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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 structure of PRRSV nsp11 reveals the nidovirus-wide conservation of the NendoU domain. (A and B) The structure of PRRSV nsp11 (subunit A, yellow) superimposed onto the structures of SARS-CoV nsp15 (PDB code 2H85, magenta) and MHV nsp15 (PDB code 2GTH, magenta). The structure of the catalytic domain (Ile108-Glu223) of PRRSV nsp11 superimposed with SARS-CoV nsp15 (Asp199-Leu345) and MHV nsp15 (Ser229-Phe369) is enlarged in panels A and B. The potential catalytic active sites are labeled with a ball-and-stick (yellow, PRRSV nsp11; magenta, SARS-CoV nsp15 and MHV nsp15) representation. The “supporting loop” and “active site loop” are highlighted with a ribbon representation (the cartoon transparency was set at 80%) according to structural data for SARS-CoV nsp15 (23). The SARS-CoV nsp15 domains are colored and marked as described for Fig. 3C.
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Figure 6: The structure of PRRSV nsp11 reveals the nidovirus-wide conservation of the NendoU domain. (A and B) The structure of PRRSV nsp11 (subunit A, yellow) superimposed onto the structures of SARS-CoV nsp15 (PDB code 2H85, magenta) and MHV nsp15 (PDB code 2GTH, magenta). The structure of the catalytic domain (Ile108-Glu223) of PRRSV nsp11 superimposed with SARS-CoV nsp15 (Asp199-Leu345) and MHV nsp15 (Ser229-Phe369) is enlarged in panels A and B. The potential catalytic active sites are labeled with a ball-and-stick (yellow, PRRSV nsp11; magenta, SARS-CoV nsp15 and MHV nsp15) representation. The “supporting loop” and “active site loop” are highlighted with a ribbon representation (the cartoon transparency was set at 80%) according to structural data for SARS-CoV nsp15 (23). The SARS-CoV nsp15 domains are colored and marked as described for Fig. 3C.

Mentions: Multiple-sequence alignment indicated that the amino acid sequence identity between arterivirus nsp11 and coronavirus nsp15 is only approximately 16.1% to 25.1%, as demonstrated by their distance on the evolutionary tree (Fig. 5). Moreover, there are distinct differences between the NTD of nsp11 and the middle domain of coronavirus nsp15 (the RSMDs with SARS and MHV are 2.41 and 2.79, respectively) (Fig. 4). However, the structures of the catalytic domains can be nearly perfectly superimposed (the RSMDs with SARS and MHV are 2.14 and 2.09, respectively), especially in the active site loop and supporting loop regions (Fig. 6). Additionally, the structural comparison demonstrated that residues His129, His144, Lys173, Thr177, Asp180, Asp204, and Tyr219 from nsp11 superimpose well onto the corresponding residues of coronavirus nsp15 (Fig. 5 and 6), indicating the relative conservation of key active site residues and similar endoribonuclease cleavage mechanisms shared among nidoviruses (families Arteriviridae and Coronaviridae). In this study, endoribonuclease activity of the wild-type and mutant nsp11 protein was measured, and the results are shown in Fig. 7C. Enzyme activity assays for the wild-type and H129A, K173A, T177A, and Y219A mutant proteins were performed under identical conditions, and the activity levels of the mutants were significantly reduced compared with the wild-type level (Fig. 7C), indicating that these residues are located in important NendoU active sites.


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 structure of PRRSV nsp11 reveals the nidovirus-wide conservation of the NendoU domain. (A and B) The structure of PRRSV nsp11 (subunit A, yellow) superimposed onto the structures of SARS-CoV nsp15 (PDB code 2H85, magenta) and MHV nsp15 (PDB code 2GTH, magenta). The structure of the catalytic domain (Ile108-Glu223) of PRRSV nsp11 superimposed with SARS-CoV nsp15 (Asp199-Leu345) and MHV nsp15 (Ser229-Phe369) is enlarged in panels A and B. The potential catalytic active sites are labeled with a ball-and-stick (yellow, PRRSV nsp11; magenta, SARS-CoV nsp15 and MHV nsp15) representation. The “supporting loop” and “active site loop” are highlighted with a ribbon representation (the cartoon transparency was set at 80%) according to structural data for SARS-CoV nsp15 (23). The SARS-CoV nsp15 domains are colored and marked as described for Fig. 3C.
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Figure 6: The structure of PRRSV nsp11 reveals the nidovirus-wide conservation of the NendoU domain. (A and B) The structure of PRRSV nsp11 (subunit A, yellow) superimposed onto the structures of SARS-CoV nsp15 (PDB code 2H85, magenta) and MHV nsp15 (PDB code 2GTH, magenta). The structure of the catalytic domain (Ile108-Glu223) of PRRSV nsp11 superimposed with SARS-CoV nsp15 (Asp199-Leu345) and MHV nsp15 (Ser229-Phe369) is enlarged in panels A and B. The potential catalytic active sites are labeled with a ball-and-stick (yellow, PRRSV nsp11; magenta, SARS-CoV nsp15 and MHV nsp15) representation. The “supporting loop” and “active site loop” are highlighted with a ribbon representation (the cartoon transparency was set at 80%) according to structural data for SARS-CoV nsp15 (23). The SARS-CoV nsp15 domains are colored and marked as described for Fig. 3C.
Mentions: Multiple-sequence alignment indicated that the amino acid sequence identity between arterivirus nsp11 and coronavirus nsp15 is only approximately 16.1% to 25.1%, as demonstrated by their distance on the evolutionary tree (Fig. 5). Moreover, there are distinct differences between the NTD of nsp11 and the middle domain of coronavirus nsp15 (the RSMDs with SARS and MHV are 2.41 and 2.79, respectively) (Fig. 4). However, the structures of the catalytic domains can be nearly perfectly superimposed (the RSMDs with SARS and MHV are 2.14 and 2.09, respectively), especially in the active site loop and supporting loop regions (Fig. 6). Additionally, the structural comparison demonstrated that residues His129, His144, Lys173, Thr177, Asp180, Asp204, and Tyr219 from nsp11 superimpose well onto the corresponding residues of coronavirus nsp15 (Fig. 5 and 6), indicating the relative conservation of key active site residues and similar endoribonuclease cleavage mechanisms shared among nidoviruses (families Arteriviridae and Coronaviridae). In this study, endoribonuclease activity of the wild-type and mutant nsp11 protein was measured, and the results are shown in Fig. 7C. Enzyme activity assays for the wild-type and H129A, K173A, T177A, and Y219A mutant proteins were performed under identical conditions, and the activity levels of the mutants were significantly reduced compared with the wild-type level (Fig. 7C), indicating that these residues are located in important NendoU active sites.

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