Tolerance of Senecavirus A to Mutations in Its Kissing-Loop or Pseudoknot Structure Computationally Predicted in 3′ Untranslated Region

Figures

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  Figure 1. Schematic representations of SVA genome, 3’ UTR sequence, and its potential structures. Positive-sense, single-stranded, and linear SVA genome (A). A typical genome is composed of 5’ UTR, polyprotein ORF, 3’ UTR, and poly(A) tail. The VPg is covalently linked to the 5′ end of genome. The proportion of elements does not exactly match them in the genome. 68-nt-long sequence of 3’ UTR (B). Putative secondary structure of 3′ UTR (C). The 3′-UTR sequence is analyzed using the UNAFold Web Server (http://www.unafold.org/) for modeling its RNA secondary structure, which contains two adjacent stem-loops. The stop codon (UGA) is underlined. Two terminal loops are predicted to have a potential “kissing interaction” with each other by means of a triple base-pairing pattern (D, red/blue circle-marked). Such an interaction refers to that of the SVV-001 strain (Hales et al., 2008). H-type-like pseudoknot (E), composed of two stems (Stem 1 and 2) and two loops (Loop 1 and 2), is predicted in 3’ UTR by the DotKnot web server. The Stem 2 contains two PFMs, separately marked with yellow and green circles.

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  Figure 2. Construction of 17 full-length SVA cDNA clones. Schematic representation of eGFP-tagged SVA cDNA clone without mutation (A). Dotted lines indicate FP1/RP1-, FP2/RP2-, FP3/RP3-, FP4/RP4-, and BamH I/Pme I-targeted sites. The proportion of elements does not exactly match them in the cD-0 plasmid. Wild-type and mutated KLFMs (B). Mutated sites are unmarked with red/blue circles. Wild-type and mutated PFMs (C). Mutated sites are unmarked with yellow/green circles. Sanger sequencing chromatograms of 17 cDNA clones (D). Mutated sites are marked with letters inside their sequencing peaks. Agarose gel electrophoresis of cD-1 to −17 after plasmid purification (E,F).

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  Figure 3. Profiles of rSVA-0- to −7-expressed eGFPs in BSR-T7/5 cells at P0, P6, and P10. Cell monolayers are separately transfected with cD-0 to −7 and subjected to one freeze and thaw cycle at 72 hpt (P0) to collect supernatants for 10 serial passages. BF, bright field.

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  Figure 4. Profiles of rSVA-8- to −17-expressed eGFPs in BSR-T7/5 cells during passaging. Cell monolayers are separately transfected with cD-8 to −17. At 72 hpt (P0), cell cultures undergo one freeze and thaw cycle to collect supernatants for three serial passages. Three eGFP-expressing groups (rSVA-9, −13, and −15) are consecutively passaged in cells until P10. BF, bright field.

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  Figure 5. Identification of replication-competent rSVAs rescued from their individual cDNA clones. RT-PCR detection of rSVA-0 to −7 at P6 (A) and P10 (B). PCR analysis is simultaneously performed to demonstrate no interference of plasmid residues, the same below. RT-PCR detection of rSVA-8 to −12 (C) and −13 to −17 (D) at P3. RT-PCR detection of rSVA-9 at P6 and P10 (E). 3’-RACE reaction of rSVA-13 and -15 at P3, P6, and P10 (F,G). Sanger sequencing chromatograms of RT-PCR products from rSVA-1 to −7 samples at P6 (H) and P10 (I). Sanger sequencing chromatograms of RT-PCR products from three rSVA-9 samples (J). Sanger sequencing chromatograms of 3’-RACE reaction products from rSVA-13 (K) and −15 (L) samples.

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  Figure 6. Multistep growth curves of replication-competent rSVAs at P6. Viral titers are measured using the Spearman–Kärber equation. Data at 0, 24, 48, and 72 hpi are representative of three independent experiments. Error bar indicates standard deviation.