Rotavirus NSP1 Requires Casein Kinase II-Mediated Phosphorylation for Hijacking of Cullin-RING Ligases

Abstract

The 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.

Biphasic regulation of RNA interference during rotavirus infection by modulation of Argonaute2

“…This was also supported by similar restoration of AGO2 protein levels in RV‐NSP1‐transfected cells in response to 5 μM of reversible proteasome inhibitor MG‐132 but not to 1 μM of lysosomal inhibitor Bafilomycin A1 (Figure S3D). In addition to the putative E3 ubiquitin ligase activity intrinsic to RV‐NSP1, co‐opted host E3 ubiquitin ligase complex Cullin has recently been implicated to be interacting with (Ding et al, ; Lutz, Pace, & Arnold, ) and be involved in mediating proteasomal degradation of host substrates (Davis, Morelli, & Patton, ; Ding et al, ). Interestingly, inhibition of E3 ubiquitin ligase activity of Cullin by MLN4924 (500 nM) failed to rescue RV‐NSP1‐mediated reduction in AGO2 levels in MA104 cells (Figure d), suggesting AGO2 degradation by RV‐NSP1 to be independent of co‐opted Cullin machinery.…”

“…AGO2 activity has been reported to be regulated by phosphorylation (Bridge et al, ; Horman et al, ). Phosphorylation of RV‐OSU‐NSP1 by caesin kinase II has also recently been shown to be a prerequisite for NSP1‐βTrCP association (Davis et al, ). To further explore whether association between RV‐NSP1 and AGO2 is influenced by phosphorylation status of either NSP1 or AGO2, coimmunoprecipitation analysis was carried out in the presence of lambda protein phosphatase (lambda PP).…”

“…RV‐NSP1 has recently been reported to interact with (Davis et al, ; Ding et al, ; Lutz et al, ) and degrade β‐TrCP by co‐opting Cullin RING E3 ubiquitin Ligases (CRLs; Ding et al, ). Reversal of AGO2 degradation, however, was observed in presence of neither dominant negative forms of Cullin1 and Cullin3 nor pan‐Cullin inhibitor MLN4924, suggesting that RV‐NSP1‐mediated degradation of AGO2 is not dependent on co‐opted CRLs (Figure d; Figure S3E).…”

Rotavirus NSP1 localizes in the nucleus to disrupt PML nuclear bodies during infection

“…The SNF and SRF viruses encode NSP1 proteins derived from K9 and RRV rotaviruses, respectively, and given their ability to induce IRF degradation are considered to have SA11-4F-like NSP1s (8, 20, 24). The SDF, SKF, and SOF viruses encode NSP1 proteins derived from DS-1, KU, and OSU rotaviruses, respectively, and are considered to have OSU-like NSP1s based on their ability to induce β-TrCP degradation (20, 24, 25). At 8 h p.i., cells were fixed and immunostained for the viral protein VP6 and PML, and nuclei were counterstained with DAPI.…”

Features of the Immune Response and Efficacy Immunotherapy for Rotavirus Infection in Children

“…Ротавирус стремится ускользнуть от воздействия направленных на него факторов врожденного иммунного ответа организма. Одним из таких защитных механизмов является синтез ротавирусом антагониста интерферонов -NSP1-протеина, который воздействует на клеточные белки, необходимые для продукции интерферонов, путем разрушения протеасом [7,8].…”