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.

MHC class I expression in intestinal cells is reduced by rotavirus infection and increased in bystander cells lacking rotavirus antigen.

Detection of viral infection by host cells leads to secretion of type I interferon, which induces antiviral gene expression. The class I major histocompatibility complex (MHCI) is required for viral antigen presentation and subsequent infected cell killing by cytotoxic T lymphocytes. STAT1 activation by interferon can induce NLRC5 expression, promoting MHCI expression. Rotavirus, an important pathogen, blocks interferon signalling through inhibition of STAT1 nuclear translocation. We assessed MHCI expression in HT-29 intestinal epithelial cells following rotavirus infection. MHCI levels were upregulated in a partially type I interferon-dependent manner in bystander cells lacking rotavirus antigen, but not in infected cells. MHCI and NLRC5 mRNA expression also was elevated in bystander, but not infected, cells, suggesting a transcriptional block in infected cells. STAT1 was activated in bystander and infected cells, but showed nuclear localisation in bystander cells only. Overall, the lack of MHCI upregulation in rotavirus-infected cells may be at least partially due to rotavirus blockade of interferon-induced STAT1 nuclear translocation. The reduced MHCI protein levels in infected cells support the existence of an additional, non-transcriptional mechanism that reduces MHCI expression. It is possible that rotavirus also may suppress MHCI expression in vivo, which might limit T cell-mediated killing of rotavirus-infected enterocytes.

Rotavirus NSP6 localizes to mitochondria via a predicted N-terminal a-helix.

Specific roles have been ascribed to each of the 12 known rotavirus proteins apart from the non-structural protein 6 (NSP6). However, NSP6 may be present at sites of viral replication within the cytoplasm. Here we report that NSP6 from diverse species of rotavirus A localizes to mitochondria via conserved sequences in a predicted N-terminal a-helix. This suggests that NSP6 may affect mitochondrial functions during rotavirus infection.

Substantial Receptor-induced Structural Rearrangement of Rotavirus VP8*: Potential Implications for Cross-Species Infection.

Rotavirus-cell binding is the essential first step in rotavirus infection. This binding is a major determinant of rotavirus tropism, as host cell invasion is necessary to initiate infection. Initial rotavirus-cell interactions are mediated by carbohydrate-recognizing domain VP8* of the rotavirus capsid spike protein VP4. Here, we report the first observation of significant structural rearrangement of VP8* from human and animal rotavirus strains upon glycan receptor binding. The structural adaptability of rotavirus VP8* delivers important insights into how human and animal rotaviruses utilize the wider range of cellular glycans identified as VP8* binding partners. Furthermore, our studies on rotaviruses with atypical genetic makeup provide information expected to be critical for understanding the mechanisms of animal rotavirus gene emergence in humans and support implementation of epidemiologic surveillance of animal reservoirs as well as future vaccination schemes.

Innate immune responses to rotavirus infection in macrophages depend on MAVS but involve neither the NLRP3 inflammasome nor JNK and p38 signaling pathways.

Rotavirus infection is a major cause of life-threatening infantile gastroenteritis. The innate immune system provides an immediate mechanism of suppressing viral replication and is necessary for an effective adaptive immune response. Innate immunity involves host recognition of viral infection and establishment of a powerful antiviral state through the expression of pro-inflammatory cytokines such as type-1 interferon (IFN). Macrophages, the front-line cells of innate immunity, produce IFN and other cytokines in response to viral infection. However, the role of macrophages during rotavirus infection is not well defined. We demonstrate here that RRV rotavirus triggers the production of proinflammatory cytokines from mouse bone marrow-derived macrophages. IFN and antiviral cytokine production was abolished in rotavirus-infected MAVS (-/-) macrophages. This indicates that rotavirus triggers innate immunity in macrophages through RIG-I and/or MDA5 viral recognition, and MAVS signaling is essential for cytokine responses in macrophages. Rotavirus induced IFN expression in both wild type and MDA5 (-/-) macrophages, showing that MDA5 is not essential for IFN secretion following infection, and RIG-I and MDA5 may act redundantly in promoting rotavirus recognition. Interestingly, rotavirus neither stimulated mitogen-activated protein kinases p38 and JNK nor activated the NLRP3 inflammasome, demonstrating that these components might not be involved in innate responses to rotavirus infection in macrophages. Our results indicate that rotavirus elicits intracellular signaling in macrophages, resulting in the induction of IFN and antiviral cytokines, and advance our understanding of the involvement of these cells in innate responses against rotavirus.

