Functional And Structural Characterization Of Human Group C Rotaviruses VP8~* Proteins Interaction With Histo-blood Group Antigens

Rotaviruses(RVs)belong to the Reoviridae family and are recognized as a major cause of acute gastroenteritis(AGE)in humans and animals,responsible for?200 000 deaths among children under five years of age worldwide each year.RVs contain an 11-segment double-stranded RNA genome within a triple-layered protein capsid.The outermost capsid spike viral protein 4(VP4)could be cleaved into two fragments,VP5*and VP8*.VP8*,the globular head of the spike,is mainly involved in the interactions with the cell receptors.RVs are currently classified into ten serogroups(A?J),among which groups A,B,C,and H RVs infect both humans and animals.Group A rotaviruses(RVAs)are the leading cause of AGE,being responsible for about 90%of cases.At present,significant advances in understanding evolution,host tropisms and receptor binding properties have been made only in RVAs.The recent discovery that RVAs recognize histo-blood group antigens(HBGAs)as potential receptors has significantly advanced our understanding of RV infection,host range,and cross-species transmission.Group C rotaviruses(RVCs)are the second most common RVs species found in humans and animals.However,little is known regarding their host-specific interaction,infection,and pathogenesis.In this study,we performed serial studies to characterize the functional and structural features of human RVC VP8*that is responsible for host receptor interaction.Objective1.To construct recombinant expression plasmid of human RVCs VP8*,and express recombinant proteins;2.To characterize the binding patterns between human RVCs VP8*proteins and HBGAs;3.To understand the structural basis of human RVCs VP8*and the receptor,to explore the structural characteristics and receptor binding mechanism of human RVCs VP8*,providing scientific basis for the development of RVC vaccines,targeted drugs and immunodiagnostic tools.Methods1.The cDNA fragments encoding VP8*domains(residues 1?231)of three human RVCs(Bristol,SZ272,SZ94)were retrived from Genbank,they were chemically synthesized by Genewiz Company and individually cloned into the pGEX-4T-1 vector with an N-terminal glutathione S-transferase(GST)tag;2.The VP8*core region(residues 64?224)primers were designed using the VP8*gene sequences as templates,and the VP8*core fragments were cloned into the pET-30a vector with a C-terminal hexahistidine tag;3.The recombinant proteins were expressed in Escherichia coli strain BL21(DE3),and the GST-and His-tagged proteins were purified by affinity chromatography.The His-tagged protein was further purified by gel filtration chromatography.The purified recombinant proteins were stored at-80?;4.The binding properties of the human RVCs VP8*and HBGAs was characterized by enzyme immunoassay and saliva binding assay;5.Crystallization screening of the His-tagged VP8*core proteins was carried out using the sitting-drop vapor diffusion method,and the VP8*core protein was co-crystallized with A-trisaccharide under the same condition as the native protein;6.The VP8*core crystal was incubated with the sodium iodide at the final concentration of 1.2 M for 1 min before loaded to the X-ray.The VP8*core,VP8*core-NaI and the complex X-ray diffraction data were collected.The structure of VP8*core-NaI was determined by single-wavelength anomalous diffraction(SAD)method,and the native VP8*core and the complex VP8*core-A were solved by molecular replacement method.Results1.The recombinant expression plasmids of human RVCs VP8*proteins and VP8*core proteins were successfully constructed,and GST-VP8*proteins and VP8*core-His proteins were expressed and purified;2.The human RVCs VP8*recognized type A HBGA in the oligosaccharide and saliva binding assays;3.The human RVC VP8*structure was solved,showing a typical galectin-like structure.The VP8*in complex with a type A trisaccharide displays a novel ligand binding site.Conclusion1.In the study,the three tested human RVC VP8*proteins were demonstrated to recognize type A HBGA,which may partly explain their low prevalence;2.The human RVC VP8*shows a typical galectin-like structure with specific features of the P-strand and loop regions,forming distinct structural characteristics;3.The human RVCs VP8*proteins interact with type A HBGA through a unique mechanism.