Functional refolding of the penetration protein on a non-enveloped virus.

A non-enveloped virus requires a membrane lesion to deliver its genome into a target cell1. For rotaviruses, membrane perforation is a principal function of the viral outer-layer protein, VP42,3. Here we describe the use of electron cryomicroscopy to determine how VP4 performs this function and show that when activated by cleavage to VP8* and VP5*, VP4 can rearrange on the virion surface from an 'upright' to a 'reversed' conformation. The reversed structure projects a previously buried 'foot' domain outwards into the membrane of the host cell to which the virion has attached. Electron cryotomograms of virus particles entering cells are consistent with this picture. Using a disulfide mutant of VP4, we have also stabilized a probable intermediate in the transition between the two conformations. Our results define molecular mechanisms for the first steps of the penetration of rotaviruses into the membranes of target cells and suggest similarities with mechanisms postulated for other viruses.

Flexibility of the Rotavirus NSP2 C-Terminal Region Supports Factory Formation via Liquid-Liquid Phase Separation.

Many viruses sequester the materials needed for their replication into discrete subcellular factories. For rotaviruses (RVs), these factories are called viroplasms, and they are formed in the host cell cytosol via the process of liquid-liquid phase separation (LLPS). The nonstructural protein 2 (NSP2) and its binding partner, nonstructural protein 5 (NSP5), are critical for viroplasm biogenesis. Yet it is not fully understood how NSP2 and NSP5 cooperate to form factories. The C-terminal region (CTR) of NSP2 (residues 291 to 317) is flexible, allowing it to participate in domain-swapping interactions that promote interoctamer interactions and, presumably, viroplasm formation. Molecular dynamics simulations showed that a lysine-to-glutamic acid change at position 294 (K294E) reduces NSP2 CTR flexibility in silico. To test the impact of reduced NSP2 CTR flexibility during infection, we engineered a mutant RV bearing this change (rRV-NSP2K294E). Single-cycle growth assays revealed a >1.2-log reduction in endpoint titers for rRV-NSP2K294E versus the wild-type control (rRV-WT). Using immunofluorescence assays, we found that rRV-NSP2K294E formed smaller, more numerous viroplasms than rRV-WT. Live-cell imaging experiments confirmed these results and revealed that rRV-NSP2K294E factories had delayed fusion kinetics. Moreover, NSP2K294E and several other CTR mutants formed fewer viroplasm-like structures in NSP5 coexpressing cells than did control NSP2WT. Finally, NSP2K294E exhibited defects in its capacity to induce LLPS droplet formation in vitro when incubated alongside NSP5. These results underscore the importance of NSP2 CTR flexibility in supporting the biogenesis of RV factories. IMPORTANCE Viruses often condense the materials needed for their replication into discrete intracellular factories. For rotaviruses, agents of severe gastroenteritis in children, factory formation is mediated in part by an octameric protein called NSP2. A flexible C-terminal region of NSP2 has been proposed to link several NSP2 octamers together, a feature that might be important for factory formation. Here, we created a change in NSP2 that reduced C-terminal flexibility and analyzed the impact on rotavirus factories. We found that the change caused the formation of smaller and more numerous factories that could not readily fuse together like those of the wild-type virus. The altered NSP2 protein also had a reduced capacity to form factory-like condensates in a test tube. Together, these results add to our growing understanding of how NSP2 supports rotavirus factory formation-a key step of viral replication.

Interaction of species A rotavirus VP4 with the cellular proteins vimentin and actin related protein 2 discovered by a proximity interactome assay.

IMPORTANCE: Rotavirus (RV) is an important zoonosis virus, which can cause severe diarrhea and extra-intestinal infection. To date, some proteins or carbohydrates have been shown to participate in the attachment or internalization of RV, including HGBAs, Hsc70, and integrins. This study attempted to indicate whether there were other proteins that would participate in the entry of RV; thus, the RV VP4-interacting proteins were identified by proximity labeling. After analysis and verification, it was found that VIM and ACTR2 could significantly promote the proliferation of RV in intestinal cells. Through further viral binding assays after knockdown, antibody blocking, and recombinant protein overexpression, it was revealed that both VIM and ACTR2 could promote RV replication.