NSP1 of human rotaviruses commonly inhibits NF-κB signalling by inducing ß-TrCP degradation.

Rotavirus is a leading cause of severe gastroenteritis in infants worldwide. Rotavirus nonstructural protein 1 (NSP1) is a virulence factor that inhibits innate host immune responses. NSP1 from some rotaviruses targets host interferon response factors (IRFs), leading to inhibition of type I interferon expression. A few rotaviruses encode an NSP1 that inhibits the NF-κB pathway by targeting ß-TrCP, a protein required for IκB degradation and NF-κB activation. Available evidence suggests that these NSP1 properties involve proteosomal degradation of target proteins. We show here that NSP1 from several human rotaviruses and porcine rotavirus CRW-8 inhibits the NF-κB pathway, but cannot degrade IRF3. Furthermore, ß-TrCP levels were much reduced in cells infected with these rotaviruses. This provides strong evidence that ß-TrCP degradation is required for NF-κB pathway inhibition by NSP1 and demonstrates the relevance of ß-TrCP degradation to rotavirus infection. C-terminal regions of NSP1, including a serine-containing motif resembling the ß-TrCP recognition motif of IκB, were required for NF-κB inhibition. CRW-8 infection of HT-29 intestinal epithelial cells induced significant levels of IFN-ß and CCL5 but not IL-8. This contrasts with monkey rotavirus SA11-4F, whose NSP1 inhibits IRF3 but not NF-κB. Substantial amounts of IL-8 but not IFN-ß or CCL5 were secreted from HT-29 cells infected with SA11-4F. Our results show that human rotaviruses commonly inhibit the NF-κB pathway by degrading ß-TrCP and thus stabilizing IκB. They suggest that NSP1 plays an important role during human rotavirus infection by inhibiting the expression of NF-κB-dependent cytokines, such as IL-8.

Revisiting the role of histo-blood group antigens in rotavirus host-cell invasion.

Histo-blood group antigens (HBGAs) have been proposed as rotavirus receptors. H type-1 and Lewis(b) antigens have been reported to bind VP8* from major human rotavirus genotypes P[4], P[6] and P[8], while VP8* from a rarer P[14] rotavirus recognizes A-type HBGAs. However, the role and significance of HBGA receptors in rotavirus pathogenesis remains uncertain. Here we report that P[14] rotavirus HAL1166 and the related P[9] human rotavirus K8 bind to A-type HBGAs, although neither virus engages the HBGA-specific α1,2-linked fucose moiety. Notably, human rotaviruses DS-1 (P[4]) and RV-3 (P[6]) also use A-type HBGAs for infection, with fucose involvement. However, human P[8] rotavirus Wa does not recognize A-type HBGAs. Furthermore, the common human rotaviruses that we have investigated do not use Lewis(b) and H type-1 antigens. Our results indicate that A-type HBGAs are receptors for human rotaviruses, although rotavirus strains vary in their ability to recognize these antigens.

VP7 of Rhesus monkey rotavirus RRV contributes to diabetes acceleration in association with an elevated anti-rotavirus antibody response.

T cell-receptor transgenic NOD8.3 mice provide a model for spontaneous type 1 diabetes development. Infection of 5 week-old NOD8.3 mice with Rhesus monkey rotavirus (RRV) accelerates the onset of their diabetes. This acceleration requires virus replication and relates to the presence and level of serum anti-rotavirus antibodies, but the role of individual RRV genes is unknown. Here we assessed the importance for diabetes acceleration of the RRV genes encoding VP4 and VP7, by infecting NOD8.3 mice with parental and reassortant rotaviruses. Diabetes was accelerated by reassortant rotaviruses containing RRV VP7 on a UK rotavirus genetic background, but not by parental UK or a UK reassortant containing RRV VP4 without VP7. Diabetes acceleration by reassortant rotaviruses containing RRV VP7 depended on the development of a high serum anti-rotavirus antibody titer. This study shows that VP7, together with an elevated anti-rotavirus antibody response, contributes to the acceleration of diabetes onset by RRV.

Rotavirus inhibits IFN-induced STAT nuclear translocation by a mechanism that acts after STAT binding to importin-α.

