The Functional Characterization of Bat and Human P[3] Rotavirus VP8*s.
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ABSTRACT:
SUBMITTER: Li D
PROVIDER: S-EPMC8165343 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Two currently licensed live oral rotavirus vaccines (Rotarix® and RotaTeq®) are highly efficacious against severe rotavirus diarrhea. However, the efficacy of such vaccines in selected low-income African and Asian countries is much lower than that in middle or high-income countries. Additionally, these two vaccines have recently been associated with rare case of intussusception in vaccinated infants. We developed a novel recombinant subunit parenteral rotavirus vaccine which may be more effective in low-income countries and also avert the potential problem of intussusception. Truncated recombinant VP8* (ΔVP8*) protein of human rotavirus strain Wa P[8], DS-1 P[4] or 1076 P[6] expressed in Escherichia coli was highly soluble and was generated in high yield. Guinea pigs hyperimmunized intramuscularly with each of the ΔVP8* proteins (i.e., P[8], P[4] or P[6]) developed high levels of homotypic as well as variable levels of heterotypic neutralizing antibodies. Moreover, the selected ΔVP8* proteins when administered to mice at a clinically relevant dosage, route and schedule, elicited high levels of serum anti-VP8* IgG and/or neutralizing antibodies. Our data indicated that the ΔVP8* proteins may be a plausible additional candidate as new parenteral rotavirus vaccines.
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Project description:Rotaviruses (RVs) of species A (RVA) are a major causative agent of acute gastroenteritis. Recently, histo-blood group antigens (HBGAs) have been reported to interact with human RVA VP8* proteins. Human P[19] is a rare P genotype of porcine origin that infects humans sporadically. The functional and structural characteristics of P[19] VP8* interaction with HBGAs are unknown. In this study, we expressed and purified the VP8* proteins of human and porcine P[19] RVs. In oligosaccharide and saliva binding assays, P[19] VP8* proteins showed obvious binding to A-, B-, and O-type saliva samples irrespective of the secretor status, implying broad binding patterns. However, they did not display specific binding to any of the oligosaccharides tested. In addition, we solved the structure of human P[19] VP8* at 2.4 Å, which revealed a typical galectin-like fold. The structural alignment demonstrated that P[19] VP8* was most similar to that of P[8], which was consistent with the phylogenetic analysis. Structure superimposition revealed the basis for the lack of binding to the oligosaccharides. Our study indicates that P[19] RVs may bind to other oligosaccharides or ligands and may have the potential to spread widely among humans. Thus, it is necessary to place the prevalence and evolution of P[19] RVs under surveillance. IMPORTANCE:Human P[19] is a rare P genotype of porcine origin. Based on phylogenetic analysis of VP8* sequences, P[19] was classified in the P[II] genogroup, together with P[4], P[6], and P[8], which have been reported to interact with HBGAs in a genotype-dependent manner. In this study, we explored the functional and structural characteristics of P[19] VP8* interaction with HBGAs. P[19] VP8* showed binding to A-, B-, and O-type saliva samples, as well as saliva of nonsecretors. This implies that P[19] has the potential to spread among humans with a broad binding range. Careful attention should be paid to the evolution and prevalence of P[19] RVs. Furthermore, we solved the structure of P[19] VP8*. Structure superimposition indicated that P[19] may bind to other oligosaccharides or ligands using potential binding sites, suggesting that further investigation of the specific cell attachment factors is warranted.
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Project description:We previously generated 32 rotavirus-specific (RV-specific) recombinant monoclonal antibodies (mAbs) derived from B cells isolated from human intestinal resections. Twenty-four of these mAbs were specific for the VP8* fragment of RV VP4, and most (20 of 24) were non-neutralizing when tested in the conventional MA104 cell-based assay. We reexamined the ability of these mAbs to neutralize RVs in human intestinal epithelial cells including ileal enteroids and HT-29 cells. Most (18 of 20) of the "non-neutralizing" VP8* mAbs efficiently neutralized human RV in HT-29 cells or enteroids. Serum RV neutralization titers in adults and infants were significantly higher in HT-29 than MA104 cells and adsorption of these sera with recombinant VP8* lowered the neutralization titers in HT-29 but not MA104 cells. VP8* mAbs also protected suckling mice from diarrhea in an in vivo challenge model. X-ray crystallographic analysis of one VP8* mAb (mAb9) in complex with human RV VP8* revealed that the mAb interaction site was distinct from the human histo-blood group antigen binding site. Since MA104 cells are the most commonly used cell line to detect anti-RV neutralization activity, these findings suggest that prior vaccine and other studies of human RV neutralization responses may have underestimated the contribution of VP8* antibodies to the overall neutralization titer.
