Dataset Information

Putative E3 ubiquitin ligase of human rotavirus inhibits NF-?B activation by using molecular mimicry to target ?-TrCP.

ABSTRACT: UNLABELLED:NF-?B plays a critical role in the induction and maintenance of innate and adaptive immune transcriptional programs. An associated inhibitor of ?B protein (I?B) regulates NF-?B activation and contains a degron motif (DSG?xS) that undergoes phosphorylation following pathogen recognition or other proinflammatory signals. The E3 ubiquitin ligase SCF(?-TrCP) recognizes this phosphodegron through its ?-transducin repeat-containing protein (?-TrCP) subunit and induces I?B degradation, allowing NF-?B to translocate to the nucleus and modulate gene expression. Rotavirus (RV), a major cause of pediatric gastroenteritis, can block NF-?B activation through the action of its nonstructural protein NSP1, a putative E3 ubiquitin ligase that mediates the degradation of ?-TrCP or other immunomodulatory proteins in a virus strain-specific manner. Here, we show that NSP1 targets ?-TrCP by mimicking the I?B phosphodegron. The NSP1 proteins of most human and porcine RV strains conserve a C-terminal phosphodegron-like (PDL) motif, DSG?S. Deletion of this motif or mutation of its serine residues disrupts NSP1-mediated degradation of ?-TrCP and inhibition of NF-?B activation. Additionally, a point mutation within the phosphodegron-binding pocket protects ?-TrCP from NSP1-mediated turnover. Fusion of the PDL motif to an NSP1 protein known to target other immunomodulatory proteins generates a chimeric NSP1 protein that can induce ?-TrCP degradation and block NF-?B activation. Other viral proteins (Epstein-Barr virus LMP1, HIV-1 Vpu, and vaccinia virus A49) also contain a PDL motif and interact with ?-TrCP to inhibit NF-?B activation. Taken together, these data suggest that targeting ?-TrCP by molecular mimicry may be a common strategy used by human viruses to evade the host immune response. IMPORTANCE:The transcription factor NF-?B, a central regulator of the host response to infection, is a frequent target of viral antagonism. Pathogen detection activates NF-?B by inducing the phosphorylation of an associated inhibitor protein (I?B), which targets I?B for degradation by the E3 ubiquitin ligase ?-TrCP. Rotavirus, a significant cause of childhood gastroenteritis, antagonizes NF-?B through the activity of its NSP1 protein, a putative E3 ubiquitin ligase that mediates ?-TrCP turnover. Here, we show that NSP1 functions by mimicking the I?B phosphodegron recognized by ?-TrCP. Nearly all human rotavirus strains conserve this motif at the NSP1 C terminus, and its removal disrupts NSP1 antagonist activity. This sequence conserves the biochemical properties of the I?B phosphodegron and can rescue antagonist activity when fused to an NSP1 protein otherwise inactive against ?-TrCP. Other viral proteins also mimic I?B to disrupt NF-?B activation, indicating that this is an important immune evasion strategy.

SUBMITTER: Morelli M

PROVIDER: S-EPMC4324316 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Putative E3 ubiquitin ligase of human rotavirus inhibits NF-κB activation by using molecular mimicry to target β-TrCP.

Morelli Marco M Dennis Allison F AF Patton John T JT

mBio 20150127 1

<h4>Unlabelled</h4>NF-κB plays a critical role in the induction and maintenance of innate and adaptive immune transcriptional programs. An associated inhibitor of κB protein (IκB) regulates NF-κB activation and contains a degron motif (DSGΦxS) that undergoes phosphorylation following pathogen recognition or other proinflammatory signals. The E3 ubiquitin ligase SCF(β-TrCP) recognizes this phosphodegron through its β-transducin repeat-containing protein (β-TrCP) subunit and induces IκB degradatio ...[more]

PMID: 25626907

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Project description:UnlabelledThe rotavirus nonstructural protein NSP1 acts as an antagonist of the host antiviral response by inducing degradation of key proteins required to activate interferon (IFN) production. Protein degradation induced by NSP1 is dependent on the proteasome, and the presence of a RING domain near the N terminus has led to the hypothesis that NSP1 is an E3 ubiquitin ligase. To examine this hypothesis, pulldown assays were performed, followed by mass spectrometry to identify components of the host ubiquitination machinery that associate with NSP1. Multiple components of cullin RING ligases (CRLs), which are essential multisubunit ubiquitination complexes, were identified in association with NSP1. The mass spectrometry was validated in both transfected and infected cells to show that the NSP1 proteins from different strains of rotavirus associated with key components of CRL complexes, most notably the cullin scaffolding proteins Cul3 and Cul1. In vitro binding assays using purified proteins confirmed that NSP1 specifically interacted with Cul3 and that the N-terminal region of Cul3 was responsible for binding to NSP1. To test if NSP1 used CRL3 to induce degradation of the target protein IRF3 or β-TrCP, Cul3 levels were knocked down using a small interfering RNA (siRNA) approach. Unexpectedly, loss of Cul3 did not rescue IRF3 or β-TrCP from degradation in infected cells. The results indicate that, rather than actively using CRL complexes to induce degradation of target proteins required for IFN production, NSP1 may use cullin-containing complexes to prevent another cellular activity.ImportanceThe ubiquitin-proteasome pathway plays an important regulatory role in numerous cellular functions, and many viruses have evolved mechanisms to exploit or manipulate this pathway to enhance replication and spread. Rotavirus, a major cause of severe gastroenteritis in young children that causes approximately 420,000 deaths worldwide each year, utilizes the ubiquitin-proteasome system to subvert the host innate immune response by inducing the degradation of key components required for the production of interferon (IFN). Here, we show that NSP1 proteins from different rotavirus strains associate with the scaffolding proteins Cul1 and Cul3 of CRL ubiquitin ligase complexes. Nonetheless, knockdown of Cul1 and Cul3 suggests that NSP1 induces the degradation of some target proteins independently of its association with CRL complexes, stressing a need to further investigate the mechanistic details of how NSP1 subverts the host IFN response.

Dataset Information

Putative E3 ubiquitin ligase of human rotavirus inhibits NF-?B activation by using molecular mimicry to target ?-TrCP.

Publications

Putative E3 ubiquitin ligase of human rotavirus inhibits NF-κB activation by using molecular mimicry to target β-TrCP.

Similar Datasets

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