Structural And Functional Studies Of The Arterivirus Nsp11 Endoribonucleases

Porcine reproductive and respiratory syndrome virus(PRRSV)and Equine arteritis virus(EAV)are two major members of the family Arteriviridae belonging to the order Nidovirales.These two viruses cause huge economic losses to animal husbandry every year all around the world.Nidoviral endonuclease specific for U(NendoU)is the most conserved non-structural protein(nsp)in the evolution of nidoviruses,being referred to as nsp11 in both PRRSV and EAV.Nsp11 is essential for virus genomic replication and subgenomic synthesis,and also involved in multiple signaling pathways including histone-related and mitogen-activated protein kinase signaling pathway,cell cycle and DNA replication pathway.In addition to NendoU activity,PRRSV nsp11 also has the activity of deubiquitinase(DUB)and can inhibit the expression of host immune-related factors through such two activities.Moreover,nsp11 significantly differs from its counterpart nsp 15 in coronavirus.Currently,however,nsp11 remains unclear in terms of various aspects such as whether PRRSV nsp11 inhibits the host immune system through the activity of NendoU or DUB,whether EAV nsp11 also has the activity of DUB,and what molecular mechanism occurs to the functional differences between nsp11 in arterivirus and nspl 5 in coronavirus.To elucidate the molecular basis of this multifunctional protein nspl 1,in-depth studies were conducted in this thesis by means of structural biology using the method of X-ray crystallography.In this study,mutant proteins were initially screened to solve the problem as to the low expression of wild type PRRSV and EAV nsp11.Subsequently,through extensive screening for crystals,the crystals of NendoU active-site mutants(specifically,EAV nsp11 H126A,H141A and K17A0 mutants,andPRRSV nsp11 H129A,H144A and K173A mutants)were obtained.By using single-wavelength anomalous diffraction(SAD)method to determine the coordinates of heavy atoms(Hg2+)soaked in the crystals,structures of a plurality of arterivirus nsp11 family proteins were systematically determined.The structures of arterivirus nsp11 contain two conserved compact domains:the N-terminal domain(NTD)and C-terminal domain(CTD),with the catalytic center of NendoU activity being located in the CTD,where a positively charged groove is next to the key catalytic residues.Structures of these mutants showed that the active-site mutations fail to pose impacts on the whole structures,and nsp11 proteins from the same arterivirus family are highly conserved in terms of the structure and amino acid residue positions at the catalytic center.Moreover,structural comparisons showed that nspll active center is remarkably flexible and adopts a variety of conformations.The intermolecular interactions in the structures of nsp11 are diverse but were further demonstrated to be weak based on the results from PISA online server.Additionally,despite the absence of the domain which mediates the formation of hexamer in its counterpart nsp 15 in coronaviruses,arterivirus nsp11 was further proved to be also present in the form of oligomers in solution with the equilibrium mixture of trimers and tetramers as the dominant form by the methods of gel filtration,sedimentation velocity(SV)and small angle X-ray scattering(SAXS)analyses.Such an oligomerization is concentration dependent and the same oligomeric states occur to the mutant proteins and the wild type ones.Structural analysis and related biochemical experiments revealed that arterivirus nsp11 is a type of NendoU possessing special catalytic properties which are greatly different from those of coronaviruses nsp15,such as unique substrates recognition mode,the involving of active centers in the oligomer formation,and unstable binding to Mn2+.What is more,it was demonstrated that arterivirus nspll has no activity of DUB.Finally,according to the data of structural biology and biochemistry,we predicted that arterivirus nsp11 exerts its catalytic function by forming tetramers in an unique manner,and thus proposed a nsp11 action mechanism that when not binding with the substrate,nsp11 is in its inactive state of trimers,adopting various conformations;in contrast,when binding with the substrate,nspll is induced to be subjected to molecule rearrangement,and then converted into its active state by forming a regular tetramer,thereby binding the substrate to its two active centers where the substrate is catalyzed and thus hydrolyzed.In addition,nsp11 in its active state is capable of forming high-order oligomers to improve its catalytic efficiency via dimerization of tetramers.Taken together,this study successfully determined several structures of PRRSV and EAV nsp11 mutants at high resolution.Structural analysis and related biochemical experiments revealed that arterivirus nsp11 is a type of NendoU possessing special catalytic properties.Our studies provide full insights into this key multifunctional NendoU family of proteins,lay a solid foundation for better understanding of the molecular mechanisms and the drug development against these deadly viruses.