Conserved Residues Adjacent to ß-Barrel and Loop Intersection among Enterovirus VP1 Affect Viral Replication: Potential Target for Anti-Enteroviral Development

Abstract

Enterovirus genus has over one hundred genotypes and could cause several kinds of severe animal and human diseases. Understanding the role of conserved residues in the VP1 capsid protein among the enterovirus genus may lead to anti-enteroviral drug development. The highly conserved residues were found to be located at the loop and ß-barrel intersections. To elucidate the role of these VP1 residues among the enterovirus genus, alanine substitution reverse genetics (rg) variants were generated, and virus properties were investigated for their impact. Six highly conserved residues were identified as located near the inside of the canyon, and four of them were close to the ß-barrel and loop intersection. The variants rgVP1-R86A, rgVP1-P193A, rgVP1-G231A, and rgVP1-K256A were unable to be obtained, which may be due to disruption in the virus replication process. In contrast, rgVP1-E134A and rgVP1-P157A replicated well and rgVP1-P157A showed smaller plaque size, lower viral growth kinetics, and thermal instability at 39.5°C when compared to the rg wild type virus. These findings showed that the conserved residues located at the ß-barrel and loop junction play roles in modulating viral replication, which may provide a pivotal role for pan-enteroviral inhibitor candidate.

Keywords: Enterovirus; VP1; antiviral; conserved residues; replication.

Figure 1

Diagram of highly conserved residues of VP1 among the Enterovirus genus. (A) After retrieving 90 reference sequences of enterovirus for alignment, amino acid conservation was demonstrated with WebLogo. Six conserved residues of this study are denoted with red arrowheads. (B) Consurf server with amino acid (PDB code 4AED) is shown in a relative steric position of the VP1 capsid protein structure of EV-A71. The six conserved residues are indicated. Highly conserved residues are shown in red and variable residues are shown in blue. (C) The representative cartoon diagram indicates the relative locations of the six conserved residues on VP1. (D) CABS-flex server was used to predict the protein backbone fluctuation profile among these conserved residues. Flexibility simulations in the VP1 protein structure of enteroviruses were produced. The 3-dimensional (3D) structures of the VP1 protein of four enterovirus strains (three EV-A71 and one echovirus 11) were generated using X-ray crystallography (PDB code 4AED, 4CDQ, 3VBS, and 1H8T). The fluctuation angles of the individual six residues are shown (***, p < 0.005).

Figure 2

Analysis of alanine substitution of reverse genetics enterovirus. (A) Immunofluorescence stain of rgVP1 variants. RNA transcript was transfected into RD cells, the viral capsid protein expression of EV-A71 was detected by mab979 monoclonal antibody three days post-transfection. The green/red fluorescence indicating viral protein expression and Evan’s blues counter stain were examined under fluorescence microscope at a 200× magnification. (B) A total number of 25 fields per slide was used to analyze the number of positive green fluorescent cells under a fluorescence microscope. (C) Viral replication titers after RNA transcript transfection at days 1, 2, and 3, viral particles in the medium containing reverse genetics variants were measured by CCID₅₀. (D) Viral RNA expression after RNA transcript transfection at days 2 and 3. The generative viral RNA from the medium was measured by real-time RT-PCR assay. The data were expressed as the mean ± SD of each group (n = 3). n.s., no significance, *, p < 0.05, **, p < 0.01, and ***, p < 0.005.

Figure 3

Viral RNA expression of rgVP1-P193A and rgVP1 wild type. Viral RNA replication after RNA transfection from reverse genetics variants were measured by real-time RT-PCR assay. The data were expressed as the mean ± SD of each group (n = 3). *, p < 0.05 and ***, p < 0.005.

Figure 4

Viral plaque size, growth kinetics, temperature sensitivity, and stability of reverse genetics variants. (A) Diagram of the plaque size of 10⁻⁵- and 10⁻⁶-fold serially diluted viruses. The plaque area was measured by Image J2. Mean of plaque area is shown in the right figure. The rgVP1-P157A, rgVP1-E134A, and rgVP1(WT) growth kinetics assays were performed in triplicate at (B) 35 °C in MOI of 10 and 0.1 after the indicated time course and (C) at 39.5 °C. The data were expressed as the mean ± SD of each group (n = 3). n.s., no significance, *, p < 0.05, **, p < 0.01, and ***, p < 0.005.

Figure 5

Effects of rgVP1-E134A and rgVP1-P157A variants on virus thermal stability. Thermostability assay was performed at 39.5 °C for 0.5, 1.0, 1.5, and 2.0 h. The data were expressed as the mean ± SD of each group (n = 3). *, p < 0.05, **, p < 0.01.

Figure 6

Effects of amino acid side-chain interaction after alanine substitution in six highly conserved residues of VP1. The hydrogen bonds interacting with other residues of six variants in UCSF chimera were shown. The wild-type residues are shown on the left and alanine substitutions on the right. The VP1 protein is shown in orange; the VP2 is shown in blue; the VP3 is shown in green; the VP4 is shown in yellow; the residues in the study are shown in red; the interaction bonds are shown as red lines.

Figure 6

Effects of amino acid side-chain interaction after alanine substitution in six highly conserved residues of VP1. The hydrogen bonds interacting with other residues of six variants in UCSF chimera were shown. The wild-type residues are shown on the left and alanine substitutions on the right. The VP1 protein is shown in orange; the VP2 is shown in blue; the VP3 is shown in green; the VP4 is shown in yellow; the residues in the study are shown in red; the interaction bonds are shown as red lines.