Rotavirus contains two outer capsid viral proteins, the spike protein VP4 and major capsid component VP7, both of which are implicated in cell entry. We show that VP4 and VP7 contain tripeptide sequences previously shown to act as recognition sites for integrins in extracellular matrix proteins. VP4 contains the ␣2␤1 integrin ligand site DGE. In VP7, the ␣x␤2 integrin ligand site GPR and the ␣4␤1 integrin ligand site LDV are embedded in a novel disintegrin-like domain that also shows sequence similarity to fibronectin and the tie receptor tyrosine kinase. Microorganism sequence homology to these ligand motifs and to disintegrins has not been reported previously. In our experiments, peptides including these rotaviral tripeptides and mAbs directed to these integrins specifically blocked rotavirus infection of cells shown to express ␣2␤1 and ␤2 integrins. Rotavirus VP4-mediated cell entry may involve the ␣2␤1 integrin, whereas VP7 appears to interact with ␣x␤2 and ␣4␤1 integrins.

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-κB, to the host cell nucleus. NF-κB 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-κB 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-κB activity. We also established that NleB, encoded directly upstream from NleE, suppressed NF-κB activation. Whereas NleE inhibited both TNFα and IL-1β stimulated p65 nuclear translocation and IκB degradation, NleB inhibited the TNFα pathway only. Neither NleE nor NleB inhibited AP-1 activation, suggesting that the modulatory activity of the effectors was specific for NF-κB 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.

Pancreatic islet autoimmunity leading to type 1 diabetes could be triggered by viruses in genetically susceptible individuals. Rotavirus (RV), the most common cause of childhood gastroenteritis, contains peptide sequences highly similar to T-cell epitopes in the islet autoantigens GAD and tyrosine phosphatase IA-2 (IA-2), suggesting T-cells to RV could trigger islet autoimmunity by molecular mimicry. We therefore sought an association between RV infection and islet autoantibody markers in children at risk for diabetes who were followed from birth. There was a specific and highly significant association between RV seroconversion and increases in any of these antibodies: 86% of antibodies to IA-2, 62% to insulin, and 50% to GAD first appeared or increased with increases in RV IgG or IgA. RV infection may therefore trigger or exacerbate islet autoimmunity in genetically susceptible children. Diabetes 49:1319-1324, 2000 T ype 1 diabetes is an autoimmune disease that results from the destruction of pancreatic islet ␤-cells in genetically predisposed individuals. A large proportion of the lifetime risk of type 1 diabetes is attributed to environmental agents (1,2), but the only virus shown unequivocally to be responsible for clinical disease is rubella following infection in utero of infants bearing the HLA haplotype B8-(DR3-DQ2) (3). Circulating insulin autoantibodies (IAA), GAD65 antibodies (GADAb), and tyrosine phosphatase IA-2 autoantibodies (IA-2Ab) are markers of islet autoimmunity that predict the T-cell-mediated destruction of ␤-cells (4-6). Peptides in these islet autoantigens that are recognized by T-cells may provide clues to environmental agents that trigger or exacerbate islet autoimmunity through the mechanism of molecular mimicry (7,8).Recently, we identified T-cell epitope peptides in tyrosine phosphatase IA-2 (IA-2) restricted by HLA-DR4 in individuals at risk for type 1 diabetes (9,10). The dominant epitope (amino acid [aa] 805-820) contains a core 9-aa sequence in which 5 aa are identical and 4 aa are homologous with a 9-aa sequence (aa 41-49) within virus protein (VP)7 of rotavirus (RV) serotype G3 and to a lesser extent in the G1 and G2 serotypes. Because the T-cell contact residues in these similar IA-2 and RV VP7 sequences appear to be identical (9), the potential exists for molecular mimicry. Furthermore, just NH 2 -terminal of this region in VP7 is a 12-aa sequence (11) strongly similar to a sequence in GAD65 (aa 117-128) (9) that is a T-cell epitope in HLA-DR4 transgenic mice (12) and DR4-DQ8 homozygous at-risk humans (13). All human RV serotypes in the GenBank database contain the GAD-related sequence. VP7, the major outer capsid protein of RV, is an important determinant of virulence and induces virus-neutralizing antibodies (14). However, elimination of RV infection is predominantly due to T-cells (15). The CD4 T-cell epitopes in VP7 are unknown, but in C57/Bl 6 and BALB/c mice, CD8 T-cell epitopes (15,16) map adjacent to the IA-2-and GAD-like sequences, confirming that this signal sequenc...

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.

