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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.

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.

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Dimerization mutants disrupt nsp11 dimerization in solution. (A) All the interactional residues between subunits A (yellow) and B (magenta) were determined using LIGPLOT. The residues that interact with Ser74, Phe76, and Arg153 are denoted with ovals (green), rectangles (red), and pentacles (black), respectively. Carbon, nitrogen, and oxygen atoms are shown as black, blue, and red circles, respectively. Hydrogen bond interactions are shown as black dashed lines between the respective donor and acceptor atoms, along with the bond distance. Hydrophobic interactions are indicated by arcs with spokes radiating toward the atoms they contact. (B) Dimerization interface of nsp11. The partial interactional residues (including interactions with Ser74, Phe76, and Arg153) between subunits A and B are shown with a stick. The figure is colored as described for panel A. (C) Calculated molecular weights of the nsp11 protein peaks with the values obtained for known calibration standards (Bio-Rad and GE Healthcare). The calculated molecular weight of nsp11 peaks was determined by fitting to the calibration curve (Kav = volumes of elution [Ves]/24); volumes of elution of 10.26 ml (approximately 58.9), 10.69 ml (approximately 48.8), and 11.46 ml (approximately 34.8) are indicated by arrows. (D) Size exclusion experiment with the nsp11 wild type and mutants (S74A, F76A, and R153A). The calculated molecular masses were determined by fitting to the calibration curve as described for panel C. The wild type is colored black, and the mutants (S74A, F76A, and R153A) are colored red, blue, and bottle green, respectively. (E) SDS-PAGE analysis of wild-type and mutant (S74A, F76A, and R153A) nsp11. The elution volume is labeled as described for panel D. Molecular mass markers are shown. (F and G) Sedimentation velocity analysis of wild-type and mutant (S74A, F76A, and R153A) nsp11. The major peaks of wild-type nsp11 and the mutants (S74A, F76A, and R153A) are shown in panel F. Panel F is colored as described for panel D. The sedimentation coefficient (S20,W) and the calculated molecular weights (MW) are shown in panel G.
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Figure 2: Dimerization mutants disrupt nsp11 dimerization in solution. (A) All the interactional residues between subunits A (yellow) and B (magenta) were determined using LIGPLOT. The residues that interact with Ser74, Phe76, and Arg153 are denoted with ovals (green), rectangles (red), and pentacles (black), respectively. Carbon, nitrogen, and oxygen atoms are shown as black, blue, and red circles, respectively. Hydrogen bond interactions are shown as black dashed lines between the respective donor and acceptor atoms, along with the bond distance. Hydrophobic interactions are indicated by arcs with spokes radiating toward the atoms they contact. (B) Dimerization interface of nsp11. The partial interactional residues (including interactions with Ser74, Phe76, and Arg153) between subunits A and B are shown with a stick. The figure is colored as described for panel A. (C) Calculated molecular weights of the nsp11 protein peaks with the values obtained for known calibration standards (Bio-Rad and GE Healthcare). The calculated molecular weight of nsp11 peaks was determined by fitting to the calibration curve (Kav = volumes of elution [Ves]/24); volumes of elution of 10.26 ml (approximately 58.9), 10.69 ml (approximately 48.8), and 11.46 ml (approximately 34.8) are indicated by arrows. (D) Size exclusion experiment with the nsp11 wild type and mutants (S74A, F76A, and R153A). The calculated molecular masses were determined by fitting to the calibration curve as described for panel C. The wild type is colored black, and the mutants (S74A, F76A, and R153A) are colored red, blue, and bottle green, respectively. (E) SDS-PAGE analysis of wild-type and mutant (S74A, F76A, and R153A) nsp11. The elution volume is labeled as described for panel D. Molecular mass markers are shown. (F and G) Sedimentation velocity analysis of wild-type and mutant (S74A, F76A, and R153A) nsp11. The major peaks of wild-type nsp11 and the mutants (S74A, F76A, and R153A) are shown in panel F. Panel F is colored as described for panel D. The sedimentation coefficient (S20,W) and the calculated molecular weights (MW) are shown in panel G.