Structural basis of P[II] rotavirus evolution and host ranges under selection of histo-blood group antigens.

Group A rotaviruses cause severe gastroenteritis in infants and young children worldwide, with P[II] genogroup rotaviruses (RVs) responsible for >90% of global cases. RVs have diverse host ranges in different human and animal populations determined by host histo-blood group antigen (HBGA) receptor polymorphism, but details governing diversity, host ranges, and species barriers remain elusive. In this study, crystal structures of complexes of the major P[II] genogroup P[4] and P[8] genotype RV VP8* receptor-binding domains together with Lewis epitope-containing LNDFH I glycans in combination with VP8* receptor-glycan ligand affinity measurements based on NMR titration experiments revealed the structural basis for RV genotype-specific switching between ßß and ßα HBGA receptor-binding sites that determine RV host ranges. The data support the hypothesis that P[II] RV evolution progressed from animals to humans under the selection of type 1 HBGAs guided by stepwise host synthesis of type 1 ABH and Lewis HBGAs. The results help explain disease burden, species barriers, epidemiology, and limited efficacy of current RV vaccines in developing countries. The structural data has the potential to impact the design of future vaccine strategies against RV gastroenteritis.

Spontaneous Interconversion between Different Narrowly Defined Shapes of Rotavirus Double-Layered Particles Studied in Real Time by High-Resolution Mobility Analysis.

Rotavirus double-layered particles (DLPs) are studied in the gas phase with a high-resolution differential mobility analyzer (DMA). DLPs were transferred to 10 mM aqueous ammonium acetate, electrosprayed into the gas phase, converted into primarily singly charged particles, and DMA-analyzed. Up to seven slightly different conformations were resolved, whose apparently random, fast (minutes), and reversible interconversions were followed in real time. They sometimes evolved into just two distinct structures, with periods of one dominating over the other and vice versa. Differences between the DLP structures in solution and in the gas phase are clearly revealed by the smaller DLP diameter found here (60 versus 70 nm). Nevertheless, we argue that the multiple gas-phase conformers observed originate in as many conformations pre-existing in solution. We further hypothesize that these conformers correspond to incomplete DLPs having lost some of the VP6 trimer quintets surrounding each of the 12 5-fold axes. Instances of this peculiar loss have been previously documented by cryoelectron microscopy for the rotavirus Wa strain, as well as via charge detection mass spectrometry for five other rotavirus strains included in the RotaTec vaccine. Evidence of this loss systematically found for all 7 rotavirus types so far studied in aqueous ammonium acetate may be a special feature of this electrolyte.

Rotavirus VP4 Epitope of a Broadly Neutralizing Human Antibody Defined by Its Structure Bound with an Attenuated-Strain Virion.

Rotavirus live-attenuated vaccines, both mono- and pentavalent, generate broadly heterotypic protection. B-cells isolated from adults encode neutralizing antibodies, some with affinity for VP5*, that afford broad protection in mice. We have mapped the epitope of one such antibody by determining the high-resolution cryo-EM structure of its antigen-binding fragment (Fab) bound to the virion of a candidate vaccine strain, CDC-9. The Fab contacts both the distal end of a VP5* ß-barrel domain and the two VP8* lectin-like domains at the tip of a projecting spike. Its interactions with VP8* do not impinge on the likely receptor-binding site, suggesting that the mechanism of neutralization is at a step subsequent to initial attachment. We also examined structures of CDC-9 virions from two different stages of serial passaging. Nearly all the VP4 (cleaved to VP8*/VP5*) spikes on particles from the earlier passage (wild-type isolate) had transitioned from the "upright" conformation present on fully infectious virions to the "reversed" conformation that is probably the end state of membrane insertion, unable to mediate penetration, consistent with the very low in vitro infectivity of the wild-type isolate. About half the VP4 spikes were upright on particles from the later passage, which had recovered substantial in vitro infectivity but had acquired an attenuated phenotype in neonatal rats. A mutation in VP4 that occurred during passaging appears to stabilize the interface at the apex of the spike and could account for the greater stability of the upright spikes on the late-passage, attenuated isolate. IMPORTANCE Rotavirus live-attenuated vaccines generate broadly heterotypic protection, and B-cells isolated from adults encode antibodies that are broadly protective in mice. Determining the structural and mechanistic basis of broad protection can contribute to understanding the current limitations of vaccine efficacy in developing countries. The structure of an attenuated human rotavirus isolate (CDC-9) bound with the Fab fragment of a broadly heterotypic protective antibody shows that protection is probably due to inhibition of the conformational transition in the viral spike protein (VP4) critical for viral penetration, rather than to inhibition of receptor binding. A comparison of structures of CDC-9 virus particles at two stages of serial passaging supports a proposed mechanism for initial steps in rotavirus membrane penetration.

