Abstract
ABSTRACTThe rotavirus nonstructural protein NSP1 repurposes cullin-RING E3 ubiquitin ligases (CRLs) to antagonize innate immune responses. By functioning as substrate adaptors of hijacked CRLs, NSP1 causes ubiquitination and proteasomal degradation of host proteins that are essential for expression of interferon (IFN) and IFN-stimulated gene products. The target of most human and porcine rotaviruses is the β-transducin repeat-containing protein (β-TrCP), a regulator of NF-κB activation. β-TrCP recognizes a phosphorylated degron (DSGΦXS) present in the inhibitor of NF-κB (IκB); phosphorylation of the IκB degron is mediated by IκB kinase (IKK). Because NSP1 contains a C-terminal IκB-like degron (ILD; DSGXS) that recruits β-TrCP, we investigated whether the NSP1 ILD is similarly activated by phosphorylation and whether this modification is required to trigger the incorporation of NSP1 into CRLs. Based on mutagenesis and phosphatase treatment studies, we found that both serine residues of the NSP1 ILD are phosphorylated, a pattern mimicking phosphorylation of IκB. A three-pronged approach using small-molecule inhibitors, small interfering RNAs, and mutagenesis demonstrated that NSP1 phosphorylation is mediated by the constitutively active casein kinase II (CKII), rather than IKK. In coimmunoprecipitation assays, we found that this modification was essential for NSP1 recruitment of β-TrCP and induced changes involving the NSP1 N-terminal RING motif that allowed formation of Cul3-NSP1 complexes. Taken together, our results indicate a highly regulated stepwise process in the formation of NSP1-Cul3 CRLs that is initiated by CKII phosphorylation of NSP1, followed by NSP1 recruitment of β-TrCP and ending with incorporation of the NSP1–β-TrCP complex into the CRL via interactions dependent on the highly conserved NSP1 RING motif.
Highlights
The rotavirus nonstructural protein NSP1 repurposes cullin-RING E3 ubiquitin ligases (CRLs) to antagonize innate immune responses
This task is accomplished through the rotavirus nonstructural protein NSP1, which incorporates itself into a CRL and serves as a substrate recognition subunit
In this study, we determined that rotavirus NSP1 is phosphorylated and that this modification represents a critical initiating event in the interaction of NSP1 with -TrCP and in the incorporation of NSP1 into Cul3 CRLs (Fig. 9)
Summary
The rotavirus nonstructural protein NSP1 repurposes cullin-RING E3 ubiquitin ligases (CRLs) to antagonize innate immune responses. A primary mechanism used by the virus to inhibit host innate immune responses is to hijack cellular cullin-RING E3 ubiquitin ligases (CRLs) and redirect their targeting activity to the degradation of cellular proteins crucial for interferon expression This task is accomplished through the rotavirus nonstructural protein NSP1, which incorporates itself into a CRL and serves as a substrate recognition subunit. We show that formation of NSP1 CRLs is a highly regulated stepwise process initiated by CKII phosphorylation of the -TrCP recognition motif in NSP1 This modification triggers recruitment of the -TrCP substrate and induces subsequent changes in a highly conserved NSP1 RING domain that allow anchoring of the NSP1–-TrCP complex to a cullin scaffold. The E3 ligase responsible for targeting IB for proteasomal degradation is assembled on a cullin-1 (Cul1) scaffold that anchors a substrate recognition complex consisting of Skp and the F-box adaptor protein -TrCP; this E3 is termed the Skp1–Cul1–F-box (SCF-TrCP) CRL [7, 13]
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