Purification, crystallization and preliminary X-ray analysis of nonstructural protein 2 (nsp2) from avian infectious bronchitis virus

Abstract

Avian infectious bronchitis virus (IBV) is a member of the group III coronaviruses, which differ from the other groups of coronaviruses in that they do not encode the essential pathogenic factor nonstructural protein 1 (nsp1) and instead start with nsp2. IBV nsp2 is one of the first replicase proteins to be translated and processed in the viral life cycle; however, it has an entirely unknown function. In order to better understand the structural details and functional mechanism of IBV nsp2, the recombinant protein was cloned, overexpressed in Escherichia coli, purified and crystallized. The crystals diffracted to 2.8 Å resolution and belonged to space group P2 1 , with unit-cell parameters a = 57. 0, b = 192.3, c = 105.7 Å , = 90.8 . Two molecules were found in the asymmetric unit; the Matthews coefficient was 3.9 Å 3 Da À1, corresponding to a solvent content of 68.2%.

Functional and evolutionary analysis of viral proteins containing a Rossmann‐like fold

“…The N‐terminal domain of Nsp2a adopts a Rossmann‐like sheet topology (2314), with β‐strand 2 being an insert to the core that is antiparallel to the rest. Although the function of Nsp2a is unknown, its lack of sequence/structure similarity to other proteins has suggested a role in host specificity …”

“…Although the function of Nsp2a is unknown, its lack of sequence/ structure similarity to other proteins has suggested a role in host specificity. 54…”

Biological and molecular characterization of ArkGA: A novel Arkansas serotype vaccine that is highly attenuated, efficacious, and protective against homologous challenge

“…Most notably, ArkGA P60 had SNPs in the nsp2, nsp3, and both S1 and S2 gene regions. Changes in nsp2 and nsp3, which are part of the viral replicase complex, have been shown to impact viral replication and pathogenesis [32][33][34][35][36][37][38]. In the spike gene, which is the major determinant of cell tropism and plays a role in viral attenuation [39,40], 5 amino acid changes occurred in the S1 and S2 subunits.…”

Atlas of coronavirus replicase structure

“…The betacoronaviruses of subgroup A, such as murine hepatitis virus (MHV) and human coronavirus OC43, encode an nsp1 protein of about 245 residues, also known as p28 (Brockway and Denison, 2005). The nsp1 of SARS-CoV and its bat Yu et al (2012) equivalents, which have been classified as the only members to date of the betacoronavirus subgroup B Gorbalenya et al, 2004;Snijder et al, 2003), have 179 residues. Nsp1 sequences are divergent between subgroups of betacoronavirus, and no sequence similarity between SARS-CoV nsp1 and betacoronavirus subgroup A nsp1 proteins could be identified using standard searching tools such as PSI-BLAST.…”

“…3). The structure of the N-terminal 359 amino acids of the IBV equivalent of nsp2 has been released, but is currently awaiting full publication, though a crystallization report is available Yu et al, 2012). Crystallization of part of the SARS-CoV nsp2 has also been reported, but the structure is not currently available pending full publication.…”