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Project description:BACKGROUND:Rotaviruses (RVs) are a major cause of acute children gastroenteritis. The rotavirus P [10] belongs to P[I] genogroup of group A rotaviruses that mainly infect animals, while the rotavirus P [10] was mainly identified from human infection. The rotavirus P [10] is an unusual genotype and the recognition pattern of cellular receptors remains unclear. METHODS:We expressed and purified the RV P [10] VP8* protein and investigated the saliva and oligosaccharide binding profiles of the protein. A homology model of the P [10] VP8* core protein was built and the superimposition structural analysis of P [10] VP8* protein on P [19] VP8* in complex with mucin core 2 was performed to explore the possible docking structural basis of P [10] VP8* and mucin cores. RESULTS:Our data showed that rotavirus P [10] VP8* protein bound to all ABO secretor and non-secretor saliva. The rotavirus P [10] could bind strongly to mucin core 2 and weakly to mucin core 4. The homology modeling indicated that RV P [10] VP8* binds to mucin core 2 using a potential glycan binding site that is the same to P [19] VP8* belonging to P[II] genogroup. CONCLUSION:Our results suggested an interaction of rotavirus P [10] VP8* protein with mucin core 2 and mucin core 4. These findings offer potential for elucidating the mechanism of RV A host specificity, evolution and epidemiology.
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Project description:The epitopes of the HRV (human rotavirus), especially those involved in virus neutralization, have not been determined in their entirety, and would have significant implications for HRV vaccine development. In the present study, we report on the epitope mapping and identification of sequential neutralization epitopes, on the Wa strain HRV subunit protein VP8, using synthetic overlapping peptides. Polyclonal antibodies against recombinant Wa VP8 were produced previously in chicken, and purified from egg yolk, which showed neutralizing activity against HRV in vitro. Overlapping VP8 peptide fragments were synthesized and probed with the anti-VP8 antibodies, revealing five sequential epitopes on VP8. Further analysis suggested that three of the five epitopes detected, M1-L10, I55-D66 and L223-P234, were involved in virus neutralization, indicating that sequential epitopes may also be important for the HRV neutralization. The interactions of the antibodies with the five epitopes were characterized by an examination of the critical amino acids involved in antibody binding. Epitopes comprised primarily of hydrophobic amino acid residues, followed by polar and charged residues. The more critical amino acids appeared to be located near the centre of the epitopes, with proline, isoleucine, serine, glutamine and arginine playing an important role in the binding of antibody to the VP8 epitopes.
| S-EPMC1223744 | biostudies-other
Project description:Group/species C rotaviruses (RVCs) have been identified as important pathogens of acute gastroenteritis (AGE) in children, family-based outbreaks, as well as animal infections. However, little is known regarding their host-specific interaction, infection, and pathogenesis. In this study, we performed serial studies to characterize the function and structural features of a human G4P[2] RVC VP8* that is responsible for the host receptor interaction. Glycan microarrays demonstrated that the human RVC VP8* recognizes type A histo-blood group antigens (HBGAs), which was confirmed by synthetic glycan-/saliva-based binding assays and hemagglutination of red blood cells, establishing a paradigm of RVC VP8*-glycan interactions. Furthermore, the high-resolution crystal structure of the human RVC VP8* was solved, showing a typical galectin-like structure consisting of two β-sheets but with significant differences from cogent proteins of group A rotaviruses (RVAs). The VP8* in complex with a type A trisaccharide displays a novel ligand binding site that consists of a particular set of amino acid residues of the C-D, G-H, and K-L loops. RVC VP8* interacts with type A HBGAs through a unique mechanism compared with that used by RVAs. Our findings shed light on the host-virus interaction and the coevolution of RVCs and will facilitate the development of specific antivirals and vaccines.IMPORTANCE Group/species C rotaviruses (RVCs), members of Reoviridae family, infect both humans and animals, but our knowledge about the host factors that control host susceptibility and specificity is rudimentary. In this work, we characterized the glycan binding specificity and structural basis of a human RVC that recognizes type A HBGAs. We found that human RVC VP8*, the rotavirus host ligand binding domain that shares only ∼15% homology with the VP8* domains of RVAs, recognizes type A HBGA at an as-yet-unknown glycan binding site through a mechanism distinct from that used by RVAs. Our new advancements provide insights into RVC-cell attachment, the critical step of virus infection, which will in turn help the development of control and prevention strategies against RVs.
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Project description:Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a "double-layer particle": DLP). Delivery is the function of a third, outer layer, which drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca2+ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca2+-stabilized trimer; loss of Ca2+ releases both VP7 and the other outer-layer protein, VP4, from the particle. VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca2+ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions ("triple-layer particles": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca2+ "leak". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry.
