Structural And Functional Study Of Replicating Essential Proteins PE165R And PEP152R Of African Swine Fever Virus

African swine fever,caused by the African swine fever virus(ASFV),is among the most significant swine diseases.ASFV was transmitted to China in August 2018,resulting in enormous economic losses to the pig industry.At present,there are no effective vaccines or drugs for the prevention or treatment of ASFV.ASFV contains two gene encoding dUTPase(E165R)and EP152R,which are required for viral replication in swine macrophages,making it an attractive target for inhibitor development.This paper focuses on the structural and functional studies of pE165R and pEP152R,mainly including as follows:1.This study revealed the the interactions with its substrates and catalytic mechanism of ASFV dUTPase.Through many comparisons between ASFV and host dUTPases,we provide guidance for the development of specific.ASFV contained a sequence of opening read frame(ORF)numbered E165R,encoding dUTPase.ASFV dUTPase is a highly specific enzyme required for efficient replication in swine macrophages.So ASFV dUTPase have been chosen as a target for inhibitor development.This article analyzes the ligand-free ASFV dUTPase structure and two complex structures.By analyzing the interaction patterns between amino-acid residues and substrates,a non-classical double-subunit catalytic mechanism of ASFV dUTPase was expounded in this study,and the key amino acid sites were verified by mutant enzyme activity experiments.In order to prevent the inhibitors targeting ASFV dUTPase affect the swine dUTPase and the growth of pigs,this study illustrated how swine dUTPase perform classical three-subunit catalytic mechanism.Through many comparisons between them,this study found:(i)They were found to assemble active sites in different manners.Swine dUTPase employs three-subunit active sites similar to the classical dUTPase.The active site of ASFV dUTPase is formed by two subunits;(ii)There are slight differences in the key amino acids involved in the catalytic substrate;(iii)The active sites of ASFV dUTPase have larger volumes;(iv)There is no significant difference in the hydrophobicity distribution of the active sites;(v)The electrostatic distribution of the active sites is significantly different.These observations should be considered in the development of inhibitors against ASFV dUTPase without affect swine dUTPase.Finally,we tested two dUTPase inhibitors against the swine and ASFV enzymes.One of these compounds inhibited the ASFV dUTPase at low micromolar concentrations(K_d=15.6μM)and with some selectivity(～2x)over swine dUTPase.In conclusion,we provided valuable structural information that will greatly facilitate the development of targeted inhibitors or specific drugs against ASFV.2.Our study provided insights into novel functions of pEP152R,revealing the involvement of pEP152R in translation,cell cycle,immune,ER stress and other cell homeostasis,these findings have important implications for understanding how pEP152R promotes ASFV replication.pEP152R was found to be an early-expressed and ER-localized membrane protein.pEP152R induced ER swelling,which triggered a significant increase in ER stress,and activates the UPR.pEP152R induces host translation shutoff through the PERK/e IF2αpathway.Furthermore,we found that ASFV pEP152R significantly reduces proliferation by inducing cell cycle arrest in G0/G1 phase and clearly inhibits the immune factors,thus establishing intracellular conditions favorable for viral replication.Moreover,virus-level experiments suggested that PERK may serve as an attractive host target to combat ASFV.In conclusion,our study provided insights into novel functions of pEP152R,revealed the involvement of pEP152R in ASF pathogenesis,and suggested PERK as an attractive host target for antiviral drugs.Overall,our study demonstrated a series of novel functions of ASFV pEP152R.In summary,this study focused on two essential proteins of ASFV,pE165R and pEP152R.Firstly,we analyzed the protein structure of pE165R,elucidated the non-classical double-subunit catalytic mechanism of ASFV dUTPase,and designed specific targeted inhibitors based on the difference between virus and host dUTPase.Secondly,we revealed the funtions of pEP152R on ER stress,immunity,cell cycle,and the molecular mechanism of pEP152R inhibiting host translation,which provided new basis and target for the development of new vaccines and the design of antiviral drugs.In general,this project focused on the replicating essential proteins,and deeply elucidated the regulatory role of viral proteins on the host environment from the structural biology and virology,which provided the structural basis and theoretical guidance for the development of target drugs.