The importance of innate immunity to rotaviruses is exemplified by the range of strategies evolved by rotaviruses to interfere with the IFN response. We showed previously that rotaviruses block gene expression induced by type I and II IFNs, through a mechanism allowing activation of signal transducer and activator of transcription (STAT) 1 and STAT2 but preventing their nuclear accumulation. This normally occurs through activated STAT1/2 dimerization, enabling an interaction with importin α5 that mediates transport into the nucleus. In rotavirus-infected cells, STAT1/2 inhibition may limit the antiviral actions of IFN produced early in infection. Here we further analysed the block to STAT1/2 nuclear accumulation, showing that activated STAT1 accumulates in the cytoplasm in rotavirus-infected cells. STAT1/2 nuclear accumulation was inhibited by rotavirus even in the presence of the nuclear export inhibitor Leptomycin B, demonstrating that enhanced nuclear export is not involved in STAT1/2 cytoplasmic retention. The ability to inhibit STAT nuclear translocation was completely conserved amongst the group A rotaviruses tested, including a divergent avian strain. Analysis of mutant rotaviruses indicated that residues after amino acid 47 of NSP1 are dispensable for STAT inhibition. Furthermore, expression of any of the 12 Rhesus monkey rotavirus proteins did not inhibit IFN-stimulated STAT1 nuclear translocation. Finally, co-immunoprecipitation experiments from transfected epithelial cells showed that STAT1/2 binds importin α5 normally following rotavirus infection. These findings demonstrate that rotavirus probably employs a novel strategy to inhibit IFN-induced STAT signalling, which acts after STAT activation and binding to the nuclear import machinery.

Relative roles of GM1 ganglioside, N-acylneuraminic acids, and α2ß1 integrin in mediating rotavirus infection.

UNLABELLED: N-acetyl- and N-glycolylneuraminic acids (Sia) and α2ß1 integrin are frequently used by rotaviruses as cellular receptors through recognition by virion spike protein VP4. The VP4 subunit VP8*, derived from Wa rotavirus, binds the internal N-acetylneuraminic acid on ganglioside GM1. Wa infection is increased by enhanced internal Sia access following terminal Sia removal from main glycan chains with sialidase. The GM1 ligand cholera toxin B (CTB) reduces Wa infectivity. Here, we found sialidase treatment increased cellular GM1 availability and the infectivity of several other human (including RV-3) and animal rotaviruses, typically rendering them susceptible to methyl α-d-N-acetylneuraminide treatment, but did not alter α2ß1 usage. CTB reduced the infectivity of these viruses. Aceramido-GM1 inhibited Wa and RV-3 infectivity in untreated and sialidase-treated cells, and GM1 supplementation increased their infectivity, demonstrating the importance of GM1 for infection. Wa recognition of α2ß1 and internal Sia were at least partially independent. Rotavirus usage of GM1 was mapped to VP4 using virus reassortants, and RV-3 VP8* bound aceramido-GM1 by saturation transfer difference nuclear magnetic resonance (STD NMR). Most rotaviruses recognizing terminal Sia did not use GM1, including RRV. RRV VP8* interacted minimally with aceramido-GM1 by STD NMR. Unusually, TFR-41 rotavirus infectivity depended upon terminal Sia and GM1. Competition of CTB, Sia, and/or aceramido-GM1 with cell binding by VP8* from representative rotaviruses showed that rotavirus Sia and GM1 preferences resulted from VP8*-cell binding. Our major finding is that infection by human rotaviruses of commonly occurring VP4 serotypes involves VP8* binding to cell surface GM1 glycan, typically including the internal N-acetylneuraminic acid. IMPORTANCE: Rotaviruses, the major cause of severe infantile gastroenteritis, recognize cell surface receptors through virus spike protein VP4. Several animal rotaviruses are known to bind sialic acids at the termini of main carbohydrate chains. Conversely, only a single human rotavirus is known to bind sialic acid. Interestingly, VP4 of this rotavirus bound to sialic acid that forms a branch on the main carbohydrate chain of the GM1 ganglioside. Here, we use several techniques to demonstrate that other human rotaviruses exhibit similar GM1 usage properties. Furthermore, binding by VP4 to cell surface GM1, involving branched sialic acid recognition, is shown to facilitate infection. In contrast, most animal rotaviruses that bind terminal sialic acids did not utilize GM1 for VP4 cell binding or infection. These studies support a significant role for GM1 in mediating host cell invasion by human rotaviruses.

Innate cellular responses to rotavirus infection.

