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The Functional Characterization of Bat and Human P[3] Rotavirus VP8*s

P[3] rotavirus (RV) has been identified in many species, including human, simian, dog, and bat. Several glycans, including sialic acid, histo-blood group antigens (HBGAs) are reported as RV attachment factors. The glycan binding specificity of different P[3] RV VP8*s were investigated in this study. Human HCR3A and dog P[3] RV VP8*s recognized glycans with terminal sialic acid and hemagglutinated the red blood cells, while bat P[3] VP8* showed neither binding to glycans nor hemagglutination. However, the bat P[3] VP8* mutant of C189Y obtained the ability to hemagglutinate the red blood cells, while human P[3] HCR3A/M2-102 mutants of Y189C lost the ability. Sequence alignment and structural analysis indicated that residue 189 played an important role in the ligand recognition and may contribute to the cross-species transmission. Structural superimposition exhibited that bat P[3] VP8* model was quite different from the simian P[3] Rhesus rotavirus (RRV) P[3] VP8*, indicating that bat P[3] RV was relatively distinct and partially contributed to the no binding to tested glycans. These results promote our understanding of P[3] VP8*/glycans interactions and the potential transmission of bat/human P[3] RVs, offering more insight into the RV infection and prevalence.


Introduction
Rotavirus (RV) is a major pathogen leading to the acute viral gastroenteritis in young children and animals. RV genome contains 11 double stranded RNA fragments, encoding 12 proteins, including 6 structural and 6 nonstructural proteins (Estes 2013). Based on VP6, RVs can be divided into ten different species by now, including A-H, one tentative species I, and a candidate species J (Banyai et al. 2017). The RV particle consists of three capsid layers. Two major surface proteins, VP7 and VP4, formed the outer most layer and played an important role in inducing neutralizing antibodies and protective immunity. VP7 and VP4 defined the RV G and P genotypes, respectively (Matthijnssens et al. 2011). The group A RVs are diverse and has been identified 51 P genotypes so far (https://rega. kuleuven.be/cev/viralmetagenomics/virus-classification/ 7th-RCWG-meeting). VP4 can be cleaved by trypsin into two fragments, VP5* and VP8* (Fiore et al.. 1991). VP8* located at the distal part of the VP4 spike and was reported to be essential in the glycan recognition and cell attachment (Dormitzer et al. 2002a).
RVs were identified in many species, including human, porcine, bovine, dog and birds. Bats, the reservoirs of many viruses, were also identified to harbor the RVs (He et al. 2013). The genetic analysis of bat RVs indicated the bat-tohuman transmission and reassortment (He et al. 2017;Komoto et al. 2020). The cross-species transmission of RVs has been reported in several RV genotypes, including P[6], P [3] RVs that were reported in both human and animal infections (Nyaga et al. 2018;Okitsu et al. 2018 He et al. 2013;Sasaki et al. 2016). Bat P[3] LZHP2 was identified in an insectivorous bat and shared high nucleotide identity with the unique human strain M2-102 that was suggested to originate from a bat RV as a result of crossspecies transmission and was divergent from other human RV strains (Dong et al. 2016;He et al. 2017). Human HCR3A was supposed to be of dog origin (Tsugawa and Hoshino 2008). The molecular mechanism of the prevalence of these P[3] RVs remained elusive. Attachment to the cell receptors is crucial for the virus infection and transmission. The illustration of the interactions between virus and ligand provided the basis for understanding host tropism, host adaptation, and zoonosis. Previous studies have shown that some animal RVs recognize sialic acid-containing glycoconjugates and are neuraminidase sensitive (Ciarlet and Estes 1999;Isa et al. 2006). Some animal RV VP8*s, including P[1] bovine Nebraska calf diarrhea virus, P[2] simian SA11, P[7] porcine CRW-8 were reported to interact with terminal sialic acids on the cell surface glycans (Ciarlet and Estes 1999). The simian P[3] Rhesus rotavirus (RRV) VP8* recognized N-acetylneuraminic acid (Neu5Ac) and the crystal structure of RRV VP8* in complex with Neu5Ac has also been determined (Dormitzer et al. 2002b). The glycan binding site located at one corner of the two beta-sheets. CRW-8 P[7] VP8* was found to bind the sialic acid using the same glycan binding site as that of RRV P[3] (Blanchard et al. 2007).
Though the interaction between RRV P[3] and sialic acid has been reported, the glycan binding specificities of the P[3] RVs of other species is not clear yet, especially the bat P[3] RVs. Here, the functional features of bat, human, and dog P[3] RVs were investigated, which deepened the understanding of P[3] RV prevalence and transmission from animals to human.

