Correction: MAVS Protein Is Attenuated by Rotavirus Nonstructural Protein 1

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

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

MAVS Protein Is Attenuated by Rotavirus Nonstructural Protein 1

<div><p>Rotavirus is the single, most important agent of infantile gastroenteritis in many animal species, including humans. In developing countries, rotavirus infection attributes approximately 500,000 deaths annually. Like other viruses it establishes an intimate and complex interaction with the host cell to counteract the antiviral responses elicited by the cell. Among various pattern recognition receptors (PAMPs) of the host, the cytosolic RNA helicases interact with viral RNA to activate the Mitochondrial Antiviral Signaling protein (MAVS), which regulates cellular interferon response. With an aim to identify the role of different PAMPs in rotavirus infected cell, MAVS was found to degrade in a time dependent and strain independent manner. Rotavirus non-structural protein 1 (NSP1) which is a known IFN antagonist, interacted with MAVS and degraded it in a strain independent manner, resulting in a complete loss of RNA sensing machinery in the infected cell. To best of our knowledge, this is the first report on NSP1 functionality where a signaling protein is targeted unanimously in all strains. In addition NSP1 inhibited the formation of detergent resistant MAVS aggregates, thereby averting the antiviral signaling cascade. The present study highlights the multifunctional role of rotavirus NSP1 and reinforces the fact that the virus orchestrates the cellular antiviral response to its own benefit by various back up strategies.</p></div

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

Proteosome-mediated MAVS degradation.

<p><b>A)</b> NSP1 induced MAVS degradation is prevented in presence of MG132. HEK293 cells were either transfected with FLAG-MAVS or co transfected with pFLAG-MAVS and pcD-NSP1 in presence or absence of 20 μM MG132 followed by immunoblot analysis with anti-FLAG, anti-GAPDH and anti-His. <b>B)</b> NSP1 induces ubiquitinylation of MAVS. HEK293 cells were transfected with expression vector encoding FLAG-tagged ubiquitin with presence or absence of pcD-NSP1. Cells were grown in DMEM containing MG132 (20 μM) for 6 h. Anti-MAVS immunoprecipitates were analyzed by immunoblotting His-tagged ubiquitin with anti-His Ab. Whole-cell lysates were subjected to immunoblotting with anti-His and anti-GAPDH was used as an equal loading control. Figure also showed that MG132 affects MAVS degradation but not ubiquitinylation.</p

List of primers designed and used in the study.

<p>List of primers designed and used in the study.</p

Mechanistic model for rotavirus NSP1-mediated attenuation of cellular proteins for improved infection.

<p>The model summarizes all the NSP1 mediated inhibitory effects during RV infection. In brief, it shows the critical role of MAVS in RLR mediated activation of type I IFN and immune response. By degrading MAVS, NSP1 can directly inhibit IRF3 and NF-κB signaling. Other cellular proteins like IRFs and β-TrCP are targeted selectively but MAVS degradation was observed in all RV strains with functional NSP1.</p

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

MAVS is degraded during rotavirus infection.

<p><b>A)</b> HT29 cells were infected with RV strain SA11 (1 M.O.I.) and cell lysates were prepared at indicated time points. Proteins were separated on 12.5% SDS-PAGE and immunoblotted using MAVS Ab. Membranes were reprobed with NSP1 NSP3, IRF3, and GAPDH antibodies as internal control. Band intensities of MAVS and NSP1 were normalized to the loading control GAPDH and expressed as a percentage of the protein in mock infected cells. <b>B)</b> HT29 cells were infected with SA11 at (1 M.O.I.) for indicated time points. RNA was isolated and the <i>nsp4</i> and <i>mavs</i> transcripts were analyzed by qRT-PCR. Fold changes were obtained by normalizing relative gene expressions to <i>gapdh</i> using the formula 2^−ΔΔCT (ΔΔC_T = ΔC_TSample-ΔC_{TUntreated control}). <b>C)</b> Expression of MAVS in HT29 cells infected with normal or psoralane-UV-irradiated replication deficient RV SA11 simian strain. Band intensities of MAVS and NSP1 were expressed in percentage. <b>D)</b> HEK293 cells were transfected with pcDNA constructs of NSP5, NSP4, NSP3, NSP2 and NSP1. The expression of MAVS protein was analyzed and expression of viral NSPs were confirmed by immunoblotting using His Ab. <b>E)</b> Dose-dependent degradation of MAVS protein in total cell extracts of HEK293 cells transfected with increasing concentration of pcD-NSP1 for 24 hours. The data represent the means ± the standard deviations (SD) of three independent experiments.</p

Degradation of MAVS in RV infected HT29 cells and NSP1 transfected HEK293 cells.

<p><b>A, B, C and D)</b> HT29 cells were infected with the indicated strains of RV (1 M.O.I.) and harvested at various time points. Total cellular protein was harvested and separated by SDS-PAGE. Immunoblot analyses were performed with antibodies to the cellular proteins MAVS, IRF3, GAPDH and the viral proteins NSP1 and NSP3. Porcine OSU, bovine A5-13, A5-16 and human Wa were harvested at 2, 4, 8 and 12 hpi. Murine EW, rhesus RRV, human KU and DS-1 were lysed at 6 and 12hpi. Band intensities of MAVS and IRF3 were normalized to the loading control GAPDH and were expressed as a percentage of the protein in mock infected cells. The data represent the means ± SD of three independent experiments. <b>E)</b> Effects of NSP1 proteins of different RV strains on human MAVS. HEK293 cells were transfected with vectors encoding indicated RV NSP1 proteins and harvested at 24 h p.i. Proteins in cell lysates were resolved by SDS-PAGE and analyzed by immunoblotting for MAVS, IRF3 and Anti-His antibody.</p