Molecular basis of P[II] major human rotavirus VP8* domain recognition of histo-blood group antigens.

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 α-helix and a ß-sheet, referred as the "ßα binding site", the P[8] and P[4] VP8*s bind Leb HBGAs in a previously undescribed pocket formed by the edges of two ß-sheets, referred to as the "ßß binding site". Importantly, the P[8] and P[4] VP8*s retain binding capability to non-Leb type 1 HBGAs using the ßα 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.

The origins of G12P[6] rotavirus strains detected in Lebanon.

The G12 rotaviruses are an increasingly important cause of severe diarrhoea in infants and young children worldwide. Seven human G12P[6] rotavirus strains were detected in stool samples from children hospitalized with gastroenteritis in Lebanon during a 2011-2013 surveillance study. Complete genomes of these strains were sequenced using VirCapSeq-VERT, a capture-based high-throughput viral-sequencing method, and further characterized based on phylogenetic analyses with global RVA and vaccine strains. Based on the complete genomic analysis, all Lebanese G12 strains were found to have Wa-like genetic backbone G12-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1. Phylogenetically, these strains fell into two clusters where one of them might have emerged from Southeast Asian strains and the second one seems to have a mixed backbone between North American and Southeast Asian strains. Further analysis of these strains revealed high antigenic variability compared to available vaccine strains. To our knowledge, this is the first report on the complete genome-based characterization of G12P[6] emerging in Lebanon. Additional studies will provide important insights into the evolutionary dynamics of G12 rotaviruses spreading in Asia.

Unraveling the role of the secretor antigen in human rotavirus attachment to histo-blood group antigens.

Rotavirus is the leading agent causing acute gastroenteritis in young children, with the P[8] genotype accounting for more than 80% of infections in humans. The molecular bases for binding of the VP8* domain from P[8] VP4 spike protein to its cellular receptor, the secretory H type-1 antigen (Fuc-α1,2-Gal-ß1,3-GlcNAc; H1), and to its precursor lacto-N-biose (Gal-ß1,3-GlcNAc; LNB) have been determined. The resolution of P[8] VP8* crystal structures in complex with H1 antigen and LNB and site-directed mutagenesis experiments revealed that both glycans bind to the P[8] VP8* protein through a binding pocket shared with other members of the P[II] genogroup (i.e.: P[4], P[6] and P[19]). Our results show that the L-fucose moiety from H1 only displays indirect contacts with P[8] VP8*. However, the induced conformational changes in the LNB moiety increase the ligand affinity by two-fold, as measured by surface plasmon resonance (SPR), providing a molecular explanation for the different susceptibility to rotavirus infection between secretor and non-secretor individuals. The unexpected interaction of P[8] VP8* with LNB, a building block of type-1 human milk oligosaccharides, resulted in inhibition of rotavirus infection, highlighting the role and possible application of this disaccharide as an antiviral. While key amino acids in the H1/LNB binding pocket were highly conserved in members of the P[II] genogroup, differences were found in ligand affinities among distinct P[8] genetic lineages. The variation in affinities were explained by subtle structural differences induced by amino acid changes in the vicinity of the binding pocket, providing a fine-tuning mechanism for glycan binding in P[8] rotavirus.

Advanced sequence optimization for the high efficient yield of human group A rotavirus VP6 recombinant protein in Escherichia coli and its use as immunogen.

