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: The crystal structure of PRRSV nsp11 (residues Gly1 to Glu223) was determined using the SAD method and was refined to 2.75-Å resolution, which was of sufficient quality to trace the entire chain (excluding the C-terminal His6 tags). The Matthews coefficient and solvent content are 2.99 and 58%, respectively, as determined by cell content analysis. The solvent content value is high, which may explain why the diffraction of the crystals was poor. The crystal belongs to the space group P41212 and consists of two subunits in an asymmetric unit (Fig. 1B). Interestingly, subunit A is visibly different from subunit B (Fig. 1C, D, and E). In subunit B, residues in the regions Asp134-Gly141, Ser166-Lys173, and Leu222-Glu223 could not be traced due to a lack of interpretable electron density (Fig. 1C). The crystal structure can be divided into two major parts: the N-terminal domain (NTD; Gly1 to Phe90) and the C-terminal catalytic domain (Arg107 to Glu223). The NTD is formed by six β-strands (β1 to β6) and two α-helices (α1 to α2) and is connected to the catalytic domain through a linker domain (LKD; Val91 to Thr106). The catalytic domain has a typical fold consisting of a compact groove region containing sequentially connected left and right parts (Fig. 1A): the left part of the catalytic domain consists of two α-helices (α3 and α4) and six β-strands (β7 to β12), and the right part is formed by three antiparallel β-strands (β13 to β15) and one α-helix (α5). Details of the data collection and structure refinement are summarized in Table 1.
Affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.