Mentions: In this study, gel filtration chromatography revealed the dimeric architecture of nsp11. Our data indicated that nsp11 eluted primarily in one peak; the calculated molecular mass is approximately 58.9 kDa, which corresponds to a dimer (Fig. 2C, D, and E). This finding is consistent with the dimeric crystal structure of nsp11 (Fig. 1B). The dimerization interface is shown in Fig. 2A and B. Residues Gly1, Leu54, Arg55, Tyr69, Ser74, Phe76, Gly137, Gly138, Arg153, Val165, and Ser166 were chosen as candidate targets to abolish the dimerization. The mutant (G1A, L54A, R55A, Y69A, G137A, G138A, V165A, and S166A) proteins eluted as a dimer; these mutations could not prevent nsp11 dimerization (data not shown). However, elution of the mutant (S74A and F76A) proteins by gel filtration yielded two 280-nm absorption peaks (Fig. 2D and E). Our results indicated that these two mutations significantly disrupt the dimerization in solution. Moreover, the R153A mutant existed mainly as an intermediate form (the calculated molecular mass is approximately 48.8 kDa) compared with the wild type (Fig. 2D and E). Meanwhile, the oligomerization of wild-type and mutant (S74A, F76A, and R153A) nsp11 proteins was further analyzed via sedimentation analytical ultracentrifugation (AUC), and the results were shown in Fig. 2F and G. The molecular weights of monomers and dimers from the wild-type nsp11 protein are approximately 29.2 (approximately 14.04% of the total population) and 63.7 (approximately 86.67%) and are essentially consistent with those of gel filtration chromatography. The sedimentation coefficient (S20,W) of the mutant (S74A, F76A, and R153A) proteins decreased significantly compared with the wild type, though the relative populations of monomers and dimers of those mutant proteins were not successfully determined. This indicated that the oligomerization of the mutant proteins had markedly changed. Therefore, our biochemical data consistently showed that nsp11 exists mainly as a dimer in solution and that the mutations in the dimerization interface, S74A, F76A, and R153A, disrupt dimerization.


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)

