseudoknot stem II(PKS-II).The results showed that any combination of point mutations could not significantly interfere with the IRES to initiate protein expression.Moreover,We constructed a full-length SVA c DNA clone,in which the PKS-II-forming c DNA motif was totally mutated for disrupting the pseudoknot structure in IRES.The mutated SVA c DNA clone was transfected into BSR-T7/5 cells.The result showed that the 5-nt-mutated PKS-II had no significant impact on the replication of SVA antigenome.However,the replication-competent recombinant SVA could not be rescued from a modified c DNA clone.It was speculated that the mutated pseudoknot possibly disrupted the viral packaging signal,causing the failure of virus recovery.Test 2: SVA pseudoknot is predicted to contain two key structures,pseudoknot stem I and II(PKS-I and-II).The SVA PKS-I is composed of two base-paired motifs(PKS-Ia and-Ib),between which there is a 2-base-unpaired spacing(Up S).In this study,we constructed five mutated minigenomes with multiple point mutations in one strand of PKS-I.Nevertheless,the dual luciferase reporter assay revealed that motif mutations in the PKS-I did not significantly impair the IRES activity to initiate NLuc expression.We constructed five mutated c DNA clones,all of which were separately transfected into BSR-T7/5 cells in an attempt to rescue replication-competent viruses.The results showed that SVA could totally tolerate PKS-Ia or Up S mutation in viral genome.In contrast,the high-fidelity sequence of PKS-Ib was possibly indispensible for virus recovery,since we failed to rescue the SVA-IV from its c DNA clone with only three point mutations.Test 3: In the present study,we constructed a total of seven e GFP-tagged SVA c DNA clones,which had different mutated kissing-loop-forming motifs(KLFMs)in the 3’ UTR.The experiment of virus recoveries showed that seven e GFP-tagged SVAs were successfully rescued from their individual c DNA clones,and moreover all mutated KLFMs were genetically stable after ten viral passages.The result demonstrated that the SVA 3’ UTR harbored a putative kissing-loop structure unnecessary for viral replication. |
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