Protein Expression and Purification
The full VP8* fragments (residues 1-231 amino acids) of human HCR3A (Genebank: EU708904), 12,638 (Genebank: LC340022), M2-102 (Genebank: KU597745), dog RV198-95 (Genebank: HQ661137), and bat LZHP2 (Genebank: KX814943) were synthesized by Genewiz company (Suzhou, China) and cloned into pGEX6P-1 with an N-terminal GST tag. The GST-fusion proteins were expressed in E.coli and induced with isopropyl-b-D-thiogalactopyranoside (IPTG) at a final concentration of 0.4 mmol/L at 22°C for 16 h. The recombinant proteins were purified as previously reported (Ma et al. 2015;Sun et al. 2018). Briefly, the bacteria were collected and resuspended, followed by ultrasonication. The supernatant of the bacterial lysates was filtered through a 0.22 lm membrane (Millipore) and then loaded onto the glutathione sepharose (GE Healthcare Life Sciences). The column was washed with PBS five times. Then the protein of interest was eluted with buffer of 50 mmol/L Tris-HCl, 10 mmol/L reduced glutathione, pH 8.0. The eluted proteins were concentrated and the buffer was changed to PBS. The protein concentration was determined using the BCA kit (BD Biosciences). Bat P[3] mutant of C189Y and human M2-102/HCR3A mutant of Y189C were cloned using the site-directed point mutation. The mutant proteins were expressed and purified as described above. The eluted proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Glycan Microarray Screening Analysis
Glycan ligand screening for human M2-102 and bat LZHP2 P[3] VP8*-GST fusion proteins were performed by the Protein-Glycan Interaction Core of the Consortium for Functional Glycomics (CFG) (http://www.functionalgly comics.org/) against a library containing 600 glycans. The identities of all glycans in Version 5.3 of the array are available at http://www.functionalglycomics.org/static/con sortium/resources/resourcecoreh8.shtml. The recombinant GST-VP8* protein was used at protein concentrations of 5 lg/mL and 50 lg/mL, respectively. The bound GST-VP8* proteins were detected using a fluorescence-labeled anti-GST monoclonal antibody (Sigma). Fluorescence was measured and quantified by a microarray scanner and relative fluorescent unit (RFU) for binding to each glycan was calculated (Cholleti et al. 2012).

Hemagglutination
The GST-VP8* proteins were twofold serially diluted with a starting concentration of 2 mg/mL using PBS buffer and added to 96-well V-bottom plates (Costar) with 50 lL per well. Animal red blood cells (RBCs) (Gelaidisi, Beijing) of porcine, rabbit, bovine, chicken, lamb, and human RBCs (A, B, O types, donated by lab colleagues) were diluted to 1% with PBS. Written informed consents were signed by the colleagues. Authorization for the sampling of human and animal blood was obtained. Then 50 lL of RBC was added to each well. Agglutination was determined after incubation at 25°C for 1 h.

Homology Modeling and Structural Analysis
A homology model of bat P[3] VP8* was constructed based on the structure of RRV P[3]VP8* (PDB identifier [ID] 1KQR) by SWISS-MODEL automated protein structure homology modeling server (http://swissmodel.expasy.org/). The structural superimposition and analysis were performed using pymol (https://pymol.org/2/). The cartoon and surface models of VP8* were shown. The electrostatic potential presentation was calculated with the pymol software.
According to the glycan microarray assay, human M2-102 and bat LZHP2 P[3] VP8* exhibited no specific binding to the tested 600 glycans including glycans with sialic acid (Fig. 3). Relative Fluorescent Units (RFUs) indicated the binding intensity. The Human M2-102 and bat LZHP2 P[3] VP8* did not showed high RFU value to these glycans (Fig. 3). For the glycan 117, the RFUs were almost the same for different concentrations and different kinds of proteins. It is suggested that the binding is regarded to be non-specific.

Hemagglutination of P[3] VP8*s and Mutants
Hemagglutination was performed to explore the interaction between VP8* and the red blood cells. Human

Structural Analysis of the Glycan Binding Site of P[3] VP8*s
The homology model of bat P[3] VP8* was constructed based on the P[3] RRV crystal structure ( PDB ID: 1KQR) by SWISS-MODEL. The structural superimposition showed that bat P[3] VP8* model presented typical galectin-like conformation, but it is quite different to RRV P[3] VP8* with the root mean square deviations (RMSD) values of 1.256 (Fig. 7A). Y189 in RRV P[3] formed hydrogen bonds with sialic acid, while C189 would lost the interaction (Fig. 7A), which may affect the ligand recognition. V144 and T191 may influence the interactions via the loop conformation, which still need further study to clarify. The surface presentation exhibited that the putative      sialic acid binding site was discontinuous in the P[3] bat VP8* model (Fig. 7B). The electrostatic potential was also different with positive charge in P[3] bat VP8* model and negative charge in P[3] RRV VP8* (Fig. 7C).