Rotavirus is the important etiological agents of infectious diarrhea among children under 5 years old. Rotaviruses are divided into 10 serogroups (A-J) and each group is based on genetic properties of major structural protein VP6. We designed a novel VP6 sequence optimization to increase the expression level of this protein. Numerous factors such as codon adaptation index, codon pair bias, and guanine-cytosine content were adapted based on Escherichiacoli codon usage. In addition, the ribosome binding site (RBS) of pET-15b was redesigned by the RBS calculator and the secondary structure of VP6 messenger RNA was optimized in the whole length of the coding sequence. Various factors including isopropyl beta- d-thiogalactoside (IPTG) concentration, temperature, and induction time were analyzed for the optimization of the best expression in E. coli by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. The recombinant VP6 (rVP6) protein was purified by the Ni-sepharose and then the hyperimmune sera were generated against rVP6 in rabbits. Among three different temperatures, IPTG concentrations, and postinductions, the level of rVP6 was higher at 37°C, 1 mM of IPTG, and 8 h, respectively. Also, the high expression level of rVP6 was obtained in the insoluble aggregate form (43.8 g/L). After purification, the yield of rVP6 was 10.83 g/L. The rVP6 specific antiserum was confirmed by both immunofluorescent and western blotting. The versatile sequence optimization was the reason to produce a high level of rVP6 compared to other reports and can potentially apply to produce cheaper commercial kits to diagnose serological tests and new rotavirus vaccines.

Crystallization of a nonreplicating rotavirus vaccine candidate.

Nonreplicating rotavirus vaccine (NRRV) candidates are being developed with the aim of serving the needs of developing countries. A significant proportion of the cost of manufacturing such vaccines is the purification in multiple chromatography steps. Crystallization has the potential to reduce purification costs and provide new product storage modality, improved operational flexibility, and reduced facility footprints. This communication describes a systematic approach for the design of the crystallization of an NRRV candidate, VP8 subunit proteins fused to the P2 epitope of tetanus toxin, using first-principles models and preliminary experimental data. The first-principles models are applied to literature data to obtain feasible crystallization conditions and lower bounds for nucleation and growth rates. Crystallization is then performed in a hanging-drop vapor diffusion system, resulting in the nucleation and growth of NRRV crystals. The crystals obtained in a scaled-up evaporative crystallization contain proteins truncated in the P2 region, but have no significant differences with the original samples in terms of antibody binding and overall conformational stability. These results demonstrate the promise of evaporative crystallization of the NRRV.

The C Terminus of Rotavirus VP4 Protein Contains an Actin Binding Domain Which Requires Cooperation with the Coiled-Coil Domain for Actin Remodeling.

The interactions between viruses and actin cytoskeleton have been widely studied. We showed that rotaviruses remodel microfilaments in intestinal cells and demonstrated that this was due to the VP4 spike protein. Microfilaments mainly occur in the apical domain of infected polarized enterocytes and favor the polarized apical exit of viral progeny. The present work aims at the identification of molecular determinants of actin-VP4 interactions. We used various deletion mutants of VP4 that were transfected into Cos-7 cells and analyzed interactions by immunofluorescence confocal microscopy. It has been established that the C-terminal part of VP4 is embedded within viral particles when rotavirus assembles. The use of specific monoclonal antibodies demonstrated that VP4 is expressed in different forms in infected cells: classically as spike on the outer layer of virus particles, but also as free soluble protein in the cytosol. The C terminus of free VP4 was identified as interacting with actin microfilaments. The VP4 actin binding domain is unable to promote microfilament remodeling by itself; the coiled-coil domain is also required in this process. This actin-binding domain was shown to dominate a previously identified peroxisomal targeting signal, located in the three last amino acids of VP4. The newly identified actin-binding domain is highly conserved in rotavirus strains from species A, B, and C, suggesting that actin binding and remodeling is a general strategy for rotavirus exit. This provides a novel mechanism of protein-protein interactions, not involving cell signaling pathways, to facilitate rotavirus exit.IMPORTANCE Rotaviruses are causal agents of acute infantile viral diarrhea. In intestinal cells, in vitro as well as in vivo, virus assembly and exit do not imply cell lysis but rely on an active process in which the cytoskeleton plays a major role. We describe here a novel molecular mechanism by which the rotavirus spike protein VP4 drives actin remodeling. This relies on the fact that VP4 occurs in different forms. Besides its structural function within the virion, a large proportion of VP4 is expressed as free protein. Here, we show that free VP4 possesses a functional actin-binding domain. This domain, in coordination with a coiled-coil domain, promotes actin cytoskeleton remodeling, thereby providing the capacity to destabilize the cell membrane and allow efficient rotavirus exit.

