Table 1.
X-ray Data collection and refinement statistics.
Fig 1.
Overview of wild-type SVA 3Cpro structure.
(A) Overall crystal structure of wild-type SVA 3Cpro, colored from blue (N-terminus) to red (C-terminus). (B) The proteolytic site of SVA 3Cpro. Unbiased omit electron density map (Fo-Fc) of the catalytic triad shown at a 1.5σ level. The key residues involved in catalysis and the β-ribbon involved in substrate specificity are highlighted in magenta. It is noted that Cys160 has an alternative conformation. (C) Structure-based sequence alignment of SVA 3Cpro with its representative homologs from various picornaviruses performed using clustal X (version 1.81) and ESPript 3. They include hepatitis A virus (HAV), foot-and-mouth disease virus (FMDV), human rhinovirus (HRV), swine vesicular disease virus (SVDV) and enterovirus 71 (EV71). The conserved residues are boxed in blue. Identical conserved and low conserved residues are highlighted in red background and red letters, respectively. The conserved catalytic triad (His48-Asp84-Cys160) and the residues involved in phospholipid-binding in SVA 3Cpro are highlighted using red arrows and red asterisks, respectively. The conserved phospholipid/RNA-binding motifs “K(R)F(V)RDI” and “V(T)GK” in most picornaviruses are labeled using green bars.
Fig 2.
Identification of an endogenous phospholipid molecule in SVA 3Cpro.
(A) Close-up view of the surface charge surrounding the phospholipid-binding region of SVA 3Cpro (blue, +6.3KT; red, -6.3KT), colored by the local electrostatic potential. The region is predominantly electropositive. Electron density map (2Fo-Fc) of the phospholipid-like molecule (modeled as a CL molecule) is shown at a 1.5σ level. (B) Contacts analysis (H-bond and charge complementary) between the CL molecule (orange sticks) and the interacting residues (magenta sticks). (C) Mutagenesis studies on the residues involved in phospholipid-binding in SVA 3Cpro. These residues were mutated to qualitatively determine the lipid content in these extracts using a CL-detection ELISA kit.
Fig 3.
Identification of phospholipids categories in SVA 3Cpro.
(A) Membrane Lipid Strips (Echelon P-6002) contain the following lipids at 100 pmol per spot: Triglyceride, Diacylglycerol (DAG), Phosphatidic acid (PA), Phosphatidylserine(PS), Phosphatidylethanolamine (PE), Phosphatidylcholine (PC), Phosphatidylglycerol (PG), Cardiolipin (CL), Phosphatidylinositol (PI), Phosphatidylinositol 4-phosphate (PI4P), Phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2), Phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3), Cholesterol, Sphingomyelin and Sulfatide. (B) A lipid-binding assay of the extracts of SVA 3Cpro C160A and wild-type using the membrane lipid strip. Duplicate experiments were performed, and one representative blot was shown. The top three abundant lipids categories (CL, PI4P and sulfatide) are highlighted using arrows. Lipid strips were incubated with His-tagged SVA 3Cpro WT (10 μg/mL) or C160A (0.5 μg/mL). The strips were then washed and developed with a mouse anti-His antibody followed by anti-mouse IgG-HRP. (C) The chemical structures of CL, PI4P and sulfatide. R1/R2/R3/R4: fatty acid chain. (D) The relative intensities of CL, PI4P and sulfatide spots that represent their relative abundances in SVA 3Cpro. The intensity of each spot was calculated using the software Image Lab. Data are presented as the average (±standard error of the mean) from duplicate experiments.
Fig 4.
The untargeted lipidomics analysis of wild-type SVA 3Cpro extract by HPLC-MS.
(A) The total ion chromatography (TIC) of lipid ions was recorded in the negative ion detection mode [M − 2H]2−. The chromatogram of phospholipid ions from accurate m/z values obtained from HPLC-MS spectra was used as input for a search in the lipid database LipidMaps. The search within CL category was accomplished by setting a mass tolerance of ± 0.01 m/z units. A CL molecule (CL 74:6) corresponds to a major peak in the TIC of lipid ions, with a retention time (RT) of 11.71 min. The chemical structure of CL 74:6 (CL 16:0_18:0_20:3_20:3) is derived from LipidMaps. (B) The MS2 spectra of the precursor m/z (upper panel) and the reference m/z (lower panel) for CL 74:6.
Table 2.
Kinetic parameters of purified SVA 3Cpro wild-type and mutants calculated by Hill equation.
Data are presented as the average (±standard error of the mean) from three independent experiments. A 95% confidence interval (CI) was provided for each parameter (Khalf, Vmax and nH) in a bracket. The parameter kcat/Khalf represents the protease activity of SVA 3Cpro.
Fig 5.
Crystallographic structure of SVA 3Cpro in complex with the physiological substrate.
(A) Crystal packing of two protein molecules (Molecule I and II in green and magenta, respectively) that generate SVA 3Cpro-substrate complex. (B) Close-up view of the C-terminus containing the autocleavage sequence (shown as magenta sticks) from Molecule II binding into the proteolytic active pocket from Molecule I (shown as surface). The catalytic triad is highlighted in green. Electron density map (2Fo-Fc) of the cleavage peptide is shown at a 1.5σ level. (C) Contacts analysis of the C-terminus of Molecule II with the active site of Molecule I. (D) Contacts analysis between substrate glutamate (or the inhibitor rupintrivir, magenta sticks) and catalytic cysteine (green sticks) in SVA 3Cpro, EV71 3Cpro (PDB ID: 3SJO) and SARS-CoV-2 Mpro (PDB ID: 7KHP). The first two groups in rupintrivir binding by EV71 3Cpro are labelled using P1’ and P1, respectively.
Fig 6.
One-step and multi-step growth curves each mutant and SVA-WT.
(A-B) BHK-21 cells were infected with mutant or WT virus at an MOI of 5 or 0.01, respectively. The resulting virus was harvested at different times, titered, and expressed as a TCID50 dose. The mean values ± SD (repeated measures ANOVA, n = 3, no significant differences identified) are shown.
Fig 7.
A supposed mechanism on the regulation of SVA 3Cpro proteolytic activity and viral infection capacity by an endogenous Cardiolipin (CL).
SVA infection may cause inflammatory insults and subsequent partial CL redistribution from the inner membrane to the cytoplasm-facing outer membrane of mitochondria. In the cytoplasm, SVA 3Cpro binds to the CL in a unique region neighboring the proteolytic site. The lipid can activate its cleavage activity to ensure its efficient replication. Meanwhile, the infectivity titer of SVA harboring the lipid-binding mutants in 3Cpro are significantly lower than the wild-type. In the lipid-free status of SVA 3Cpro, its enzymatic activity is inhibited and the cleavage capacity for the viral polyprotein and several host proteins is decreased. The phospholipid may function as an allosteric activator to regulate SVA 3Cpro proteolytic activity, and as a switch to keep a balance between replication and infection.