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...

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Autores principales:	Kaitlin A. Davis, Marco Morelli, John T. Patton
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	CKII E3 ligase NF-kB cullin ring ligase innate immunity rotavirus Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/4927981d85ec4c64843ee382e006d0da
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spelling	oai:doaj.org-article:4927981d85ec4c64843ee382e006d0da2021-11-15T15:51:43ZRotavirus NSP1 Requires Casein Kinase II-Mediated Phosphorylation for Hijacking of Cullin-RING Ligases10.1128/mBio.01213-172150-7511https://doaj.org/article/4927981d85ec4c64843ee382e006d0da2017-09-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01213-17https://doaj.org/toc/2150-7511ABSTRACT 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. IMPORTANCE Rotavirus is a segmented double-stranded RNA virus that causes severe diarrhea in young children. 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. The substrate recognized by the NSP1 of many human and porcine rotaviruses is β-TrCP, a protein that regulates the transcription factor NF-κB. In this study, we show that formation of NSP1 CRLs is a highly regulated stepwise process initiated by CKII phosphorylation of the β-TrCP recognition motif in NSP1. This modification triggers recruitment of the β-TrCP substrate and induces subsequent changes in a highly conserved NSP1 RING domain that allow anchoring of the NSP1–β-TrCP complex to a cullin scaffold.Kaitlin A. DavisMarco MorelliJohn T. PattonAmerican Society for MicrobiologyarticleCKIIE3 ligaseNF-kBcullin ring ligaseinnate immunityrotavirusMicrobiologyQR1-502ENmBio, Vol 8, Iss 4 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	CKII E3 ligase NF-kB cullin ring ligase innate immunity rotavirus Microbiology QR1-502
spellingShingle	CKII E3 ligase NF-kB cullin ring ligase innate immunity rotavirus Microbiology QR1-502 Kaitlin A. Davis Marco Morelli John T. Patton Rotavirus NSP1 Requires Casein Kinase II-Mediated Phosphorylation for Hijacking of Cullin-RING Ligases
description	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. IMPORTANCE Rotavirus is a segmented double-stranded RNA virus that causes severe diarrhea in young children. 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. The substrate recognized by the NSP1 of many human and porcine rotaviruses is β-TrCP, a protein that regulates the transcription factor NF-κB. In this study, we show that formation of NSP1 CRLs is a highly regulated stepwise process initiated by CKII phosphorylation of the β-TrCP recognition motif in NSP1. This modification triggers recruitment of the β-TrCP substrate and induces subsequent changes in a highly conserved NSP1 RING domain that allow anchoring of the NSP1–β-TrCP complex to a cullin scaffold.
format	article
author	Kaitlin A. Davis Marco Morelli John T. Patton
author_facet	Kaitlin A. Davis Marco Morelli John T. Patton
author_sort	Kaitlin A. Davis
title	Rotavirus NSP1 Requires Casein Kinase II-Mediated Phosphorylation for Hijacking of Cullin-RING Ligases
title_short	Rotavirus NSP1 Requires Casein Kinase II-Mediated Phosphorylation for Hijacking of Cullin-RING Ligases
title_full	Rotavirus NSP1 Requires Casein Kinase II-Mediated Phosphorylation for Hijacking of Cullin-RING Ligases
title_fullStr	Rotavirus NSP1 Requires Casein Kinase II-Mediated Phosphorylation for Hijacking of Cullin-RING Ligases
title_full_unstemmed	Rotavirus NSP1 Requires Casein Kinase II-Mediated Phosphorylation for Hijacking of Cullin-RING Ligases
title_sort	rotavirus nsp1 requires casein kinase ii-mediated phosphorylation for hijacking of cullin-ring ligases
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/4927981d85ec4c64843ee382e006d0da
work_keys_str_mv	AT kaitlinadavis rotavirusnsp1requirescaseinkinaseiimediatedphosphorylationforhijackingofcullinringligases AT marcomorelli rotavirusnsp1requirescaseinkinaseiimediatedphosphorylationforhijackingofcullinringligases AT johntpatton rotavirusnsp1requirescaseinkinaseiimediatedphosphorylationforhijackingofcullinringligases
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Rotavirus NSP1 Requires Casein Kinase II-Mediated Phosphorylation for Hijacking of Cullin-RING Ligases

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