Identification of a Small Molecule That Compromises the Structural Integrity of Viroplasms and Rotavirus Double-Layered Particles.

Despite the availability of two attenuated vaccines, rotavirus (RV) gastroenteritis remains an important cause of mortality among children in developing countries, causing about 215,000 infant deaths annually. Currently, there are no specific antiviral therapies available. RV is a nonenveloped virus with a segmented double-stranded RNA genome. Viral genome replication and assembly of transcriptionally active double-layered particles (DLPs) take place in cytoplasmic viral structures called viroplasms. In this study, we describe strong impairment of the early stages of RV replication induced by a small molecule known as an RNA polymerase III inhibitor, ML-60218 (ML). This compound was found to disrupt already assembled viroplasms and to hamper the formation of new ones without the need for de novo transcription of cellular RNAs. This phenotype was correlated with a reduction in accumulated viral proteins and newly made viral genome segments, disappearance of the hyperphosphorylated isoforms of the viroplasm-resident protein NSP5, and inhibition of infectious progeny virus production. In in vitro transcription assays with purified DLPs, ML showed dose-dependent inhibitory activity, indicating the viral nature of its target. ML was found to interfere with the formation of higher-order structures of VP6, the protein forming the DLP outer layer, without compromising its ability to trimerize. Electron microscopy of ML-treated DLPs showed dose-dependent structural damage. Our data suggest that interactions between VP6 trimers are essential, not only for DLP stability, but also for the structural integrity of viroplasms in infected cells.IMPORTANCE Rotavirus gastroenteritis is responsible for a large number of infant deaths in developing countries. Unfortunately, in the countries where effective vaccines are urgently needed, the efficacy of the available vaccines is particularly low. Therefore, the development of antivirals is an important goal, as they might complement the available vaccines or represent an alternative option. Moreover, they may be decisive in fighting the acute phase of infection. This work describes the inhibitory effect on rotavirus replication of a small molecule initially reported as an RNA polymerase III inhibitor. The molecule is the first chemical compound identified that is able to disrupt viroplasms, the viral replication machinery, and to compromise the stability of DLPs by targeting the viral protein VP6. This molecule thus represents a starting point in the development of more potent and less cytotoxic compounds against rotavirus infection.

Structural basis of glycan specificity of P[19] VP8*: Implications for rotavirus zoonosis and evolution.

Recognition of specific cell surface glycans, mediated by the VP8* domain of the spike protein VP4, is the essential first step in rotavirus (RV) infection. Due to lack of direct structural information of virus-ligand interactions, the molecular basis of ligand-controlled host ranges of the major human RVs (P[8] and P[4]) in P[II] genogroup remains unknown. Here, through characterization of a minor P[II] RV (P[19]) that can infect both animals (pigs) and humans, we made an important advance to fill this knowledge gap by solving the crystal structures of the P[19] VP8* in complex with its ligands. Our data showed that P[19] RVs use a novel binding site that differs from the known ones of other genotypes/genogroups. This binding site is capable of interacting with two types of glycans, the mucin core and type 1 histo-blood group antigens (HBGAs) with a common GlcNAc as the central binding saccharide. The binding site is apparently shared by other P[II] RVs and possibly two genotypes (P[10] and P[12]) in P[I] as shown by their highly conserved GlcNAc-interacting residues. These data provide strong evidence of evolutionary connections among these human and animal RVs, pointing to a common ancestor in P[I] with a possible animal host origin. While the binding properties to GlcNAc-containing saccharides are maintained, changes in binding to additional residues, such as those in the polymorphic type 1 HBGAs may occur in the course of RV evolution, explaining the complex P[II] genogroup that mainly causes diseases in humans but also in some animals.

