Information-Entropy-Based Single Amino Acid Polymorphism Analysis Reveals Functional Variance of Enterovirus 2A Proteases

Figures

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  Figure 3. Effect of IC threshold selection on clustering of kSAPs. PCA clustering results of kSAPs under six different IC thresholds: (A) 1.677–4.322, (B) 1.761–4.022, (C) 2.735–4.022, (D) 3.022–4.022, (E) 3.274–4.022, and (F) 3.274–4.322.

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  Figure 1. Overview of the SAAPS analysis pipeline. The SAAPS (Single Amino Acid Polymorphism Statistics) pipeline is built with Python 3.10, integrating multiple functional modules for the analysis of amino acid sequences. The workflow consists of six main modules: (1) Data Preprocessing; (2) Amino Acid Polymorphism Calculation; (3) Information Content Computation; (4) Key Polymorphic Site Selection; (5) Feature Encoding; (6) Dimensionality Reduction and Clustering. Color coding: blue, input; green, processing; yellow, decision; orange, output.

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  Figure 2. Evolutionary relationships and clusters of EV-A 2A proteases. (A) Sequence identity heatmap of the 2A protease among 25 EV-A serotypes. Pairwise sequence identities were calculated using BioAider and visualized using the R package ggplot2. The color gradient ranges from blue (low similarity) to red (high similarity). Hierarchical clustering dendrograms are displayed above the matrix. (B) Neighbor-Joining (NJ) phylogenetic tree of the 25 EV-A serotypes based on 2A protease amino acid sequences. The tree was constructed using the Poisson model with bootstrap values (500 replicates) shown at the nodes. The scale bar represents 0.10 substitutions per site. (C) Maximum Likelihood (ML) phylogenetic tree of the 25 EV-A serotypes based on 2A protease amino acid sequences. The tree was constructed using the LG + G substitution model (selected by ProtTest and MEGA) with bootstrap values shown at the nodes. The scale bar represents the number of substitutions per site.

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  Figure 4. Distribution of polymorphic sites in the 2A protease. Distribution of polymorphic sites across the 2A protease sequence. A total of 88 polymorphic sites were identified; amino acid types are distinguished by unique colors. Secondary structure elements are annotated below the sequence, with α-helices highlighted in red and β-strands highlighted in yellow.

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  Figure 5. Polymorphism and information content analyses of the EV-A 2A protease. (A) Distribution of polymorphic sites by variant number. Among 150 total positions, 88 were polymorphic (≥2 variants). (B) Secondary structure distribution of polymorphic sites. For each secondary structure type, the percentages of sites with 1–9 variants are shown. (C) IC values for each site. Dashed lines indicate the selected threshold range (2.735–4.022). (D) Secondary structure distribution of the 56 kSAPs. The percentages of sites located in α-helices, β-strands, and random coils are presented.

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  Figure 6. Dimensionality reduction and clustering analyses of kSAPs. (A) Principal Component Analysis (PCA) of the selected polymorphic sites revealed three major clusters. (B) OPTICS clustering identified EV-A89 as a noise point, aligning with its phylogenetic position. In the OPTICS clustering color legend, “−1”, ”0”, “1” and “2” represent noise point, group 1, group 2, and group 3, respectively.

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  Figure 7. Transcriptomic phenotypic analyses of EV-A 2A protease. (A) Western blot of HEK-293T cells expressing 2A protease from 25 EV-A serotypes. Empty vector was used as a negative control. Top membrane (lanes 1–14): vector, CVA2-CVA8, CVA10, CVA12, CVA14, CVA16, EV-A71, and EV-A76. Bottom membrane (lanes 1–13): vector, EV-A89, EV-A90, EV-A91, EV-A92, EV-A114, EV-A119, EV-A120, EV-A121, EV-A122, EV-A123, EV-A124, and EV-A125. The expected molecular weights of EV-A 2A (30 kDa) and β-actin (42 kDa) are indicated on the right. Representative of three independent experiments. (B) PCA clustering of 12 serotypes from Group 1 (CVA2–CVA8, CVA10, CVA12, CVA14, CVA16, and EV-A71). (C) Venn diagram of shared KEGG pathways among EV-A76, EV-A91, and EV-A121.

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  Figure 8. Experimental verification of the kSAPs responsible for the unique expression pattern of EV-A89 2A. (A) Western blot of HEK-293T cells transfected with empty vector, C-terminal Myc-tagged EV-A89 2A (89-2A-Myc), or N-terminal Myc-tagged EV-A89 2A (Myc-89-2A). Lanes 1–3: vector, 89-2A-Myc, and Myc-89-2A. The positions of the 89-2A-Myc band (~25 kDa) and β-actin (42 kDa) are indicated on the right. (B) Schematic representation of four 2A fusion chimeras: 89-2AΔ71(1–38), 89-2AΔ71(39–76), 89-2AΔ71(77–120), and 89-2AΔ71(121–150). Amino acid boundaries (1, 38, 76, 120, 150) are indicated above the diagrams. All constructs carry a C-terminal 6×Myc tag. (C) Western blot of HEK-293T cells expressing the four fusion chimeras and controls. Lanes 1–7: vector, 89-2AΔ71(1–38), 89-2AΔ71(39–76), 89-2AΔ71(77–120), 89-2AΔ71(121–150), 89-2A-Myc, and Myc-89-2A. The 89-2A-Myc control migrates at ~25 kDa. Replacement of residues 77–120 shifts the band to ~27.5 kDa (lane 4), whereas the other chimeras retain the ~25 kDa band. Observed molecular weights are indicated on the right. (D) Amino acid sequence alignment of EV-A71 2A and EV-A89 2A within the region spanning residues 77–120. The positions of…