A Dimerization-Dependent Mechanism Drives the Endoribonuclease Function of Porcine Reproductive and Respiratory Syndrome Virus nsp11.
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.
Affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.Show MeSH
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Mentions: To clarify the relationship between dimerization and catalytic activity, we performed FRET assays using fluorescence-labeled RNA as the substrate. As predicted, the activity levels of the mutants (S74A and F76A) were significantly decreased (being at least 4-fold less than wild-type levels) but not completely abolished (see Fig. 7C) because the mutant proteins were not purely monomeric. In addition, gel filtration chromatography revealed that the 280-nm absorption peak of the mutant S74A protein was obviously lower than that of the mutant F76A protein with the same amount of total protein (Fig. 2D and E), which indicated that the mutant S74A protein is very unstable. This may be the reason why the NendoU activity of the S74A mutant is lower than that of the F76A mutant. In conclusion, the S74A and F76A mutations severely diminished the catalytic activity, indicating that the dimer is the biologically functional unit.
Affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.