Molecular Basis For Interaction Between The Host Histidine Methyl Transferase SETD3 And The Enteroviral 2A Protease

Enteroviruses are a genus of small positive-sense single-stranded RNA viruses that comprises numerous clinically significant human pathogens,including polioviruses,coxsackieviruses,numbered enteroviruses,echoviruses,and rhinoviruses.The infections caused by enteroviruses range in their presentation and severity from asymptomatic cases to mild diseases such as hand-foot-and-mouth disease and common cold,to severe or even fatal neurological,cardiovascular,and pulmonary conditions.Currently,there is an urgent need for broad-range antiviral drugs and vaccines to treat and prevent enteroviral infections.Recent discovery that the interaction between the enteroviral 2A protease(2A^pro)and the host actin histidine methyltransferase SETD3 is essential for replication of multiple strains of enteroviruses suggested that this interaction may serve as an attractive target for antiviral drugs and therapies.Although the 2A^pro residues required for SETD3 binding have already been mapped with the help of mutational analysis,the structural basis for the SETD3-2A^pro interaction remains elusive,thus limiting our understanding of how is SETD3 exploited by the enteroviral 2A proteases.In this study,crystal structures of SETD3 in complex with coxsackievirus B3 2A^pro(CV-B3 2A^pro)and CV-A10 2A^pro were determined,followed by systematic structural and bioinformatical analysis,as well as biochemical and mutational experiments.In both complexes,a single copy of 2A^pro was found to bind within the V-shaped cleft formed between the two SETD3 lobes.As neither2A^pro variants nor SETD3 underwent an observable conformational change,the binding of 2A^pro to SETD3 is likely based on multiple weak interactions that rely on shape complementarity of the two proteins.Detailed analysis of the structural interface detected three main groups of intermolecular interactions,among which the two groups formed between the structural elements surrounding the zinc-binding site of 2A^pro and the SET domain of SETD3 were conserved in both complexes.Protein binding assays performed with relevant CV-B3 2A^pro and SETD3 mutant variants demonstrated that the amino acid residues V62,the K68-N69-K70 patch（particularly K70）,and Y72 from CV-B3 2A^proand V266,Y288,and E296 from SETD3 are the key mediators of SETD3-CV-B3 2A^prointeractions.In addition,multiple sequence alignment indicated that the majority of enteroviral 2A proteases share the key SETD3-interacting residues,suggesting strong similarities in the way they bind to SETD3.Finally,based on the structural data and review of related literature,this study discusses possible functional roles of SETD3-2A^prointeractions in viral replication.The findings of this study elucidate the molecular basis for the interaction between the enteroviral 2A proteases and SETD3 and lay the groundwork for the development of broad-spectrum antiviral drugs and therapies targeting this interaction.