An intermolecular salt bridge linking substrate binding and P1 substrate specificity switch of arterivirus 3C-like proteases

Figures

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  Fig. 1. Sequence logos of the polyprotein junctions cleaved by PRRSV 3CLpro and EAV 3CLpro from different isolates. (a) Schematic diagrams of the PRRSV and EAV polyproteins. The PRRSV 3CLpro and EAV 3CLpro encoded by nsp4 are marked by orange rectangular; all the 3CLpro-mediated cleavage sites in polyproteins are shown in the orange triangle. (b) Conservation of the polyprotein junctions cleaved by PRRSV 3CLpro from 1037 PRRSV strains and EAV 3CLpro from 96 EAV strains. Amino acid sequence logos of the substrates were created using PSSMSearch (http://slim.icr.ac.uk/pssmsearch/), and the height of the letters represented the relative frequency of the amino acid. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 2. Structure comparison of the S1 subsites in PRRSV 3CLpro and EAV 3CLpro. (a) The overall structures of PRRSV 3CLpro (PDB id: 5Y4L) and EAV 3CLpro (PDB id: 1MBM) and details of their catalytic centers. The surface view of PRRSV 3CLpro and EAV 3CLpro comprised three domains (domain I marked in green, domain II marked in blue, and domain III marked in yellow). The canonical Ser-His-Asp triad responsible for recognizing cleavage sites was presented in the middle of the image, which is located in the cleft between domains I and II. (b) The S1 subsites of PRRSV 3CLpro (grey) and EAV 3CLpro (orange) in complex with its substrates are shown as surface and cartoon, respectively. The amino acid residues composited the S1 subsite and the catalytic triad Gly116-His39-Ser118 (PRRSV 3CLpro numbering is used) are displayed as sticks. Enlarged view of residues Ser136 and Lys138 in PRRSV 3CLpro and residues Ser137 and Thr139 in EAV 3CLpro. Based on the structure of arterivirus 3CLpro (PRRSV, PDB id: 5Y4L; EAV, PDB id: 1MBM), the molding structure of the 3CLpro-substrate complex was generated. (c) Sequence alignment for the S1 subsites of the PRRSV 3CLpro and EAV 3CLpro amongst all PRRSV and EAV…

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  Fig. 3. Exploiting the biosensor assay to evaluate the protease-substrate specificity of arterivirus 3CLproin vivo. (a) Diagram of the generation of luciferase-based biosensors. The purple and the orange structures represent the recombined firefly luciferase. The green rectangle indicates the different recognition sequences that were used to evaluate the activity of PRRSV 3CLpro or EAV 3CLpro. (b and c) HEK293T cells in 24-well plates were transfected with the pRL-TK plasmid, the plasmid encoding PRRSV 3CLproor EAV 3CLpro, and corresponding luciferase-based biosensors plasmids, or their control 233D. After 28 h transfection, cell lysates were prepared and analyzed by dual-luciferase assays (b) and western blotting (c). ns, not significant. ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 4. Identifying the amino acid residues involved in the S1 subsites of arterivirus 3CLpro. (a-c) HEK293T cells were cotransfected with the indicated plasmids encoding WT or mutants of arterivirus 3CLpro, pRL-TK and corresponding biosensor plasmids (a. PRRSV-nsp3/4 biosensor (with P1-Glu); b. EAV-nsp3/4 biosensor (with P1-Glu); c. EAV-nsp10/11 biosensor (with P1-Gln)). Dual-luciferase assays and western blotting were performed at 28 h after the transfection. (d-f) Heatmaps of the variation in H-bond occupancy between P1 residue and S1 subsite of WT or mutants of arterivirus 3CLpro. All H-bonds between S1 subsite and P1 residue occurring with an occupancy rate of at least 10% during MD simulations were investigated. Heatmaps were plotted using GraphPad Prism Version 9.3 (www.graphpad.com). The first one or two characters of the atom name of H-bond comprise the chemical symbol for the atom type. All the atom names beginning with C are carbon atoms, N shows nitrogen and O shows oxygen. The next character is the remoteness indicator code, which follows the Greek alphabet, starting with the alpha carbon (A) and moving on to beta (B), gamma (G), delta (D), epsilon (E), zeta (Z), and eta…

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  Fig. 5. Identifying an intermolecular salt bridge linking P1 substrate specificity shift of arterivirus 3CLpro. (a-b) HEK293T cells were transfected with plasmid encoding WT or the mutant T139K of EAV 3CLpro (salt bridge formation), pRL-TK and corresponding luciferase-based biosensors plasmids. The cells were harvested after 28 h transfection and analyzed by dual-luciferase assays and western blotting. (c-d) HEK293T cells were cotransfected with a plasmid encoding PRRSV 3CLpro WT or the mutant K138T (salt bridge disruption), pRL-TK and corresponding luciferase-based biosensors plasmids. The cells were harvested after 28 h transfection and analyzed by dual-luciferase assays and western blotting. *p < 0.05; ***p < 0.001

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  Fig. 6. Electrostatic and vdW interaction energies between P1 residue and S1 subsite of WT arterivirus 3CLpro or its mutants. (a) Heatmaps represent the different binding free energies between P1 residue and S1 subsite of WT arterivirus 3CLpro or its mutants, and the contributions from two individual energetic components: electrostatics (Eelectro) and van der Waals (EvdW) energy. Cooler (blue) and warmer (red) colors indicate higher or lower energies contributions to the interaction between P1 residue and S1 subsite, respectively. Heatmaps were plotted using GraphPad Prism Version 9.3 (www.graphpad.com). (b) Comparison of energies surface diagrams of WT arterivirus 3CLpro and its mutants from MD simulations. The surface representation of the S1 subsite is colored according to the energy of the respective residue positions. Energetically favorable residues are shown as red, neutral as yellow, and unfavorable as green. The P6-P6′ positions of substrate are displayed as a white cartoon. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 7. Evolutionary mechanism of salt-bridge-triggered P1 substrate specificity switch of PRRSV 3CLpro. (a) HEK293T cells were cotransfected with PRRSV-nsp3/4-Q biosensor plasmid, pRL-TK and a plasmid encoding PRRSV 3CLpro WT or the mutant K138T. After 28 h transfection, cell lysates were prepared and analyzed by dual-luciferase assays and western blotting. **p < 0.01. (b) HEK293T cells were cotransfected with NEMO-Q205 biosensor plasmid, pRL-TK and a plasmid encoding PRRSV 3CLpro WT or the mutant K138T. Cells lysates were processed as described above for panel A. ***p < 0.001. (c) HEK293T cells were cotransfected with PRRSV 3CLpro WT or the mutant K138T, along with Flag-tagged NEMO (E166A-E171A-E349A). Cell lysates were prepared 30 h post transfection and analyzed by western blotting. (d-e) HEK293T cells were cotransfected with plasmids encoding WT PRRSV 3CLpro or its mutants, pRL-TK and corresponding biosensor plasmid. After 28 h transfection, cell lysates were prepared and analyzed by dual-luciferase assays and western blotting.