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Project description:Rotaviruses (RVs), an important cause of severe diarrhea in children, have been found to recognize sialic acid as receptors for host cell attachment. While a few animal RVs (of P[1], P[2], P[3], and P[7]) are sialidase sensitive, human RVs and the majority of animal RVs are sialidase insensitive. In this study, we demonstrated that the surface spike protein VP8* of the major P genotypes of human RVs interacts with the secretor histo-blood group antigens (HBGAs). Strains of the P[4] and P[8] genotypes shared reactivity with the common antigens of Lewis b (Le(b)) and H type 1, while strains of the P[6] genotype bound the H type 1 antigen only. The bindings between recombinant VP8* and human saliva, milk, or synthetic HBGA oligosaccharides were demonstrated, which was confirmed by blockade of the bindings by monoclonal antibodies (MAbs) specific to Le(b) and/or H type 1. In addition, specific binding activities were observed when triple-layered particles of a P[8] (Wa) RV were tested. Our results suggest that the spike protein VP8* of RVs is involved in the recognition of human HBGAs that may function as ligands or receptors for RV attachment to host cells.
| S-EPMC3347384 | biostudies-literature
Project description:Initial cell attachment of rotavirus (RV) to specific cell surface glycan receptors, which is the essential first step in RV infection, is mediated by the VP8* domain of the spike protein VP4. Recently, human histo-blood group antigens (HBGAs) have been identified as receptors or attachment factors for human RV strains. RV strains in the P[4] and P[8] genotypes of the P[II] genogroup share common recognition of the Lewis b (Leb) and H type 1 antigens, however, the molecular basis of receptor recognition by the major human P[8] RVs remains unknown due to lack of experimental structural information. Here, we used nuclear magnetic resonance (NMR) spectroscopy-based titration experiments and NMR-derived high ambiguity driven docking (HADDOCK) methods to elucidate the molecular basis for P[8] VP8* recognition of the Leb (LNDFH I) and type 1 HBGAs. We also used X-ray crystallography to determine the molecular details underlying P[6] recognition of H type 1 HBGAs. Unlike P[6]/P[19] VP8*s that recognize H type 1 HBGAs in a binding surface composed of an ?-helix and a ?-sheet, referred as the "?? binding site", the P[8] and P[4] VP8*s bind Leb HBGAs in a previously undescribed pocket formed by the edges of two ?-sheets, referred to as the "?? binding site". Importantly, the P[8] and P[4] VP8*s retain binding capability to non-Leb type 1 HBGAs using the ?? binding site. The presence of two distinct binding sites for Leb and non-Leb HBGA glycans in the P[8] and P[4] VP8* domains suggests host-pathogen co-evolution under structural and functional adaptation of RV pathogens to host glycan polymorphisms. Assessment and understanding of the precise impact of this co-evolutionary process in determining RV host ranges and cross-species RV transmission should facilitate improved RV vaccine development and prediction of future RV strain emergence and epidemics.
| S-EPMC7122821 | biostudies-literature
Project description:Rotaviruses (RVs), which cause severe gastroenteritis in infants and children, recognize glycan ligands in a genotype-dependent manner via the distal VP8* head of the spike protein VP4. However, the glycan binding mechanisms remain elusive for the P[II] genogroup RVs, including the widely prevalent human RVs (P[8], P[4], and P[6]) and a rare P[19] RV. In this study, we characterized the glycan binding specificities of human and porcine P[6]/P[19] RV VP8*s and found that the P[II] genogroup RV VP8*s could commonly interact with mucin core 2, which may play an important role in RV evolution and cross-species transmission. We determined the first P[6] VP8* structure, as well as the complex structures of human P[19] VP8*, with core 2 and lacto-N-tetraose (LNT). A glycan binding site was identified in human P[19] VP8*. Structural superimposition and sequence alignment revealed the conservation of the glycan binding site in the P[II] genogroup RV VP8*s. Our data provide significant insight into the glycan binding specificity and glycan binding mechanism of the P[II] genogroup RV VP8*s, which could help in understanding RV evolution, transmission, and epidemiology and in vaccine development.IMPORTANCE Rotaviruses (RVs), belonging to the family Reoviridae, are double-stranded RNA viruses that cause acute gastroenteritis in children and animals worldwide. Depending on the phylogeny of the VP8* sequences, P[6] and P[19] RVs are grouped into genogroup II, together with P[4] and P[8], which are widely prevalent in humans. In this study, we characterized the glycan binding specificities of human and porcine P[6]/P[19] RV VP8*s, determined the crystal structure of P[6] VP8*, and uncovered the glycan binding pattern in P[19] VP8*, revealing a conserved glycan binding site in the VP8*s of P[II] genogroup RVs by structural superimposition and sequence alignment. Our data suggested that mucin core 2 may play an important role in P[II] RV evolution and cross-species transmission. These data provide insight into the cell attachment, infection, epidemiology, and evolution of P[II] genogroup RVs, which could help in developing control and prevention strategies against RVs.
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