Rotavirus is a leading cause of severe dehydrating diarrhoea in infants and young children. Following rotavirus infection in the intestine an innate immune response is rapidly triggered. This response leads to the induction of type I and type III interferons (IFNs) and other cytokines, resulting in a reduction in viral replication. Here we review the current literature describing the detection of rotavirus infection by pattern recognition receptors within host cells, the subsequent molecular mechanisms leading to IFN and cytokine production, and the processes leading to reduced rotavirus replication and the development of protective immunity. Rotavirus countermeasures against innate responses, and their roles in modulating rotavirus replication in mice, also are discussed. By linking these different aspects of innate immunity, we provide a comprehensive overview of the host's first line of defence against rotavirus infection. Understanding these processes is expected to be of benefit in improving strategies to combat rotavirus disease.

The type III effectors NleE and NleB from enteropathogenic E. coli and OspZ from Shigella block nuclear translocation of NF-kappaB p65.

Many bacterial pathogens utilize a type III secretion system to deliver multiple effector proteins into host cells. Here we found that the type III effectors, NleE from enteropathogenic E. coli (EPEC) and OspZ from Shigella, blocked translocation of the p65 subunit of the transcription factor, NF-kappaB, to the host cell nucleus. NF-kappaB inhibition by NleE was associated with decreased IL-8 expression in EPEC-infected intestinal epithelial cells. Ectopically expressed NleE also blocked nuclear translocation of p65 and c-Rel, but not p50 or STAT1/2. NleE homologues from other attaching and effacing pathogens as well OspZ from Shigella flexneri 6 and Shigella boydii, also inhibited NF-kappaB activation and p65 nuclear import; however, a truncated form of OspZ from S. flexneri 2a that carries a 36 amino acid deletion at the C-terminus had no inhibitory activity. We determined that the C-termini of NleE and full length OspZ were functionally interchangeable and identified a six amino acid motif, IDSY(M/I)K, that was important for both NleE- and OspZ-mediated inhibition of NF-kappaB activity. We also established that NleB, encoded directly upstream from NleE, suppressed NF-kappaB activation. Whereas NleE inhibited both TNFalpha and IL-1beta stimulated p65 nuclear translocation and IkappaB degradation, NleB inhibited the TNFalpha pathway only. Neither NleE nor NleB inhibited AP-1 activation, suggesting that the modulatory activity of the effectors was specific for NF-kappaB signaling. Overall our data show that EPEC and Shigella have evolved similar T3SS-dependent means to manipulate host inflammatory pathways by interfering with the activation of selected host transcriptional regulators.

Death mechanisms in epithelial cells following rotavirus infection, exposure to inactivated rotavirus or genome transfection.

Intestinal epithelial cell death following rotavirus infection is associated with villus atrophy and gastroenteritis. Roles for both apoptosis and necrosis in cytocidal activity within rotavirus-infected epithelial cells have been proposed. Additionally, inactivated rotavirus has been reported to induce diarrhoea in infant mice. We further examined the death mechanisms induced in epithelial cell lines following rotavirus infection or inactivated rotavirus exposure. Monolayer integrity changes in MA104, HT-29 and partially differentiated Caco-2 cells following inactivated rotavirus exposure or RRV or CRW-8 rotavirus infection paralleled cell metabolic activity and viability reductions. MA104 cell exposure to rotavirus dsRNA also altered monolayer integrity. Inactivated rotaviruses induced delayed cell function losses that were unrelated to apoptosis. Phosphatidylserine externalization, indicating early apoptosis, occurred in MA104 and HT-29 but not in partially differentiated Caco-2 cells by 11 h after infection. Rotavirus activation of phosphatidylinositol 3-kinase partially protected MA104 and HT-29 cells from early apoptosis. In contrast, activation of the stress-activated protein kinase JNK by rotavirus did not influence apoptosis induction in these cells. RRV infection produced DNA fragmentation, indicating late-stage apoptosis, in fully differentiated Caco-2 cells only. These studies show that the apoptosis initiation and cell death mechanism induced by rotavirus infection depend on cell type and degree of differentiation. Early stage apoptosis resulting from rotavirus infection is probably counter-balanced by virus-induced phosphatidylinositol 3-kinase activation. The ability of inactivated rotaviruses and rotavirus dsRNA to perturb monolayer integrity supports a potential role for these rotavirus components in disease pathogenesis.

Rotavirus antagonizes cellular antiviral responses by inhibiting the nuclear accumulation of STAT1, STAT2, and NF-kappaB.

