Enterovirus A Shows Unique Patterns of Codon Usage Bias in Conventional Versus Unconventional Clade

Figures

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  Figure 1. Relative dinucleotide analysis and ENC plot. (A) Dinucleotide composition of EV-A clades. (B) Clade-level comparison of ENC values of EV-A CDS and individual gene segments. CDS is short for coding sequences.

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  Figure 2. AU3 and GC3 in codons of P1 and P2P3 regions of EV-A clade 1, clade 2 and clade 3. Numbers in the square array indicate the RSCU values of preferred codon by each amino acid. Yellow shade indicate AU3 and blue shade indicate GC3 for the codon ending.

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  Figure 3. Correspondence analysis (COA). COA plots were constructed for coding sequences of each EV-A strains.

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  Figure 4. Correlation analysis between nucleotide compositions and the two principal axes of COA plot. The numbers in lower triangle matrix show the slope of regression line. The upper triangle matrix shows the information by the color and size of circles; Dark blue means positive correlation and dark red means negative correlation; The bigger circle means more significant correlation; NS means non-significant (p > 0.05), * represents p < 0.05, ** represents p < 0.01.

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  Figure 5. Parity rule 2 (PR2) bias plots. PR2 plots were constructed for the coding sequences, P1 and P2P3 regions of EV-A strains respectively. (A) coding sequences, (B) P1 region, (C) P2P3 region. The blue dots, red squares, and green triangles indicate clade 1, clade 2 and clade 3 strains.

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  Figure 6. ENC-GC3 plots. The curve indicates the expected codon usage if GC compositional constraints alone account for the codon usage bias. (A) coding sequences, (B) P1 region, (C) P2P3 region. The blue dots, red squares, and green triangles indicate clade 1, clade 2 and clade 3 strains.

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  Figure 7. Neutrality plot analysis. Neutrality plots (GC12 and GC3) was constructed for the coding sequences, P1 and P2P3 regions of EV-A strains. (A) coding sequences, (B) P1 region, (C) P2P3 region, (D) VP1 gene. The blue dots, red squares, and green triangles indicate clade 1, clade 2 and clade 3 strains.

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  Figure 8. CAI analysis of the EV-A coding sequences against model organisms. CDS stands for the coding sequences of EV-A strains.

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  Figure 9. Codon optimization and protein expression levels for EV-A strains. (A) Schematic map of P1 protein expression plasmid. (B) Post plasmid transfection, GFP-tagged P1 protein levels of different EV-A strains in HEK293T cells was monitored by fluorescence microscopy. (C) Comparison of EVA76 and EVA89 P1 protein levels before and after codon optimization. Expression of GFP-tagged P1 protein in the cell culture was monitored through fluorescence microscopy (left panel). Furthermore, the expression of GFP-tagged P1 proteins was examined by anti-GFP antibodies using Western Blot (right panel). Housekeeping gene GAPDH was used for loading control.