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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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The potential cytotoxicity of wild-type nsp11 may inhibit IFN-β promoter activation. (A and B) HEK293T cells were cotransfected with IRF3-Luc plasmid (0.1 μg) (A) of IFN-β–Luc (B) and pRL-TK plasmid (0.02 μg), together with the wild-type and mutant nsp11 plasmids (0.4 μg). At 24 h after the initial transfection, the cells were infected with Sendai virus (SEV). At 40 h posttransfection, the cells were harvested and the luciferase activity was measured. The firefly luciferase activity was normalized to Renilla reniformis luciferase, and the untreated empty vector control value was set to 1. *, P < 0.05 (considered significant compared with the luciferase activity of the cells expressing the wild-type protein); **, P < 0.01 (considered highly significant); ***, P < 0.001 (considered extremely significant). (C) Relative luciferase activity of pRL-TK. Relative luciferase activity = 100 × (the luciferase value for the wild-type and mutant strains/the luciferase value for the control group). (D) Western blot analysis of the expression levels from wild-type and mutant nsp11. (E and F) Cell viability analysis of wild-type and mutant nsp11 in HEK293T cells. Cell viability of wild-type and mutant nsp11 was evaluated via the use of a CellTiter-Glo luminescent cell viability assay. Percent cell viability = 100 × (luminescence of the experimental group/luminescence of the control group).
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Figure 8: The potential cytotoxicity of wild-type nsp11 may inhibit IFN-β promoter activation. (A and B) HEK293T cells were cotransfected with IRF3-Luc plasmid (0.1 μg) (A) of IFN-β–Luc (B) and pRL-TK plasmid (0.02 μg), together with the wild-type and mutant nsp11 plasmids (0.4 μg). At 24 h after the initial transfection, the cells were infected with Sendai virus (SEV). At 40 h posttransfection, the cells were harvested and the luciferase activity was measured. The firefly luciferase activity was normalized to Renilla reniformis luciferase, and the untreated empty vector control value was set to 1. *, P < 0.05 (considered significant compared with the luciferase activity of the cells expressing the wild-type protein); **, P < 0.01 (considered highly significant); ***, P < 0.001 (considered extremely significant). (C) Relative luciferase activity of pRL-TK. Relative luciferase activity = 100 × (the luciferase value for the wild-type and mutant strains/the luciferase value for the control group). (D) Western blot analysis of the expression levels from wild-type and mutant nsp11. (E and F) Cell viability analysis of wild-type and mutant nsp11 in HEK293T cells. Cell viability of wild-type and mutant nsp11 was evaluated via the use of a CellTiter-Glo luminescent cell viability assay. Percent cell viability = 100 × (luminescence of the experimental group/luminescence of the control group).

Mentions: In addition, previous studies reported that the endoribonuclease activity of PRRSV nsp11 is essential to inhibit IFN-β induction (38). We found that the overexpression of wild-type nsp11 markedly inhibited the activity of the IFN-β luciferase reporter induced by Sendai virus (SEV), while the mutants (S74A, S76A, H129A, K173A, T177A, and Y219A) lost the capacity to block the activation of IFN-β promoter (Fig. 8A and B). Because the recombinant arterivirus nsp11 protein displays broad substrate specificity in vitro and is extremely toxic to prokaryotic and eukaryotic cells (20), it is possible that the suppression of IFN-β induction by wild-type nsp11 is due to its cytotoxicity. HEK293T cells expressing wild-type nsp11 appeared to be in good shape and showed no obvious cytotoxicity in detection experiments performed with the CellTiter-Glo luminescent cell viability assay (Fig. 8E and F). However, when we analyzed the ability of wild-type and mutant nsp11 to inhibit IFN-β induction by the use of a Dual-Luciferase reporter assay system (Promega), we found that the value for pRL-TK, an internal control reporter, was significantly lower in cells expressing wild-type nsp11 than in cells expressing nsp11 mutants or in cells that had received mock treatment (Fig. 8C), indicating that wild-type nsp11 inhibits host gene expression. Coincidentally, none of the tested nsp11 mutants without cytotoxicity significantly inhibited IFN-β induction (Fig. 8A, B, and C). Therefore, we could not exclude the possibility that the potential cytotoxicity of wild-type nsp11 inhibits IFN-β induction. It should be noted that this study involved the individual expression of nsp11, outside the context of infection. Whether the endoribonuclease function of nsp11 specifically contributes to the decline of innate immune functions in PRRSV infection requires further investigation.


