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