Rotavirus NSP1 as a phosphorylated substrate adaptor of hijacked Cullin‐RING ligases

Abstract

Rotavirus is a segmented, double‐stranded RNA virus responsible for significant diarrheal disease worldwide, primarily in infants and young children. To circumvent host interferon (IFN) responses, rotavirus NSP1 proteins hijack host Cullin‐RING E3 ubiquitin ligases (CRLs), acting as substrate adaptors to redirect their activity towards proteasomal degradation of IFN signaling proteins. For the NSP1 proteins of many human and porcine virus strains, this activity facilitates the degradation of β‐TrCP, a key regulator of NF‐κB signaling. NF‐κB, a transcription factor required for IFN induction, is activated when inhibitor of NF‐κB (IκB) is phosphorylated on a conserved degron motif by IκB kinase complex (IKK). Phosphorylation leads to recognition by β‐TrCP, which subsequently targets IκB for degradation. NSP1 contains a C‐terminal IκB‐like degron motif that competitively recruits β‐TrCP. Because the IκB degron requires phosphorylation to mediate interactions with β‐TrCP, we sought to address whether the NSP1 degron is also phosphorylated, and whether phosphorylation regulates NSP1 activity. Interestingly, antibodies specific for the phosphorylated IκB degron bound to NSP1, and this recognition was ablated by mutagenesis or phosphatase treatment, suggesting the degron is phosphorylated. Similarly, inhibition of casein kinase II (CKII) activity targeted by siRNAs, small‐molecule inhibitors, or motif mutagenesis disrupted NSP1 degron phosphorylation. Mutagenesis of an N‐terminal RING domain thought to mediate recruitment of CRLs had no effect on NSP1 degron phosphorylation. Our data indicates that binding of β‐TrCP to NSP1 hinges upon degron phosphorylation, and that phosphorylation by CKII occurs independently of binding to hijacked CRLs. The reliance of NSP1 on CKII, rather than IKK, suggests a distinct vulnerability and new potential target for therapeutic intervention.