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A Dimerization-Dependent Mechanism Drives the Endoribonuclease Function of Porcine Reproductive and Respiratory Syndrome Virus nsp11.

Shi Y, Li Y, Lei Y, Ye G, Shen Z, Sun L, Luo R, Wang D, Fu ZF, Xiao S, Peng G - J. Virol. (2016)

Bottom Line: The PRRSV nsp11 endoribonuclease plays a vital role in arterivirus replication, but its precise roles and mechanisms of action are poorly understood.Structural and biochemical experiments demonstrated that nsp11 exists mainly as a dimer in solution and that nsp11 may be fully active as a dimer.Mutagenesis and structural analysis revealed NendoU active site residues, which are conserved throughout the order Nidovirales(families Arteriviridae and Coronaviridae) and the major determinants of dimerization (Ser74 and Phe76) in Arteriviridae Importantly, these findings may provide a new structural basis for antiviral drug development.

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Affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.

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The dimerization mechanism of PRRSV nsp11. (A) Surface representation of dimeric PRRSV nsp11. By analogy with the monomeric structure of SARS-CoV nsp15, the loops consisting of residues His129 to His144 and Val162 to Thr179 are identified as the “active site loop” and “supporting loop,” respectively (23). These two loops are highlighted with red and blue ribbons, respectively (the cartoon transparency was set at 40%). The potential NendoU active sites are shown. (B) Detailed molecular interactions of the supporting loop (subunit A, magenta) with the active site loop (subunit A, red) and the N-terminal domain (subunit B, red) were determined using LIGPLOT. Hydrogen bond interactions and hydrophobic interactions are shown as described for Fig. 2A. (C) Surface representation of hexameric SARS-CoV nsp15 (PDB code 2RHB). The individual subunits are colored and marked A to F. The active site loop (residues His234 to His249) and the supporting loop (residues Lys276 to IIe295) are highlighted as described for panel A. The description of SARS-CoV nsp15 domains is based on a previous report (23). The monomer-monomer buried surface areas of PRRSV nsp11 and SARS-CoV nsp15 were analyzed using PDBePISA.
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Figure 3: The dimerization mechanism of PRRSV nsp11. (A) Surface representation of dimeric PRRSV nsp11. By analogy with the monomeric structure of SARS-CoV nsp15, the loops consisting of residues His129 to His144 and Val162 to Thr179 are identified as the “active site loop” and “supporting loop,” respectively (23). These two loops are highlighted with red and blue ribbons, respectively (the cartoon transparency was set at 40%). The potential NendoU active sites are shown. (B) Detailed molecular interactions of the supporting loop (subunit A, magenta) with the active site loop (subunit A, red) and the N-terminal domain (subunit B, red) were determined using LIGPLOT. Hydrogen bond interactions and hydrophobic interactions are shown as described for Fig. 2A. (C) Surface representation of hexameric SARS-CoV nsp15 (PDB code 2RHB). The individual subunits are colored and marked A to F. The active site loop (residues His234 to His249) and the supporting loop (residues Lys276 to IIe295) are highlighted as described for panel A. The description of SARS-CoV nsp15 domains is based on a previous report (23). The monomer-monomer buried surface areas of PRRSV nsp11 and SARS-CoV nsp15 were analyzed using PDBePISA.

Mentions: In our crystal structure, a total binding surface of 1,309 Å2 is buried at the interface (Fig. 3A), which is smaller than the subunit A-subunit B binding surface of SARS-CoV nsp15, which is 2,253.3 Å2 (Fig. 3C). Regardless, this smaller binding surface may be sufficient to stabilize monomer-monomer interactions because the molecular weight of nsp11 (approximately 25.6) is lower than that of SARS-CoV nsp15 (approximately 38.5). In addition, the catalytic domain of subunit A and the NTD of subunit B are associated with a largely hydrophobic and hydrogen-bonding network (Fig. 2A and B). A total of 16 residues in the NTD of subunit B interact with 17 residues in the catalytic domain of subunit A. Residue Phe76 interacts with residues Tyr150, Leu151, Pro152, Gly164, Val165, and Ser166 via the hydrophobic forces (Fig. 2A and B); thus, Phe76 is a key residue within the dimer interface. Moreover, residue Ser74 interacts with residues Pro152, Val163, and Gly164 and is thus also a key residue within the dimer interface (Fig. 2A and B). In addition, interactional residues Leu77, Val110, and Cys112 were also observed with residue Arg153 (Fig. 2A and B). Therefore, these mutations (S74A, F76A, and R153A) may disturb both hydrophilic and hydrophobic interactions between monomers and prevent the formation of stable dimers.