Structural basis for concerted recruitment and activation of IRF-3 by innate immune adaptor proteins.

Type I IFNs are key cytokines mediating innate antiviral immunity. cGMP-AMP synthase, ritinoic acid-inducible protein 1 (RIG-I)-like receptors, and Toll-like receptors recognize microbial double-stranded (ds)DNA, dsRNA, and LPS to induce the expression of type I IFNs. These signaling pathways converge at the recruitment and activation of the transcription factor IRF-3 (IFN regulatory factor 3). The adaptor proteins STING (stimulator of IFN genes), MAVS (mitochondrial antiviral signaling), and TRIF (TIR domain-containing adaptor inducing IFN-ß) mediate the recruitment of IRF-3 through a conserved pLxIS motif. Here we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF binds to IRF-3 in a similar manner, whereas residues upstream of the motif confer specificity. The structure of the IRF-3 phosphomimetic mutant S386/396E bound to the cAMP response element binding protein (CREB)-binding protein reveals that the pLxIS motif also mediates IRF-3 dimerization and activation. Moreover, rotavirus NSP1 (nonstructural protein 1) employs a pLxIS motif to target IRF-3 for degradation, but phosphorylation of NSP1 is not required for its activity. These results suggest a concerted mechanism for the recruitment and activation of IRF-3 that can be subverted by viral proteins to evade innate immune responses.

Complete genome characterization of a rotavirus B (RVB) strain identified in Alpine goat kids with enteritis reveals inter-species transmission with RVB bovine strains.

Rotavirus B (RVB) has been associated with enteric disease in many animal species. An RVB strain was identified in pooled intestinal samples from Alpine caprine kids (between 2 and 3 days of age) experiencing high (>90 %) morbidity, and the complete caprine RVB genome was characterized. Histology revealed villus atrophy, the samples tested positive for RVB by real-time RT-PCR and metagenomic next-generation sequencing identified only RVB and orf virus. In the VP4 gene segment, the caprine RVB strain had a higher percentage nucleotide identity to the Indian bovine RVB strains than to the Japanese bovine RVB strains, but the VP7, VP6, VP2, NSP1, NSP2 and NSP5 gene segments of the American caprine RVB strain were genetically related to the Japanese bovine RVB strains. The results indicate a lack of RVB sequences to understand reassortment or the evolutionary relationship of RVB strains from cattle and goats.

Specificity and affinity of neuraminic acid exhibited by canine rotavirus strain K9 carbohydrate-binding domain (VP8*).

The outer capsid spike protein VP4 of rotaviruses is a major determinant of infectivity and serotype specificity. Proteolytic cleavage of VP4 into 2 domains, VP8* and VP5*, enhances rotaviral infectivity. Interactions between the VP4 carbohydrate-binding domain (VP8*) and cell surface glycoconjugates facilitate initial virus-cell attachment and subsequent cell entry. Our saturation transfer difference nuclear magnetic resonance (STD NMR) and isothermal titration calorimetry (ITC) studies demonstrated that VP8*64-224 of canine rotavirus strain K9 interacts with N-acetylneuraminic and N-glycolylneuraminic acid derivatives, exhibiting comparable binding epitopes to VP8* from other neuraminidase-sensitive animal rotaviruses from pigs (CRW-8), cattle (bovine Nebraska calf diarrhoea virus, NCDV), and Rhesus monkeys (Simian rhesus rotavirus, RRV). Importantly, evidence was obtained for a preference by K9 rotavirus for the N-glycolyl- over the N-acetylneuraminic acid derivative. This indicates that a VP4 serotype 5A rotavirus (such as K9) can exhibit a neuraminic acid receptor preference that differs from that of a serotype 5B rotavirus (such as RRV) and the receptor preference of rotaviruses can vary within a particular VP4 genotype.

New tetrameric forms of the rotavirus NSP4 with antiparallel helices.