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 potential cytotoxicity of wild-type nsp11 may inhibit IFN-β promoter activation. (A and B) HEK293T cells were cotransfected with IRF3-Luc plasmid (0.1 μg) (A) of IFN-β–Luc (B) and pRL-TK plasmid (0.02 μg), together with the wild-type and mutant nsp11 plasmids (0.4 μg). At 24 h after the initial transfection, the cells were infected with Sendai virus (SEV). At 40 h posttransfection, the cells were harvested and the luciferase activity was measured. The firefly luciferase activity was normalized to Renilla reniformis luciferase, and the untreated empty vector control value was set to 1. *, P < 0.05 (considered significant compared with the luciferase activity of the cells expressing the wild-type protein); **, P < 0.01 (considered highly significant); ***, P < 0.001 (considered extremely significant). (C) Relative luciferase activity of pRL-TK. Relative luciferase activity = 100 × (the luciferase value for the wild-type and mutant strains/the luciferase value for the control group). (D) Western blot analysis of the expression levels from wild-type and mutant nsp11. (E and F) Cell viability analysis of wild-type and mutant nsp11 in HEK293T cells. Cell viability of wild-type and mutant nsp11 was evaluated via the use of a CellTiter-Glo luminescent cell viability assay. Percent cell viability = 100 × (luminescence of the experimental group/luminescence of the control group).
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Figure 8: The potential cytotoxicity of wild-type nsp11 may inhibit IFN-β promoter activation. (A and B) HEK293T cells were cotransfected with IRF3-Luc plasmid (0.1 μg) (A) of IFN-β–Luc (B) and pRL-TK plasmid (0.02 μg), together with the wild-type and mutant nsp11 plasmids (0.4 μg). At 24 h after the initial transfection, the cells were infected with Sendai virus (SEV). At 40 h posttransfection, the cells were harvested and the luciferase activity was measured. The firefly luciferase activity was normalized to Renilla reniformis luciferase, and the untreated empty vector control value was set to 1. *, P < 0.05 (considered significant compared with the luciferase activity of the cells expressing the wild-type protein); **, P < 0.01 (considered highly significant); ***, P < 0.001 (considered extremely significant). (C) Relative luciferase activity of pRL-TK. Relative luciferase activity = 100 × (the luciferase value for the wild-type and mutant strains/the luciferase value for the control group). (D) Western blot analysis of the expression levels from wild-type and mutant nsp11. (E and F) Cell viability analysis of wild-type and mutant nsp11 in HEK293T cells. Cell viability of wild-type and mutant nsp11 was evaluated via the use of a CellTiter-Glo luminescent cell viability assay. Percent cell viability = 100 × (luminescence of the experimental group/luminescence of the control group).
Mentions: In addition, previous studies reported that the endoribonuclease activity of PRRSV nsp11 is essential to inhibit IFN-β induction (38). We found that the overexpression of wild-type nsp11 markedly inhibited the activity of the IFN-β luciferase reporter induced by Sendai virus (SEV), while the mutants (S74A, S76A, H129A, K173A, T177A, and Y219A) lost the capacity to block the activation of IFN-β promoter (Fig. 8A and B). Because the recombinant arterivirus nsp11 protein displays broad substrate specificity in vitro and is extremely toxic to prokaryotic and eukaryotic cells (20), it is possible that the suppression of IFN-β induction by wild-type nsp11 is due to its cytotoxicity. HEK293T cells expressing wild-type nsp11 appeared to be in good shape and showed no obvious cytotoxicity in detection experiments performed with the CellTiter-Glo luminescent cell viability assay (Fig. 8E and F). However, when we analyzed the ability of wild-type and mutant nsp11 to inhibit IFN-β induction by the use of a Dual-Luciferase reporter assay system (Promega), we found that the value for pRL-TK, an internal control reporter, was significantly lower in cells expressing wild-type nsp11 than in cells expressing nsp11 mutants or in cells that had received mock treatment (Fig. 8C), indicating that wild-type nsp11 inhibits host gene expression. Coincidentally, none of the tested nsp11 mutants without cytotoxicity significantly inhibited IFN-β induction (Fig. 8A, B, and C). Therefore, we could not exclude the possibility that the potential cytotoxicity of wild-type nsp11 inhibits IFN-β induction. It should be noted that this study involved the individual expression of nsp11, outside the context of infection. Whether the endoribonuclease function of nsp11 specifically contributes to the decline of innate immune functions in PRRSV infection requires further investigation.

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