Evolution Of Major Capsid Protein And Molecular Mechanism Of Interaction Between Major Capsid Protein And Minor Capsid Protein Of Norovirus G?.17

BackgroundNorovirus(NoV)is one of the most predominant viral agents of acute gastroenteritis.With the diversity of variants,low infectious dose,easy transmissibility,and no approved vaccines,it is hard to manage NoV burden around the world and the prevalence does not decrease in recent years.Giving the absence of effective antiviral drugs,surveillance studies can provide valuable information for the prevention and control of NoV.Due to a large amount of labor and economic burden,it has been a long time lacking a large-scale surveillance study.Therefore,meta-analysis is a promising tool to analyze the prevalence of NoV and provide a global picture for virus prevention and control.Over the last 30 years,G?.4 genotype has been dominant worldwide,leading to?80%of noroviruses infections.However,the emergence of G?.17 kawasaki2014 caught the publics'attention.G?.17 seldom leads outbreaks before 2014 but outcompeted G?.4 as the predominant cause of NoV outbreaks in Southeast Asia in late 2014.Until now,the reason for the sudden emergence of G?.17 remains unknown.The P protein in the NoV major capsid protein is responsible for immunogenicity,which could bind to histo-blood group antigens(HBGAs)and mediate the infection process.Amino acid-related immunogenicity and receptor binding site mutations are well-accepted as the driving force of virus evolution.These changes occur in the major capsid protein VP1 of Norovirus.Therefore,the mutation of VP1 is likely to promote the outbreaks of G?.17.In addition,the functions of VP2 could not be ignored.It has been reported that VP2 could assist the assembly of viral capsids,encapsidate the genome,promote VLPs(virus-like particles,VLPs)expression and stabilize the structure of VLPs.VP2 enhances the stability of the capsid by interacting with VP1 and make the NoV more stable in the environment.Analyzing the interaction as well as the key sites can provide valuable information for the molecular evolution of NoV.In brief,this study intended to analyze the global NoV prevalence using meta-analysis;selected G?.17 NoV as the research object and found out the evolutionary mechanisms behind the sudden outbreaks.We first focused on the VP1of G?.17,trying to explore the substitutions of the P domain of VP1 during the evolution and how the mutation contributed to the molecular mechanism of G?.17.Then we investigated the interaction between VP1 and VP2 and mapped the interaction sites.These findings would lay a theoretical foundation for further understanding of VP2.Method(1)Four databases(Pub Med,EMBASE,Cochrane Library,and Web of Science)were searched for epidemiological papers from 1997-2021 using the MESH and other search items.Studies were selected based on the pre-designed inclusion and exclusion criteria.Statistical analysis was conducted using R-4.0.0software.(2)G?.17-VP1 nucleic acid sequence alignment and phylogenetic tree model calculation were used MAFFT and j Model software,respectively.BEAST software and Phy ML software were used to build a phylogenetic tree.G?.17 P protein was cloned into p ET-22b containing the T7 promoter,then extracted the recombinant plasmid and added into the cell-free protein synthesis system(CFPS).Enzyme-linked immunosorbent assay(ELISA)was performed to distinguish antigenic characteristics of wild-type and mutant cell-free protein.The protein expressed in the cell-based expression system was used as a positive control.(3)Recombinant p GADT7-VP1 and the bait vector pGBKT7-VP2 were co-transformed into the Y2H Gold strain to explore the cross interaction of different VP2 and different VP1.The p Fast Bac-VP1-FLAG and p Fast Bac-VP2-HA recombinant vectors were constructed for transfecting and co-transfecting Sf9 cells,for the reason of investigating the expression levels as well as the intracellular location of VP1 and VP2.Results(1)A total of 12,155 articles were retrieved,and 405 studies were included,237 new studies from 24 countries more than the previous report.There were 16%(95%CI 15,17,I~2=98.8%)patients with acute gastroenteritis caused by NoV.The prevalence stayed the same as the 2014 meta-analysis and did not decrease in the past six years.(2)The G?.17-VP1 phylogenetic tree was constructed and was divided into four clusters,namely G?.17-A,G?.17-B,G?.17-C,and G?.17-D.A representative strain was selected from each of the clusters to explore the variation of VP1.The recombinant expression vector p ET-22b-G?.17-P with T7 promoter was constructed.The results of Elisa showed that the CFPS system could translate NoV P protein precisely.We found that MAb(monoclonal antibody,MAb)2D11 could only recognize B/D clades of G?.17.We then introduced alanine to six potential epitopes in P2 domain.Reduced binding ability to MAb 2D11 indicated the unique sequence in residues 293-300.A panel of mutants with alanine substitution in 293-300aa were constructed to confirm key residues affecting the conformation for antibody binding.Among these residues,amino acid 298 seemed to be the key residue binding to MAb2D11,while 295,299,and 300aa were more like subordinate and should not be neglected.(3)G?.17-/B/C/D VP2 and G?.17-A/B/C/D VP1 were cloned into pGBKT7and p GADT7 vector,respectively.These recombinant plasmids were used for yeast two-hybrid assays.The results showed that VP1 cross interacted with VP2 in G?.17.Then amino acid sequence of G?.17-/B/C/D VP2 was aligned,and eight domains were selected for alanine introduction.It was found that 174-179A were unable to interact with G?.17-D VP1.However,the G?.17-B/C VP2 174-179A could interact with the corresponding VP1.Expression of VP1,VP2,and VP1+VP2 was observed in the Sf9 cytoplasm.Conclusion(1)Over the last 30 years,16%of patients with acute gastroenteritis were caused by NoV globally.These findings provided insights on the global burden of norovirus,which suggested that the continuous surveillance of norovirus infection was important to provide a global picture for virus prevention and control.(2)The peptide selected by MAb 2D11 matched four continuous residues in P domain from 295 to 300aa,among which residue 298 played the most important role.We proposed a hypothesis:norovirus epitopes might repeatedly form several conformations during the evolution.CFPS system can offer rapid synthesizing protein,which bypasses the requirements for time-consuming procedure of the cell-based system.In the absence of a useful vitro or vivo culture method,it was confirmed in this study that the CFPS system was a promising tool for screening mutants of norovirus P proteins in mutational analysis in the future.(3)Like VP1,a hypervariable region was found in the VP2.The region was responsible for VP1 interaction.174-179aa in the VP2 were key sites for interaction with VP1.But 174-179aa was not the universal interaction site with VP1 in different clusters of G?.17.The results of our mutagenesis studies could be used for the confirmation of the crystal structure of VP2.In addition,VP2 did not have the function of nucleus localization.Other possibilities are needed to be further explored.