| Human noroviruses(HuNoVs)are the main foodborne pathogen causing non-bacterial gastroenteritis worldwide.The VP1 is the main capsid protein of the HuNoVs.It consists of S(Shell)and P(Protruding)domains.The P domain is the main functional region of the virus binding to its receptor/ligand.HuNoVs can be classified into different genotypes according to the encoding sequences of VP1.Moreover,the genotype GⅡ.4 is the main epidemic strain worldwide.Due to the lack of mature in vitro culturing model and small animal model for HuNoVs,the traditional virus-receptor/ligand study method is not feasible.As a filter feeder,oyster is one of the main carriers of HuNoVs.Recent studies have shown that oyster heat shock protein 70(oHSP70)was an important ligand in oyster tissue for HuNoVs enrichment,but the molecular mechanism of the binding remains unclear.Therefore,clarifying the molecular mechanism of interaction between HuNoVs and oHSP70 has become an important breakthrough for the prevention and control of HuNoVs in oysters.On the basis of previous research in the laboratory,forty-three representative VP1 sequences were downloaded from NCBI database for bioinformatics analysis.DNAMAN software was used to group and compare the amino acid sequences at position 276-420 of VP1 P protein.And 14 potential conserved sites of HuNoVs were predicted for binding to the receptor/ligand.PCR site-directed mutagenesis was used to construct recombinant mutant plasmids.Then the HuNoVs(GⅡ.4)P protein mutant library was established by bacterial surface display technique.Secondly,the HuNoVs(GⅡ.4)P mutant proteins were purified.The mouse anti-oHSP70 and HRP labeled sheep anti-mouse IgG were used as the primary antibody and the secondary antibody,respectively.Then,11 amino acid sites related to oHSP70 binding with HuNoVs(GⅡ.4)P protein were identified by ELISA.The binding capacity of two essential amino acids(P318 and D325)were decreased to 0% and 11%,respectively.The binding capacity of the other 9 binding amino acid sites(G322,P324,P360,G363,P406,Y408,L418,A419,P420)were all less than 50% after mutation.Finally,according to the experimental results,SWISS-Model and PyMOL software were used to analyze the position of binding related amino acid sites on the spatial structure of HuNoVs(GⅡ.4)P protein.Two key amino acids were found to be exposed in two deep“pockets” on the surface of the P protein,adjacent to an exposed surface formed by five amino acids(P406,Y408,L418,A419,and P420).Therefore,we hypothesized that these 7 amino acids constituted the binding interface between HuNoVs(GⅡ.4)P protein and oHSP70,and were directly involved in the interaction between VP1 and oHSP70.Although P324,P360,G322 and G363 were not directly participated in the binding of VP1 to oHSP70,they might maintain the integrity and stability of binding interface.These mutation sites indirectly affected the binding of VP1 to oHSP70.In this study,the interaction between P domain of HuNoVs capsid protein and oHSP70 was studied.The key amino acid sites of P protein binding to oHSP70 were initially revealed.The conclusion in this study enriched the exploration of the binding mechanism between HuNoVs and their receptor/ligand.It can provide a theoretical basis for the HuNoVs prevention in oysters and the formulation of propagation strategies. |