Dimerization mutants disrupt nsp11 dimerization in solution. (A) All the interactional residues between subunits A (yellow) and B (magenta) were determined using LIGPLOT. The residues that interact with Ser74, Phe76, and Arg153 are denoted with ovals (green), rectangles (red), and pentacles (black), respectively. Carbon, nitrogen, and oxygen atoms are shown as black, blue, and red circles, respectively. Hydrogen bond interactions are shown as black dashed lines between the respective donor and acceptor atoms, along with the bond distance. Hydrophobic interactions are indicated by arcs with spokes radiating toward the atoms they contact. (B) Dimerization interface of nsp11. The partial interactional residues (including interactions with Ser74, Phe76, and Arg153) between subunits A and B are shown with a stick. The figure is colored as described for panel A. (C) Calculated molecular weights of the nsp11 protein peaks with the values obtained for known calibration standards (Bio-Rad and GE Healthcare). The calculated molecular weight of nsp11 peaks was determined by fitting to the calibration curve (Kav = volumes of elution [Ves]/24); volumes of elution of 10.26 ml (approximately 58.9), 10.69 ml (approximately 48.8), and 11.46 ml (approximately 34.8) are indicated by arrows. (D) Size exclusion experiment with the nsp11 wild type and mutants (S74A, F76A, and R153A). The calculated molecular masses were determined by fitting to the calibration curve as described for panel C. The wild type is colored black, and the mutants (S74A, F76A, and R153A) are colored red, blue, and bottle green, respectively. (E) SDS-PAGE analysis of wild-type and mutant (S74A, F76A, and R153A) nsp11. The elution volume is labeled as described for panel D. Molecular mass markers are shown. (F and G) Sedimentation velocity analysis of wild-type and mutant (S74A, F76A, and R153A) nsp11. The major peaks of wild-type nsp11 and the mutants (S74A, F76A, and R153A) are shown in panel F. Panel F is colored as described for panel D. The sedimentation coefficient (S20,W) and the calculated molecular weights (MW) are shown in panel G.
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Figure 2: Dimerization mutants disrupt nsp11 dimerization in solution. (A) All the interactional residues between subunits A (yellow) and B (magenta) were determined using LIGPLOT. The residues that interact with Ser74, Phe76, and Arg153 are denoted with ovals (green), rectangles (red), and pentacles (black), respectively. Carbon, nitrogen, and oxygen atoms are shown as black, blue, and red circles, respectively. Hydrogen bond interactions are shown as black dashed lines between the respective donor and acceptor atoms, along with the bond distance. Hydrophobic interactions are indicated by arcs with spokes radiating toward the atoms they contact. (B) Dimerization interface of nsp11. The partial interactional residues (including interactions with Ser74, Phe76, and Arg153) between subunits A and B are shown with a stick. The figure is colored as described for panel A. (C) Calculated molecular weights of the nsp11 protein peaks with the values obtained for known calibration standards (Bio-Rad and GE Healthcare). The calculated molecular weight of nsp11 peaks was determined by fitting to the calibration curve (Kav = volumes of elution [Ves]/24); volumes of elution of 10.26 ml (approximately 58.9), 10.69 ml (approximately 48.8), and 11.46 ml (approximately 34.8) are indicated by arrows. (D) Size exclusion experiment with the nsp11 wild type and mutants (S74A, F76A, and R153A). The calculated molecular masses were determined by fitting to the calibration curve as described for panel C. The wild type is colored black, and the mutants (S74A, F76A, and R153A) are colored red, blue, and bottle green, respectively. (E) SDS-PAGE analysis of wild-type and mutant (S74A, F76A, and R153A) nsp11. The elution volume is labeled as described for panel D. Molecular mass markers are shown. (F and G) Sedimentation velocity analysis of wild-type and mutant (S74A, F76A, and R153A) nsp11. The major peaks of wild-type nsp11 and the mutants (S74A, F76A, and R153A) are shown in panel F. Panel F is colored as described for panel D. The sedimentation coefficient (S20,W) and the calculated molecular weights (MW) are shown in panel G.
Mentions: In this study, gel filtration chromatography revealed the dimeric architecture of nsp11. Our data indicated that nsp11 eluted primarily in one peak; the calculated molecular mass is approximately 58.9 kDa, which corresponds to a dimer (Fig. 2C, D, and E). This finding is consistent with the dimeric crystal structure of nsp11 (Fig. 1B). The dimerization interface is shown in Fig. 2A and B. Residues Gly1, Leu54, Arg55, Tyr69, Ser74, Phe76, Gly137, Gly138, Arg153, Val165, and Ser166 were chosen as candidate targets to abolish the dimerization. The mutant (G1A, L54A, R55A, Y69A, G137A, G138A, V165A, and S166A) proteins eluted as a dimer; these mutations could not prevent nsp11 dimerization (data not shown). However, elution of the mutant (S74A and F76A) proteins by gel filtration yielded two 280-nm absorption peaks (Fig. 2D and E). Our results indicated that these two mutations significantly disrupt the dimerization in solution. Moreover, the R153A mutant existed mainly as an intermediate form (the calculated molecular mass is approximately 48.8 kDa) compared with the wild type (Fig. 2D and E). Meanwhile, the oligomerization of wild-type and mutant (S74A, F76A, and R153A) nsp11 proteins was further analyzed via sedimentation analytical ultracentrifugation (AUC), and the results were shown in Fig. 2F and G. The molecular weights of monomers and dimers from the wild-type nsp11 protein are approximately 29.2 (approximately 14.04% of the total population) and 63.7 (approximately 86.67%) and are essentially consistent with those of gel filtration chromatography. The sedimentation coefficient (S20,W) of the mutant (S74A, F76A, and R153A) proteins decreased significantly compared with the wild type, though the relative populations of monomers and dimers of those mutant proteins were not successfully determined. This indicated that the oligomerization of the mutant proteins had markedly changed. Therefore, our biochemical data consistently showed that nsp11 exists mainly as a dimer in solution and that the mutations in the dimerization interface, S74A, F76A, and R153A, disrupt dimerization.

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