Putative E3 Ubiquitin Ligase of Human Rotavirus Inhibits NF-kappa B Activation by Using Molecular Mimicry To Target beta-TrCP

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