Crystal Structure Research For The C Terminal Domain Of VP3 Protein From Porcine Rotavirus

Rotavirus(RV) is one of the important zoonosis pathogens, and the typical symptoms of disease caused by RV are diarrhea and vomiting. The existence of RV not only affects the development of animal husbandry, but also threatens human health and causes serious economic losses. Therefore, RV has become the focus of scientific research. In this study, the role of structural VP3 protein C terminal domain(VP3-CTD) of Procine Rotavirus(RV) plays in viral pathogenicity and immune evasion was investigated through structural analysis. Human VP3-CTD has been found to be important for viral pathogenicity. In addition, it was reported that VP3-CTD has the 2’, 5’-phosphodiesterase activity, which can cut 2’-5’A synthesized by 2’, 5’-oligoadenylate synthetase(OAS) to inhibit OAS-RNase L signaling pathway and result in immune evasion and disease development. Therefore, the structural analysis of VP3-CTD can lead to a further understanding of the PRV immune evasion mechanism, which can lay a foundation for the design of anti-PRV drugs.In this study, the VP3-CTD gene was codon optimized and cloned into prokaryotic expression vector p ET30 a.The expression conditions of E.coli strain Rosseta containing recombinant vector was optimized, and the optimum condition is 0.5 mmol/L of IPTG, 18 ℃,180 rpm/min, and 18 h. The non-monomer form of VP3-CTD protein with high purity and high concentration was obtained by using Ni2+ column affinity chromatography, molecular sieve chromatography for purification, and ultrafiltration, and the purified protein was then used for crystallization.To optimize the crystal growth condition, the sitting drop method was performed. After testing a set of conditions, the 36 th condition of HR2-110 kit(0.1 mol/L Tris-HCl, p H 8.5, and 8% PEG8000) was considered as the suitable one for crystal growth. Finally, the optimum crystal growth condition(0.15 mol/L Tris-HCl p H 8.3, 4% PEG8000, 8% EG, 20 ℃, and for 60 days) was determined through further optimization. The regular cuboid crystal(15 μm×15 μm×200 μm) was observed under the optimum condition by the X diffraction resolution, and the VP3-CTD protein crystal is 2.3 ?. The data was then analysed by using the macular replacement method and a variety of crystal analysis softwares. The momomer structure of VP3-CTD is composed of two alpha helix and seven beta sheets. The beta sheets at C terminal of VP3-CTD formed an obvious groove, which is considered to be positive charge through the surface electrostatic potential analysis. Two VP3-CTD molecules form a homologous dimmer, and their grooves are complementary and chimeric. The charge of the groove is suggested as positive, where could be the domain that binds and cuts the 2’-5’A with negative charge.Together, the VP3-CTD of PRV was expressed and purified, and the crystal structure of VP3-CTD was obtained as well in this study. The structural analysis of VP3-CTD further reveal the mechanism of cutting 2’-5’A by VP3-CTD, which would provide theoretical basis for screening of anti-PRV drugs.