Integrins ␣2␤1, ␣X␤2, and ␣V␤3 have been implicated in rotavirus cell attachment and entry. The virus spike protein VP4 contains the ␣2␤1 ligand sequence DGE at amino acid positions 308 to 310, and the outer capsid protein VP7 contains the ␣X␤2 ligand sequence GPR. To determine the viral proteins and sequences involved and to define the roles of ␣2␤1, ␣X␤2, and ␣V␤3, we analyzed the ability of rotaviruses and their reassortants to use these integrins for cell binding and infection and the effect of peptides DGEA and GPRP on these events. Many laboratory-adapted human, monkey, and bovine viruses used integrins, whereas all porcine viruses were integrin independent. The integrin-using rotavirus strains each interacted with all three integrins. Integrin usage related to VP4 serotype independently of sialic acid usage. Analysis of rotavirus reassortants and assays of virus binding and infectivity in integrin-transfected cells showed that VP4 bound ␣2␤1, and VP7 interacted with ␣X␤2 and ␣V␤3 at a postbinding stage. DGEA inhibited rotavirus binding to ␣2␤1 and infectivity, whereas GPRP binding to ␣X␤2 inhibited infectivity but not binding. The truncated VP5* subunit of VP4, expressed as a glutathione S-transferase fusion protein, bound the expressed ␣2 I domain. Alanine mutagenesis of D308 and G309 in VP5* eliminated VP5* binding to the ␣2 I domain. In a novel process, integrin-using viruses bind the ␣2 I domain of ␣2␤1 via DGE in VP4 and interact with ␣X␤2 (via GPR) and ␣V␤3 by using VP7 to facilitate cell entry and infection.Rotaviruses are leading causes of acute gastroenteritis in human infants and young animals. Their restricted tropism suggests that very specific virus-host cell interactions are necessary to establish infection. The viral spike protein VP4, the major cell attachment protein, is cleaved by trypsin for enhanced infectivity into two subunits, VP5* (60 kDa) and VP8* (28 kDa). VP4 and the major outer capsid protein VP7 independently define serotype specificities. The inner capsid protein VP6 contains group-specific antigenic determinants. Reassortant rotaviruses containing combinations of the 11 double-stranded RNA genes from two parental viruses can be generated (27) which occasionally show unexpected phenotypes due to VP4-VP7 interactions (43).Integrins ␣2␤1, ␣X␤2, and ␣4␤1 were implicated in group A rotavirus cell attachment and entry (11, 23), and ␣V␤3 was proposed to mediate rotavirus cell entry (20). Integrin usage by rotaviruses was discovered when VP5* was shown to contain the type I collagen-derived, ␣2␤1 ligand sequence DGE at amino acids 308 to 310, and the fibrinogen-derived ␣X␤2 integrin ligand sequence GPR was identified in VP7 at amino acids 253 to 255 (11). Peptides containing these viral integrin ligand sequences (GPRP and RDGEE), monoclonal antibodies (MAbs) to ␣2␤1 and MAbs to ␤2, inhibited the infection of monolayers of MA104 and Caco-2 cells by simian rotavirus SA11 and/or human rotavirus RV-5 by 30 to 90% in additive fashion (9, 11). Infectivity blockade in MA104 cell monolayers...

Most mammalian rotaviruses contain tripeptide amino acid sequences in outer capsid proteins VP4 and VP7 which have been shown to act as ligands for integrins ␣2␤1 and ␣4␤1. Peptides containing these sequences and monoclonal antibodies directed to these integrins block rotavirus infection of cells. Here we report that SA11 rotavirus binding to and infection of K562 cells expressing ␣2␤1 or ␣4␤1 integrins via transfection is increased over virus binding to and infection of cells transfected with ␣3 integrin or parent cells. The increased binding and growth were specifically blocked by a monoclonal antibody to the transfected integrin subunit but not by irrelevant antibodies. In our experiments, integrin activation with phorbol ester did not affect virus binding to cells. However, phorbol ester treatment of K562 parent and transfected cells induced endogenous gene expression of ␣2␤1 integrin, which was detectable by flow cytometry 16 h after treatment and quantitatively correlated with the increased level of SA11 virus growth observed after this time. Virus binding to K562 cells treated with phorbol ester 24 h previously and expressing ␣2␤1 was elevated over binding to control cells and was specifically blocked by the anti-␣2 monoclonal antibody AK7. Virus growth in ␣4-transfected K562 cells which had also been induced to express ␣2␤1 integrin with phorbol ester occurred at a level approaching that in the permissive MA104 cell line. We therefore have demonstrated that two integrins, ␣2␤1 and ␣4␤1, are capable of acting as cellular receptors for SA11 rotavirus.Rotaviruses, members of the family Reoviridae, are the major etiological agents of severe acute gastroenteritis in infants and young children worldwide and are important pathogens in most mammalian species. It is anticipated that the longawaited introduction of the first vaccine against human rotavirus into use in North America in 1998 will lead to a reduction in the most severe human illness associated with this virus, but other vaccination and therapeutic approaches are required. Novel strategies may be devised following the identification of cellular receptors for rotavirus and from an understanding of the process of viral entry into cells, particularly into intestinal epithelial cells. These are essential steps for productive rotavirus infection and major determinants of host cell tropism.The nonenveloped, icosahedral rotavirus particle consists of a genome of 11 segments of double-stranded RNA (48) in a triple-layered protein capsid (66). The outermost layer of each virion is composed of trimers of the 37-kDa glycoprotein VP7 and 60 spikes of the 88-kDa protein VP4, probably as dimers, which extend about 12 nm above the VP7 surface and interact extensively with VP7 throughout the outer surface (47,65,66).Both VP4 and VP7 independently elicit neutralizing, protective antibodies and are virulence determinants (26, 46). VP4 is an important determinant of host cell tropism (27), receptor binding, and cell penetration (37). Proteolytic cleavage of VP4 into two...

Histo-blood group antigens (HBGAs)

An enzyme immunoassay for serotyping human rotaviruses in stools and in cell culture was developed. Hyperimmune rabbit antisera to rotaviruses were used as capture antibodies, and rotavirus-neutralizing mouse monoclonal antibodies specific for serotypes 1, 2, 3, and 4 were used as detection reagents. Partial purification of monoclonal antibodies and inclusion of skim milk powder in antibody diluents contributed to assay specificity. The sensitivity of this assay was greater than that of a direct enzyme immunoassay in which rotaviruses of the appropriate serotype were adsorbed directly to the solid phase. When fecal extracts were concentrated threefold, this serotyping enzyme immunoassay was of equal specificity and approached the sensitivity of electron microscopy for rotavirus detection. This assay is simple and rapid and is suitable for serotyping the large numbers of isolates obtained from epidemiological studies and vaccine trials. again in 7 days with virus in saline. Serum was collected 14 5(9