Rotavirus NSP1 Associates with Components of the Cullin RING Ligase Family of E3 Ubiquitin Ligases

Abstract

The 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 ␤-TrCP, Cul3 levels were knocked down using a small interfering RNA (siRNA) approach. Unexpectedly, loss of Cul3 did not rescue IRF3 or ␤-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. P rotein ubiquitination is among the most widely used posttranslational modifications and regulates many aspects of cell biology, including cell signaling, DNA damage responses, and protein degradation (1). Conjugation of ubiquitin to a target protein occurs by the sequential activities of three types of enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-ligating enzyme (E3). E3 ubiquitin ligases are classified on the basis of the presence of either a...

A paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection

“…In several in vitro models, rotavirus NSP1 inhibits the host IFN response by preventing IFN from being transcribed (35,51). Recent studies have shown that NSP1 acts as an adaptor that links the host Cullin-E3 ligase complex with β-transducin repeat-containing protein (β-TrCP), which is a necessary component for NF-κB-mediated induction of IFN transcripts (77,78). Through this interaction with the Cullin-E3 ligase complex, NSP1 facilitates the proteasomal degradation of itself and β-TrCP (78).…”

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

“…Reciprocal coimmunoprecipitation studies subsequently showed RV‐NSP1 to associate with the PAZ domain containing span of AGO2 via its own N‐terminal domain independent of any RNA intermediates and residue phosphorylation, justifying degradation of AGO2 in presence of N‐terminal domain of RV‐NSP1 (Figure a–d, F; Figure S5A, C). This is in contrast to the fact that substrate binding specificity of RV‐NSP1 generally lies in its highly variable C‐terminal half (Lutz et al, ; Morelli et al, ). The association between AGO2 and RV‐NSP1 (Figure e, Figure S5B) as well as ubiquitin enrichment of AGO2 (Figure S3F) were also observed at early hour of RV‐SA11 infection.…”

“…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.…”

“…Detailed mechanistic investigations revealed overexpression of RV‐NSP1, especially the N‐terminal region of RV‐NSP1 (Figure a,f; Figure S3A, B, G), to trigger protein level reduction of AGO2. RV‐NSP1 is a multifunctional protein involved in IFN antagonism, activation of pro‐survival PI3K/Akt pathways, denucleating cytoplasmic P bodies, and also harbouring RNA binding as well as cytoskeleton localisation domain (Bagchi et al, ; Bhowmick et al, ; Ding et al, ; Lutz et al, ; Morelli, Ogden, & Patton, ). IFN antagonistic properties of RV‐NSP1 lies in its ability to trigger proteasomal destruction of IRF3, 5, 7, 9, β‐TrCP, TRAF2, and RIG‐I, whereas P body destabilising property of NSP1 includes degradation of Pan3 by ubiquitin–proteasome system.…”

“…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).…”