Structural Basis And Molecular Mechanism Of I215L Protein Of African Swine Fever Virus Participating In Ubiquitin Binding

African swine fever virus is a highly infectious double-stranded DNA virus with a mortality rate of nearly 100% for domestic pigs.ASFV was first observed in Kenya in 1921,and then spread to Africa,Europe,South America and other regions.Currently It is mainly monitored in Europe and Asia.African swine fever(ASF)was first reported in the northeastern region of China in August 2018,and then spread rapidly to most parts of the country,causing great impact to the breeding industry in China.However,there is still no commercial vaccine or drug on the market to target this virus.The ASFV genome encoded150-200 proteins,of which,the I215 L protein present as the core role of ubiquitinproteasome system,which is used to regulate the virus infection and the replication by E2 ubiquitin binding function.However,the structural basis and molecular mechanism of ASFV participating in the ubiquitination of host proteins are still unclear.In this study,we chose the ASFV I215 L protein as the research object,attempting to clarify the structural basis and molecular mechanism for its regulation of viral replication or infection.The in vitro ubiquitination experiment was successfully established,and it was verified that ASFV I215 L had the function of E2 ubiquitin-binding enzyme.The carboxy terminal of I215 L was confirmed to be capable of affecting the structural stability of the protein by bioinformatics analysis and structural biology.Through the processes of primary screening and optimization of crystals,data collection and processing,the truncated highresolution crystal structure of ASFV I215 L protein,consisting of five ?-helices and four ?-sheets,was successfully resolved for the very first time.Although ASFV I215 L shares low homology with the amino acid sequence of the host E2 ubiquitin-binding enzyme,they share high homology in the tertiary structure.Based on their structural similarities,stacking analysis was performed on the ASFV I215 L structure with the reported E2-Ub and E1-E2 complex structures,and the potential interaction sites of ASFV I215 L with Ub and E1 were found.Finally Ribopuromycylation and Renilla luciferase reporter assay confirmed that C85 was not only the active site of ASFV I215 L that exerted the ubiquitin binding function,but also the active site inhibiting the host protein synthesis function.In this paper,we successfully established an I215 L biochemical experimental platform.By first analyzing the crystal structure of ASFV-I215 L protein,we identified its interaction interface that played as ubiquitin binding function,and interpreted the structural basis of I215 L participating in ubiquitination,which laid a scientific foundation for elucidating the molecular mechanism of I215 L participating in virus replication or infection.