The Primary Study On The Interaction Mechanism Between Rotavirus VP8*and VP5* Proteins

Rotaviruses are the main pathogens causing diarrhea in infants and yonug animals,and its infection is a multi-step process which requires a variety of receptors.Rotavirus VP4,the spike protein,is sensitive to trypsin and can be digested into VP8?and VP5*,and activate the infection of rotavirus.During the infection of rotavirus,VP8*head should dissociate from the top of VP5*to expose the hydrophobic membrane fusion region,which will miediate the penetration of rotavirus into the cytoplasm.The cryo-EM structure of rotavirus showed that after trypsin digestion,VP8*was still located on the tope of VP5*,and the VP8*head was linked to the N-terminal ?-helix by a 40aa peptide,which could help maintain the stability of VP4.However,the interaction and dissociation mechanism between VP8*and VP5*are still unclear.In previous studies,we have found that immunization with the recombinantly expressed rotavirus VP4*proteins(aa 26-476)could inhibit the virus shedding and development of diarrhea after rotavirus infection in a mice model.In addition,similar to VP4 proteins in the viruses,VP4*could be digested into VP8*and VP5*proteins,which lays the foundation for the study of the interaction and dissociation mechanism between VP8*and VP5*.Firstly,the amino acids participated in VP8*and VP5*was analyzed by molecular docking,and it was found that VP8*interact with VP5*protein out of the a helix structure.Then,the critical regions and amino acids for the interaction between VP8*and VP5*proteins were analyzed by N-terminal trucation and site-directed mutation.The results suggested that the key amino acids are P37,T43,Y45,Y69,S257,G286,V474,and P475 that participate in the interaction between VP8*and VP5*proteins.According to the results of molecular docking,and site-directed mutation,we found that the major interaction between VP8*and VP5*were hydrophobic interaction and hydrogen bonds.Forthe study of the dissociation mechanism,the effects of receptor,pH,the intracellular proteinases and their combinations on the interaction between VP8*and VP5*proteins were analyzed.The results suggested that there was some differences in the dissociation of VP8*and VP5*:receptor binding could lead to the dissociation between VP8*and VP5*proteins for SA11 and DS-1 strains;acidification can lead to the dissociation of VP8*from VP5*for LLR and DS-1 strains;while the intracellular proteinases has no obvious effect on the interaction between VP8*and VP5*.In addition,the receptor and low pH has synergistic effects on the dissociation of VP8*fromthe top of VP5*for rotavirus LLR and DS-1.It was speculated that the differences in the dissocation mechanism may be related to the cytoplasm release of rotavirus particles for different strains of rotavirus.In summary,this is a preliminary study on interation and dissociation mechanism between rotavirus VP8*and VP5*proteins.This study provide basis for elucidation of the mechanism in rotavirus infection and could promote basis for the development of rotavirus vaccines.