| The aim of this thesis is to investigate the molecular mechanism of important non-structural proteins in viruses.This thesis is divided into two parts,the first part focuses on the structural and functional analysis of the 3C protease of Senecavirus A(SVA),and the second part focuses on the structural and functional analysis of the isoprenoid synthase pB318L of African swine fever virus(ASFV).Senecavirus A is a novel picornavirus that causes vesicular disease in pigs,and the 3C protease plays a critical role in the processing of viral precursor polyprotein and in antagonizing the host antiviral response.Recent research on the picornavirus 3C protease suggests that the KFRDI motif involved in RNA-binding has a dual function of binding phospholipids,however,the molecular mechanism and function of phospholipid binding by the 3C protease has not been thoroughly investigated.In the first part of this thesis,the structure of the 3C wild-type protein at 1.99 A and the inactivated mutant C160A at 1.61 A were determined by X-ray crystallography method.During the process of resolving the SVA 3C wild-type structure,an unknown electron density similar to a phospholipid molecule was found in the area adjacent to the hydrolytic active site.By phospholipid binding assay and lipidomics research method,we identified that the phospholipid mainly bound by SVA 3C is cardiolipin(CL),and the unknown electron density in the above structure represents the cardiolipin 74:6.Combining the structural information of 3C protease binding to phospholipids,this study identified the key amino acid residues of 3C protease involved in binding to cardiolipin as His75 and His78 through enzyme linked immunosorbent assay(ELISA).In addition,this study found that SVA 3C can be located in the mitochondrial inner membrane by binding to cardiolipin.More importantly,the study showed that phospholipid binding can not only affect the protease activity of 3C protease,but also have an impact on virus replication.Based on the above research,this study provides structural biology support for the study of picornavirus 3C protease binding to phospholipids.African swine fever virus is a virulent pathogen capable of causing extremely high infection and lethality rates in domestic and wild pigs.pB318L is a unique isoprenoid synthase encoded by ASFV and is an essential protein for virus replication.However,the detailed mechanism of pB318L’s function in the virus life cycle is still unclear.In the second part of this thesis,the crystal structure of pB318L at 3.20 A was resolved by X-ray crystallography method.In the process of resolving the structure,multiple known structures of homologous proteins were used as templates but phase could not be resolved.We then attempted to use AlphaFold2 predicted models as templates for molecular replacement and successfully resolved the structure.Based on the crystal structure of pB318L,this study conducted an in-depth analysis of the accuracy of AlphaFold2 predicted protein structures,especially the accuracy of predicting amino acid side chains,indicating that the accuracy of predicting non-polar residues in the AlphaFold2 predicted model is better than that of polar residues.At the same time,this study combined AlphaFold2 predicted models with small-angle scattering experiments to propose the conformational characteristics of the homologous dimer model formed by pB318L in solution.Furthermore,in order to elucidate the function of pB318L in virus replication,this study used cell biology and immunology methods to study the subcellular localization of pB318L in eukaryotic cells and found that pB318L can enter the nucleus,suggesting that pB318L may be involved in immune evasion.Subsequently,this study preliminarily explored the mechanism by which pB318L inhibits the JAK-STAT signaling pathway and proposed a possible mechanism for pB318L’s negative regulation of the type Ⅰ interferon signaling pathway,providing a foundation for understanding the role of pB318L in virus replication. |
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