Motif mutations in pseudoknot stem I upstream of start codon in Senecavirus A genome: Impacts on activity of viral IRES and on rescue of recombinant virus

•Pseudoknot stem I (PKS-I)-disrupting SVA IRESs still initiate protein expression.•Mutation of PKS-Ia motif does not inhibit SVA rescue from cDNA clone.•Mutation of unpaired spacing in PKS-I also does not inhibit SVA rescue.•Mutations of PKS-Ib motif completely interfere with SVA rescue. Senecavirus A (SVA), formerly known as Seneca Valley virus, is classified into the genus Senecavirus in the family Picornaviridae. Mature virion harbors an approximately 7 300-nt-long, positive-sense, and single-stranded RNA genome, which contains 5′ and 3′ untranslated regions (UTRs). Internal ribosome entry site (IRES) is identified in the SVA 5′ UTR, and includes a RNA pseudoknot upstream of the start codon. This pseudoknot contains two stem structures, pseudoknot stem I and II (PKS-I and -II). The PKS-I is composed of two base-paired motifs (PKS-Ia and -Ib), between which there is an unpaired spacing (UpS). We reported previously that motif mutation in the PKS-II did not abolish the IRES activity, but interfered with SVA recovery from cDNA clone. In this study, we constructed five SVA minigenomes with point mutations in the PKS-I motif. Dual-luciferase reporter assay showed that motif mutations in PKS-I did not significantly interfere with the IRES activity to initiate protein expression. Correspondingly, we constructed five SVA cDNA clones with point mutations in the PKS-I motif. These genetically modified cDNA clones were separately transfected into BSR-T7/5 cells in attempting to rescue competent SVAs. However, only two viruses, namely PKS-Ia- and UpS-mutated recombinants, could be recovered from their individual cDNA clones. It can be concluded that the PKS-Ib is indispensable for viral growth.