A vital arm of the innate immune response to viral infection is the induction and subsequent antiviral effects of interferon (IFN). Rotavirus reduces type I IFN induction in infected cells by the degradation of IFN regulatory factors. Here, we show that the monkey rotavirus RRV and human rotavirus Wa also block gene expression induced by type I and II IFNs through a mechanism allowing signal transducer and activator of transcription 1 (STAT1) and STAT2 activation but preventing their nuclear accumulation. In infected cells, this may allow rotavirus to block the antiviral actions of IFN produced early in infection or by activated immune cells. As the intracellular expression of rotavirus nonstructural proteins NSP1, NSP3, and NSP4 individually did not inhibit IFN-stimulated gene expression, their involvement in this process is unlikely. RRV and Wa rotaviruses also prevented the tumor necrosis factor alpha-stimulated nuclear accumulation of NF-kappaB and NF-kappaB-driven gene expression. In addition, NF-kappaB was activated by rotavirus infection, confirming earlier findings by others. As NF-kappaB is important for the induction of IFN and other cytokines during viral infection, this suggests that rotavirus prevents cellular transcription as a means to evade host responses. To our knowledge, this is the first report of the use of this strategy by a double-stranded RNA virus.

Sialic acid dependence in rotavirus host cell invasion.

We used NMR spectroscopy, molecular modeling and infectivity competition assays to investigate the key interactions between the spike protein (VP8(*)) from 'sialidase-insensitive' human Wa and 'sialidase-sensitive' porcine CRW-8 rotaviruses and the glycans of gangliosides G(M1) and G(D1a). Our data provide strong evidence that N-acetylneuraminic acid is a key determinant for binding of these rotaviruses. This is in contrast to the widely accepted paradigm that sialic acids are irrelevant in host cell recognition by sialidase-insensitive rotaviruses.

Rotavirus replication in intestinal cells differentially regulates integrin expression by a phosphatidylinositol 3-kinase-dependent pathway, resulting in increased cell adhesion and virus yield.

Changes in the interactions between intestinal cells and their surrounding environment during virus infection have not been well documented. The growth and survival of intestinal epithelial cells, the main targets of rotavirus infection, are largely dependent on the interaction of cell surface integrins with the extracellular matrix. In this study, we detected alterations in cellular integrin expression following rotavirus infection, identified the signaling components required, and analyzed the subsequent effects on cell binding to the matrix component collagen. After rotavirus infection of intestinal cells, expression of alpha2beta1 and beta2 integrins was up-regulated, whereas that of alphaVbeta3, alphaVbeta5, and alpha5beta1 integrins, if present, was down-regulated. This differential regulation of integrins was reflected at the transcriptional level. It was unrelated to the use of integrins as rotavirus receptors, as both integrin-using and integrin-independent viruses induced integrin regulation. Using pharmacological agents that inhibit kinase activity, integrin regulation was shown to be dependent on phosphatidylinositol 3-kinase (PI3K) but independent of the activities of the mitogen-activated protein kinases p38 and ERK1/2, and cyclooxygenase-2. Replication-dependent activation of the PI3K/Akt pathway was observed following infection of intestinal and nonintestinal cell lines. Rotavirus activation of PI3K was important for regulation of alpha2beta1 expression. Blockade of integrin regulation by PI3K inhibition led to decreased adherence of infected intestinal cells to collagen and a concomitant decrease in virus titer. These findings indicate that rotavirus-induced PI3K activation causes regulation of integrin expression in intestinal cells, leading to prolonged adherence of infected cells to collagen and increased virus production.

STD NMR spectroscopy and molecular modeling investigation of the binding of N-acetylneuraminic acid derivatives to rhesus rotavirus VP8* core.

