Rotavirus NSP1 inhibits interferon induced non-canonical NFκB activation by interacting with TNF receptor associated factor 2

AbstractTNF receptor associated factor 2 (TRAF2) plays a very important role in cellular innate immune as well as inflammatory responses. Previous studies have reported TRAF2 mediated regulation of TNF and Interferon (IFN) induced canonical and non-canonical activation of NFκB. In this study, we show that rotavirus NSP1 targets TRAF2 to regulate IFN induced non-canonical NFκB activation. Here we found that rotavirus Non-Structural Protein-1 (NSP1) interacts with TRAF2 and degrades it in a proteasome dependent manner. C-terminal part of NSP1 was sufficient for interacting with TRAF2 but it alone could not degrade TRAF2. This inhibition of interferon mediated non-canonical NFκB activation by NSP1 may modulate inflammatory cytokine production after rotavirus infection to help the virus propagation

The molecular chaperone heat shock protein-90 positively regulates rotavirus infection

AbstractRotaviruses are the major cause of severe dehydrating gastroenteritis in children worldwide. In this study, we report a positive role of cellular chaperone Hsp90 during rotavirus infection. A highly specific Hsp90 inhibitor, 17-allylamono-demethoxygeldanamycin (17-AAG) was used to delineate the functional role of Hsp90. In MA104 cells treated with 17-AAG after viral adsorption, replication of simian (SA11) or human (KU) strains was attenuated as assessed by quantitating both plaque forming units and expression of viral genes. Phosphorylation of Akt and NFκB observed 2–4 hpi with SA11, was strongly inhibited in the presence of 17-AAG. Direct Hsp90–Akt interaction in virus infected cells was also reduced in the presence of 17-AAG. Anti-rotaviral effects of 17-AAG were due to inhibition of activation of Akt that was confirmed since, PI3K/Akt inhibitors attenuated rotavirus growth significantly. Thus, Hsp90 regulates rotavirus by modulating cellular signaling proteins. The results highlight the importance of cellular proteins during rotavirus infection and the possibility of targeting cellular chaperones for developing new anti-rotaviral strategies

Hospital based surveillance and genetic characterization of rotavirus strains in children (<5 years) with acute gastroenteritis in Kolkata, India, revealed resurgence of G9 and G2 genotypes during 2011–2013

AbstractIntroductionIndia accounts for an estimated 457,000–884,000 hospitalizations and 2 million outpatient visits for diarrhea. In spite of the huge burden of rotavirus (RV) disease, RV vaccines have not been introduced in national immunization programme of India. Therefore, continuous surveillance for prevalence and monitoring of the circulating genotypes is needed to assess the disease burden prior to introduction of vaccines in this region.MethodsDuring January 2011 through December 2013, 830 and 1000 stool samples were collected from hospitalized and out-patient department (OPD) patients, respectively, in two hospitals in Kolkata, Eastern India. After primary screening, the G-P typing was done by multiplex semi-nested PCR using type specific primers followed by sequencing. Phylogenetic analysis for the VP7 gene of 25 representative strains was done.ResultsAmong hospitalized and OPD patients, 53.4% and 47.5% cases were positive for rotaviruses, respectively. Unlike previous studies where G1 was predominant, in hospitalized cases G9 rotavirus strains were most prevalent (40%), followed by G2 (39.6%) whereas G1 and G12 occurred at 16.4% and 5.6% frequency. In OPD cases, the most prevalent strain was G2 (40.3%), followed by G1, G9 and G12 at 25.5%, 22.8%, 9.3%, respectively. Phylogenetically the G1, G2 and G9 strains from Kolkata did not cluster with corresponding genotypes of Rotarix, RotaTeq and Rotavac (116E) vaccine strains.ConclusionThe study highlights the high prevalence of RV in children with gastroenteritis in Kolkata. The circulating genotypes have changed over the time with predominance of G9 and G2 strains during 2011-2013. The current G2, G9 and G1 Kolkata strains shared low amino acid homologies with current vaccine strains. Although there is substantial evidence for cross protection of vaccines against a variety of strains, still the strain variation should be monitored post vaccine introduction to determine if it has any impact on vaccine effectiveness

NSP1 inhibits IFN-β induction irrespective of IRF3 degradation.

<p><b>A)</b> HEK293 cells were transfected with FLAG-MAVS and pcD-OSU-NSP1 vector in order to assess the MAVS mediated inhibition of IRF3 phosphorylation. Cell lysates were analyzed for pIRF3, IRF3, Anti-His, Anti-FLAG and GAPDH specific antibodies. <b>B)</b> Fold change of IFN-β transcripts was assessed in cells overexpressing human TBK1 and pFLAG-MAVS vectors, in presence or absence of pcD-NSP1. The data shown are means ± the SD (n = 3). * Significantly different in comparison to human TBK1 and NSP1 transfected and pFLAG-MAVS untransfected condition. P<0.05 <b>C)</b> Activation of IRF3 was assessed in absence or presence of pcD-NSP1 in cells transfected with TBK1 and FLAG-MAVS. <b>D)</b> Association of MAVS with TBK1 was studied by Co-IP in presence or absence of pcD-NSP1 in cells overexpressing TBK1 and MAVS. The MAVS degradation was controlled by proteosomal inhibitor MG132 Results reveal reduced interaction between MAVS-TBK1 in presence of NSP1.</p

Correction: MAVS Protein Is Attenuated by Rotavirus Nonstructural Protein 1

<p>Correction: MAVS Protein Is Attenuated by Rotavirus Nonstructural Protein 1</p

Formation of MAVS aggregates during Rotavirus infection.

<p><b>A)</b> Crude mitochondrial extracts were prepared from HEK293 cells infected with A5-16 strain (3 M.O.I.) at increasing time points (4, 8 and 12). Extracts were analyzed by SDD-AGE to assess the MAVS aggregate formation. Results revealed MAVS aggregate formation from 4 hpi. <b>B)</b> Role of NSP1 on MAVS aggregation and ubiquitinylation was observed by overexpressing FLAG-MAVS in absence or presence of NSP1. Infection of A5-16 was used for inducing MAVS aggregation, as overexpression of MAVS alone is insufficient for inducing aggregate formation. Results show inhibition of MAVS aggregates in presence of NSP1, which gets restored following MG132 (20 μM) treatment. A fraction of SDD-AGE lysate was analyzed by SDS-PAGE followed by immunoblotting of MAVS, p-IRF3 and COX IV.</p

La cité des femmes (1980) de Federico Fellini

<p><b>Three-dimensional quaternary structure of HA (A) and NA (B) proteins of 2015 A(H1N1)pdm09 strains from this study identifying mutations compared to A/California/04/2009 strain.</b> The three mutations at the antigenic epitopes within the receptor-binding pocket of HA protein were indicated. Probable binding position of oseltamivir in the NA protein of 2015 A(H1N1)pdm09 strain is shown in purple colour. The mutations are indicated in red and the amino acids responsible for oseltamivir binding are shown in black.</p