Discussion
Many rotavirus genotypes can infect different species and species-specific RV strains are frequently identified (Papp et al. 2013). The zoonotic potential has led to the increased burden of RV control and prevention (Doro et al. 2015). The glycan binding specificity of different virus strains are vital for understanding the host infection and tropism (Ramani et al. 2016). VP8*, located at the distal terminal of the VP4 spike, was involved in the glycan recognition and played an important role in the virus attachment (Huang et al. 2012;Ramani et al. 2016). Bats are recognized as the reservoir of many viruses, including the lifethreatening Ebola virus, SARS, influenza virus. Rotaviruses in bats were identified by several studies (Esona et al. 2010;He et al. 2013He et al. , 2017. P[3] RVs are also found in cats/dogs and some feline/canine-like RVA strains are identified to infect humans sporadically (Tsugawa and Hoshino 2008). The glycan specificity and molecular mechanism of the cross-species transmission is important for understanding the virus prevalence. Here, we delineated the functional features of P[3] RV VP8*s of different species.
Bat LZHP2 P[3] VP8* showed no hemagglutination to all the erythrocytes tested, while other VP8* hemagglutinated the cells, indicating that bat LZHP2 P[3] VP8* probably do not interact with classical ligands that were recognized by Rhesus P[3] RV (Dormitzer et al. 2002a). Glycan binding assay verified that human HCR3A, 12,638, and dog RV198-95 P[3] VP8* bound to glycans with sialic acid while bat P[3] VP8* did not. Human M2-102 hemagglutinated the red blood cells, while showed no obvious binding to the glycans with sialic acid, providing more evidence that human M2-102 was originated from bat P[3] RVs. Human HCR3A and dog  exhibited similar binding to both Neu5Aca2-3Gal and Neu5Aca2-6Gal, indicating that these P[3] RV bound sialic acid irrespective of the a2-3 or a2-6 linkage and   Fig. 4 Biolayer interometry (BLI) analysis. PAA-biotin labeled oligosaccharides, Neu5Ac, Neu5Gc, Neu5Aca2-3Galb1-4GlcNA-(3'SLN), Neu5Aca2-6Gal, H1, H2, H3, Neu5Acb2-6(Galb1-3) B disaccharide,A disaccharide,B tetrasaccharide,core1,core2,core8,siaLea,siaLex,siaLec,lea,leb,lex,ley were loaded on the biosensor at 10 lg/mL. Human HCR3A, M2-102, and bat LZHP2 P[3] VP8* proteins were diluted at 10 lmol/L and applied to the association and dissociation procedures. The glycan binding data was analyzed by the Octet analyzing software.  possessed the potential of cross-species transmission. These results demonstrated that different P[3] VP8*s had various hemagglutination ability and glycan binding specificity. Bat P[3] VP8*s displayed no binding to sialic acid and probably could not infect human, while human/dog origin P[3] VP8*s bound sialic acid and could infect both human and animals. Sequence alignment showed that several amino acids were different between bat LZHP2 and human RV VP8*s, including the residue 189. Bat LZHP2 C189Y mutant obtained the ability of hemagglutination while human P[3] Y189C mutant had no hemagglutination, implying that residue 189 was one of the key residues for the ligand recognition and the C189Y alteration in M2-102 RV probably facilitate the sporadic bat-origin RV infection in human. Meanwhile, other amino acids, including residue 144, 155, and 191 may contribute to the different glycan binding capacity of P[3] VP8*.
The structural analysis revealed that the putative glycan binding site in bat P[3] VP8* model was quite different to that in the P[3] RRV VP8*, which partially explained that bat VP8* did not bind sialic acid glycans. These indicated that species barrier probably existed for bat LZHP2 P[3] RVs. Bat LZHP2 P[3] RV could not transmit to humans at present but may infect human sporadically with the C189Y alteration. Analysis of all the reported bat P[3] RV VP8* sequences showed that bat LZHP2 and YSSK5 (He et al. 2017) possessed 189C, while other 8 strains contained 189Y (Fig. 8), implying that some bat P[3] RV strains may have the potential to infect human like the M2-102 strain. Therefore, the consistent surveillance of bat and animal RVs is needed.
Animal and human rights statement All institutional and national guidelines for the care and use of laboratory animals were followed.