The VP8* subunit of rotavirus spike protein VP4 contains a sialic acid (Sia)-binding domain important for host cell attachment and infection. In this study, the binding epitope of the N-acetylneuraminic acid (Neu5Ac) derivatives has been characterized by saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy. From this STD NMR data, it is proposed that the VP8* core recognizes an identical binding epitope in both methyl alpha-D-N-acetylneuraminide (Neu5Acalpha2Me) and the disaccharide methyl S-(alpha-D-N-acetylneuraminosyl)-(2-->6)-6-thio-beta-D-galactopyranoside (Neu5Ac-alpha(2,6)-S-Galbeta1Me). In the VP8*-disaccharide complex, the Neu5Ac moiety contributes to the majority of interaction with the protein, whereas the galactose moiety is solvent-exposed. Molecular dynamics calculations of the VP8*-disaccharide complex indicated that the galactose moiety is unable to adopt a conformation that is in close proximity to the protein surface. STD NMR experiments with methyl 9-O-acetyl-alpha-D-N-acetylneuraminide (Neu5,9Ac(2)alpha2Me) in complex with rhesus rotavirus (RRV) VP8* revealed that both the N-acetamide and 9-O-acetate moieties are in close proximity to the Sia-binding domain, with the N-acetamide's methyl group being saturated to a larger extent, indicating a closer association with the protein. RRV VP8* does not appear to significantly recognize the unsaturated Neu5Ac derivative [2-deoxy-2,3-didehydro-D-N-acetylneuraminic acid (Neu5Ac2en)]. Molecular modeling of the protein-Neu5Ac2en complex indicates that key interactions between the protein and the unsaturated Neu5Ac derivative when compared with Neu5Acalpha2Me would not be sustained. Neu5Acalpha2Me, Neu5Ac-alpha(2,6)-S-Galbeta1Me, Neu5,9Ac(2)alpha2Me, and Neu5Ac2en inhibited rotavirus infection of MA104 cells by 61%, 35%, 30%, and 0%, respectively, at 10 mM concentration. NMR spectroscopic, molecular modeling, and infectivity inhibition results are in excellent agreement and provide valuable information for the design of inhibitors of rotavirus infection.

Rotavirus activates JNK and p38 signaling pathways in intestinal cells, leading to AP-1-driven transcriptional responses and enhanced virus replication.

Rotavirus infection is known to regulate transcriptional changes in many cellular genes. The transcription factors NF-kappaB and AP-1 are activated by rotavirus infection, but the upstream processes leading to these events are largely unidentified. We therefore studied the activation state during rotavirus infection of c-Jun NH2-terminal kinase (JNK) and p38, which are kinases known to activate AP-1. As assessed by Western blotting using phospho-specific antibodies, infection with rhesus rotavirus (RRV) or exposure to UV-psoralen-inactivated RRV (I-RRV) resulted in the activation of JNK in HT-29, Caco-2, and MA104 cells. Activation of p38 during RRV infection was observed in Caco-2 and MA104 cells but not in HT-29 cells, whereas exposure to I-RRV did not lead to p38 activation in these cell lines. Rotavirus strains SA11, CRW-8, Wa, and UK also activated JNK and p38. Consistent with the activation of JNK, a corresponding increase in the phosphorylation of the AP-1 component c-Jun was shown. The interleukin-8 (IL-8) and c-jun promoters contain AP-1 binding sequences, and these genes have been shown previously to be transcriptionally up-regulated during rotavirus infection. Using specific inhibitors of JNK (SP600125) and p38 (SB203580) and real-time PCR, we showed that maximal RRV-induced IL-8 and c-jun transcription required JNK and p38 activity. This highlights the importance of JNK and p38 in RRV-induced, AP-1-driven gene expression. Significantly, inhibition of p38 or JNK in Caco-2 cells reduced RRV growth but not viral structural antigen expression, demonstrating the potential importance of JNK and p38 activation for optimal rotavirus replication.

Crystallization and preliminary X-ray diffraction analysis of the sialic acid-binding domain (VP8*) of porcine rotavirus strain CRW-8.

Rotavirus recognition and attachment to host cells involves interaction with the spike protein VP4 that projects outwards from the surface of the virus particle. An integral component of these spikes is the VP8* domain, which is implicated in the direct recognition and binding of sialic acid-containing cell-surface carbohydrates and facilitates subsequent invasion by the virus. The expression, purification, crystallization and preliminary X-ray diffraction analysis of VP8* from porcine CRW-8 rotavirus is reported. Diffraction data have been collected to 2.3 A resolution, enabling the determination of the VP8* structure by molecular replacement.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* carbohydrate-binding protein of the human rotavirus strain Wa.

Rotaviruses exhibit host-specificity and the first crystallographic information on a rotavirus strain that infects humans is reported here. Recognition and attachment to host cells, leading to invasion and infection, is critically linked to the function of the outer capsid spike protein of the rotavirus particle. In some strains the VP8* component of the spike protein is implicated in recognition and binding of sialic-acid-containing cell-surface carbohydrates, thereby enabling infection by the virus. The cloning, expression, purification, crystallization and initial X-ray diffraction analysis of the VP8* core from human Wa rotavirus is reported. Two crystal forms (trigonal P3(2)21 and monoclinic P2(1)) have been obtained and X-ray diffraction data have been collected, enabling determination of the VP8*(64-223) structure by molecular replacement.