A Dimerization-Dependent Mechanism Drives the Endoribonuclease Function of Porcine Reproductive and Respiratory Syndrome Virus nsp11.

Shi Y, Li Y, Lei Y, Ye G, Shen Z, Sun L, Luo R, Wang D, Fu ZF, Xiao S, Peng G - J. Virol. (2016)

The dimerization mechanism of PRRSV nsp11. (A) Surface representation of dimeric PRRSV nsp11. By analogy with the monomeric structure of SARS-CoV nsp15, the loops consisting of residues His129 to His144 and Val162 to Thr179 are identified as the “active site loop” and “supporting loop,” respectively (23). These two loops are highlighted with red and blue ribbons, respectively (the cartoon transparency was set at 40%). The potential NendoU active sites are shown. (B) Detailed molecular interactions of the supporting loop (subunit A, magenta) with the active site loop (subunit A, red) and the N-terminal domain (subunit B, red) were determined using LIGPLOT. Hydrogen bond interactions and hydrophobic interactions are shown as described for Fig. 2A. (C) Surface representation of hexameric SARS-CoV nsp15 (PDB code 2RHB). The individual subunits are colored and marked A to F. The active site loop (residues His234 to His249) and the supporting loop (residues Lys276 to IIe295) are highlighted as described for panel A. The description of SARS-CoV nsp15 domains is based on a previous report (23). The monomer-monomer buried surface areas of PRRSV nsp11 and SARS-CoV nsp15 were analyzed using PDBePISA.
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Figure 3: The dimerization mechanism of PRRSV nsp11. (A) Surface representation of dimeric PRRSV nsp11. By analogy with the monomeric structure of SARS-CoV nsp15, the loops consisting of residues His129 to His144 and Val162 to Thr179 are identified as the “active site loop” and “supporting loop,” respectively (23). These two loops are highlighted with red and blue ribbons, respectively (the cartoon transparency was set at 40%). The potential NendoU active sites are shown. (B) Detailed molecular interactions of the supporting loop (subunit A, magenta) with the active site loop (subunit A, red) and the N-terminal domain (subunit B, red) were determined using LIGPLOT. Hydrogen bond interactions and hydrophobic interactions are shown as described for Fig. 2A. (C) Surface representation of hexameric SARS-CoV nsp15 (PDB code 2RHB). The individual subunits are colored and marked A to F. The active site loop (residues His234 to His249) and the supporting loop (residues Lys276 to IIe295) are highlighted as described for panel A. The description of SARS-CoV nsp15 domains is based on a previous report (23). The monomer-monomer buried surface areas of PRRSV nsp11 and SARS-CoV nsp15 were analyzed using PDBePISA.
Mentions: In our crystal structure, a total binding surface of 1,309 Å2 is buried at the interface (Fig. 3A), which is smaller than the subunit A-subunit B binding surface of SARS-CoV nsp15, which is 2,253.3 Å2 (Fig. 3C). Regardless, this smaller binding surface may be sufficient to stabilize monomer-monomer interactions because the molecular weight of nsp11 (approximately 25.6) is lower than that of SARS-CoV nsp15 (approximately 38.5). In addition, the catalytic domain of subunit A and the NTD of subunit B are associated with a largely hydrophobic and hydrogen-bonding network (Fig. 2A and B). A total of 16 residues in the NTD of subunit B interact with 17 residues in the catalytic domain of subunit A. Residue Phe76 interacts with residues Tyr150, Leu151, Pro152, Gly164, Val165, and Ser166 via the hydrophobic forces (Fig. 2A and B); thus, Phe76 is a key residue within the dimer interface. Moreover, residue Ser74 interacts with residues Pro152, Val163, and Gly164 and is thus also a key residue within the dimer interface (Fig. 2A and B). In addition, interactional residues Leu77, Val110, and Cys112 were also observed with residue Arg153 (Fig. 2A and B). Therefore, these mutations (S74A, F76A, and R153A) may disturb both hydrophilic and hydrophobic interactions between monomers and prevent the formation of stable dimers.

Bottom Line: The PRRSV nsp11 endoribonuclease plays a vital role in arterivirus replication, but its precise roles and mechanisms of action are poorly understood.Structural and biochemical experiments demonstrated that nsp11 exists mainly as a dimer in solution and that nsp11 may be fully active as a dimer.Mutagenesis and structural analysis revealed NendoU active site residues, which are conserved throughout the order Nidovirales(families Arteriviridae and Coronaviridae) and the major determinants of dimerization (Ser74 and Phe76) in Arteriviridae Importantly, these findings may provide a new structural basis for antiviral drug development.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.

Show MeSH
Related in: MedlinePlus