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    Specificity and affinity of neuraminic acid exhibited by canine rotavirus strain K9 carbohydrate-binding domain (VP8*)
    Mishra, R ; Yu, X ; Kishor, C ; Holloway, G ; Lau, K ; von Itzstein, M ; Coulson, BS ; Blanchard, H (WILEY, 2018-09)
    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.
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    The Type III Effectors NleE and NleB from Enteropathogenic E. coli and OspZ from Shigella Block Nuclear Translocation of NF-κB p65
    Newton, HJ ; Pearson, JS ; Badea, L ; Kelly, M ; Lucas, M ; Holloway, G ; Wagstaff, KM ; Dunstone, MA ; Sloan, J ; Whisstock, JC ; Kaper, JB ; Robins-Browne, RM ; Jans, DA ; Frankel, G ; Phillips, AD ; Coulson, BS ; Hartland, EL ; Tran Van Nhieu, G (PUBLIC LIBRARY SCIENCE, 2010-05)
    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.
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    MHC class I expression in intestinal cells is reduced by rotavirus infection and increased in bystander cells lacking rotavirus antigen
    Holloway, G ; Fleming, FE ; Coulson, BS (NATURE PUBLISHING GROUP, 2018-01-08)
    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.
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    Rotavirus acceleration of murine type 1 diabetes is associated with a T helper 1-dependent specific serum antibody response and virus effects in regional lymph nodes
    Pane, JA ; Webster, NL ; Graham, KL ; Holloway, G ; Zufferey, C ; Coulson, BS (SPRINGER, 2013-03)
    AIMS/HYPOTHESIS: Rotavirus infection in at-risk children correlates with production of serum autoantibodies indicative of type 1 diabetes progression. Oral infection with rhesus monkey rotavirus (RRV) accelerates diabetes onset in mice. This relates to their rotavirus-specific serum antibody titre and local pro-inflammatory cytokine induction without pancreatic infection. Our aim was to further investigate the roles of serum antibodies and viral extra-intestinal spread in diabetes acceleration by rotavirus. METHODS: Rotavirus-specific serum antibody production was detected by ELISA in diabetes-prone mice given either inactivated or low-dose RRV, in relation to their diabetes development. Serum anti-rotavirus antibody titres and infectious virus in lymph nodes were measured in mice given RRV or porcine rotavirus CRW-8. In lymph node cells, rotavirus antigen presence and immune activation were determined by flow cytometry, in conjunction with cytokine mRNA levels. RESULTS: Acceleration of diabetes by RRV required virus replication, which correlated with antibody presence. CRW-8 induced similar specific total immunoglobulin and IgA titres to those induced by RRV, but did not accelerate diabetes. RRV alone elicited specific serum IgG antibodies with a T helper (Th)1 bias, spread to regional lymph nodes and activated antigen-presenting cells at these sites. RRV increased Th1-specific cytokine expression in pancreatic lymph nodes. Diabetes onset was more rapid in the RRV-infected mice with the greater Th1 bias. CONCLUSIONS/INTERPRETATION: Acceleration of murine diabetes by rotavirus is virus strain-specific and associated with virus spread to regional lymph nodes, activation of antigen-presenting cells at these sites and induction of a Th1-dominated antibody and cytokine response.