Rotavirus nonstructural protein 4, the first viral enterotoxin to be identified, is a multidomain, multifunctional glycoprotein. Earlier, we reported a Ca2+-bound coiled-coil tetrameric structure of the diarrhea-inducing region of NSP4 from the rotavirus strains SA11 and I321 and a Ca2+-free pentameric structure from the rotavirus strain ST3, all with a parallel arrangement of α-helices. pH was found to determine the oligomeric state: a basic pH favoured a tetramer, whereas an acidic pH favoured a pentamer. Here, we report two novel forms of the coiled-coil region of NSP4 from the bovine rotavirus strains MF66 and NCDV. These crystallized at acidic pH, forming antiparallel coiled-coil tetrameric structures without any bound Ca2+ ion. Structural and mutational studies of the coiled-coil regions of NSP4 revealed that the nature of the residue at position 131 (Tyr/His) plays an important role in the observed structural diversity.

Performance of pre-RT-qPCR treatments to discriminate infectious human rotaviruses and noroviruses from heat-inactivated viruses: applications of PMA/PMAxx, benzonase and RNase.

AIMS: Detection/Quantification of RNA viruses is mostly done by reverse-transcriptase (RT)-(q)PCR, but it does not distinguish between infectious and noninfectious viruses. Our aim was to test, how different pretreatments before RT-qPCR could eliminate positivity originated from external nucleic acids or genomes of damaged particles. METHODS AND RESULTS: Heat-inactivated (80°C for 10 min) rotavirus Wa strain and faecal samples containing rotavirus or norovirus were treated with PMA/PMAxx, benzonase or crude extract RNase prior to RT-qPCR. PMA/PMAxx pretreatments were not consistently efficient for RV, although they seemed to work to some extent for heat-inactivated norovirus. Benzonase and RNase provided consistently 2·2-2·8 log10 reductions in the titre of faecal rotavirus. CONCLUSIONS: All pretreatments need to be further validated for each virus separately, taking into account sample matrix and inactivation conditions. Although none of the pretreatments could completely render inactivated viruses undetectable, RNase worked most consistently for both rota- and norovirus. SIGNIFICANCE AND IMPACT OF THE STUDY: This study sheds light on capacity of the most common pre-RT-qPCR treatments to eliminate damaged, noninfectious rotaviruses and noroviruses after thermal treatment. To our knowledge, this is the first time, when benzonase has been used in this context.

Cell attachment protein VP8* of a human rotavirus specifically interacts with A-type histo-blood group antigen.

As with many other viruses, the initial cell attachment of rotaviruses, which are the major causative agent of infantile gastroenteritis, is mediated by interactions with specific cellular glycans. The distally located VP8* domain of the rotavirus spike protein VP4 (ref. 5) mediates such interactions. The existing paradigm is that 'sialidase-sensitive' animal rotavirus strains bind to glycans with terminal sialic acid (Sia), whereas 'sialidase-insensitive' human rotavirus strains bind to glycans with internal Sia such as GM1 (ref. 3). Although the involvement of Sia in the animal strains is firmly supported by crystallographic studies, it is not yet known how VP8* of human rotaviruses interacts with Sia and whether their cell attachment necessarily involves sialoglycans. Here we show that VP8* of a human rotavirus strain specifically recognizes A-type histo-blood group antigen (HBGA) using a glycan array screen comprised of 511 glycans, and that virus infectivity in HT-29 cells is abrogated by anti-A-type antibodies as well as significantly enhanced in Chinese hamster ovary cells genetically modified to express the A-type HBGA, providing a novel paradigm for initial cell attachment of human rotavirus. HBGAs are genetically determined glycoconjugates present in mucosal secretions, epithelia and on red blood cells, and are recognized as susceptibility and cell attachment factors for gastric pathogens like Helicobacter pylori and noroviruses. Our crystallographic studies show that the A-type HBGA binds to the human rotavirus VP8* at the same location as the Sia in the VP8* of animal rotavirus, and suggest how subtle changes within the same structural framework allow for such receptor switching. These results raise the possibility that host susceptibility to specific human rotavirus strains and pathogenesis are influenced by genetically controlled expression of different HBGAs among the world's population.