key: cord-0303766-7ter8iud
authors: Li, Jiaying; Yu, Haixiao; Jiang, Wenjin; Ma, Ping; Feng, Zezhong; Lu, Yang; Tu, Changchun; Sun, Jinfu
title: Classical swine fever virus NS5A inhibits NF-κB signaling by targeting NEMO
date: 2021-06-22
journal: bioRxiv
DOI: 10.1101/2021.06.21.449351
sha: a387c79cef6315f71bfc92a81ab0c8449a4d25b9
doc_id: 303766
cord_uid: 7ter8iud

The NS5A non-structural protein of classical swine fever virus (CSFV) is a multifunctional protein involved in viral genomic replication, protein translation and regulation of cellular signaling pathways, and assembly of infectious virus particles. Previous report showed that NS5A inhibited nuclear factor kappa B (NF-κB) signaling induced by poly(I:C); however, the mechanism was not elucidated. Here, we report that NS5A interacts with NF-κB essential modulator (NEMO),a regulatory subunit of the IκB kinase (IKK) complex, and that the zinc finger domain of NEMO essential for NEMO ubiquitination and IKK activation is required for the interaction of NEMO with NS5A. Viral infection or NS5A expression by itself reduced the protein level of NEMO. Mechanistic analysis revealed that NS5A mediated proteasomal degradation of NEMO. Ubiquitination assay showed that NS5A induced K27-but not K48-linked polyubiquitination of NEMO. In addition, NS5A blocked k63-linked polyubiquitination of NEMO, thereby inhibiting activation of IKK and NF-κB. These findings indicate that NS5A inhibits NF-κB signaling by mediating proteasomal degradation of NEMO and blocking k63-linked polyubiquitination of NEMO, thereby revealing a novel mechanism by which CSFV inhibits host innate immunity. Importance Classical swine fever (CSF) is a economically important swine viral disease leading to hemorrhagic fever and immuno-suppression. In order to successfully infect and replicate in a host cell, viruses have evolved various strategies to antagonize host innate immunity. It is known that CSFV non-structural protein Npro interacts with interferon regulatory factor 3 (IRF3) and mediates its proteasomal degradation, thereby inhibiting the production of type I interferon. However, no other mechanism by which CSFV antagonizes host innate immunity has so far been reported. Here, we show that NS5A inhibits NF-κB signaling by mediating proteasomal degradation of NEMO and by blocking k63-linked polyubiquitination of NEMO, thereby revealing a novel mechanism by which CSFV antagonizes host innate immunity.

Transfected 293T cells were used to evaluate the effect of NS5A or the NS5A truncated 201 mutants on NF-κB dependent IFNα mRNA expression. At 48 h the transfected cells were 202 incubated with TNFα for 15 mins, then harvested and assayed for IFNα mRNA expression. As 203 before, only the NS5Am2 mutant failed to inhibit IFNα mRNA expression (Fig.4 B) , indicating 204 again that the 126-250 aa region was implicated in the inhibition of NF-κB signaling by NS5A. 205 co-transfected with the indicated vectors (Fig. 10A, B) . At 48 h post-transfection, cells were 346 incubated with TNFα for 15 min before immunopreciptation with mouse anti-His monoclonal 347 antibody (Fig. 10A ) or anti-NEMO antibody and unspecific IgG (Fig. 10B) . Precipitated proteins 348 and whole cell lysates were subjected to western blot analysis using the indicated antibodiess. 349 Fig 2B, polyunbiquitination, 293T cells were co-transfected with the indicated vectors (Fig.10A, B) , 355 incubated with TNFα for 15 min at 48 h post-transfection, then immunoprecipitated with anti-Myc 356 antibody (Fig.10A) or rabbit anti-NEMO monoclonal antibody (Abcam, USA) and nonspecific 357 IgG (Fig.10B) . Precipitated proteins were subjected to western blotting with anti-HA antibody. 358

Results showed that K63-linked polyunbiquition of exogenous NEMO (Fig.10A) and levels of 359 endogenous NEMO (Fig.10B) were decreased significantly in NS5A-expressing cells. shown that levels of NEMO protein are reduced in CSFV-infected or NS5A-expressing cells 395 ( Fig.5) , but recover in NS5A-expressing cells in the presence of proteasome inhibitor MG132 396 ( Fig.6) , indicating that NS5A mediated proteasomal degradation of NEMO. 397 20 K48-linked polyubiquitination being the major signal of proteasomal degradation, we therefore 398 tested whether NS5A induced K48-linked polyubiquitination of NEMO. Ubiquitination assay 399 demonstrated that NS5A induced K27-linked but not K48-linked polyubiquitination of NEMO for 400 its proteasomal degradation (Fig.9) . Further study is needed to clarify how NS5A induces NEMO and blocks its K63-linked ubiquitination, leading to inhibition of NF-κB signaling and 413 IFN production. Here, we have demonstrated that CSFV NS5A interacts with NEMO and that the 414 C-terminal ZF domain of NEMO is required for the interaction (Fig. 2B) . Significantly, the ZF 415 domain of NEMO is a functional ubiquitin-binding domain (29) and an intact zinc finger of 416 NEMO is essential for NEMO ubiquitination and the activation of IKK and NF-κB (28). 417

Considering the important role of ZF in NEMO ubiquitination, the potential effects of NS5A on 418 NEMO ubiquitination were analysed. Fig.10 shows that K63-linked polyubiquitination of NEMO 419 21 is decreased in NS5A-expressing cells, indicating that NS5A inhibits K63-linked 420 polyubiquitination of NEMO. 421

In summary, this study has identified CSFV NS5A as a novel antagonist of innate immunity and 422 has shown that NS5A inhibits NF-κB signaling and type I IFN production by mediating 423 proteasomal degradation of NEMO and blocking K63-linked polyubiquitination of NEMO. These 424 findings reveal a novel mechanism by which CSFV evades host innate immunity and contribute to 425 better understand of the pathogenesis of CSFV. 

Postnatal 506 persistent infection with classical swine fever virus and its immunological implications

Family Flaviridae

Virus Taxonomy. Eighth Report of the 511 International Committee on Taxonomy of Virus

The molecular biology of pestiviruses

Classical swine fever virus

NS5A protein interacts with 3'-untranslated region and regulates viral RNA synthesis

Classical swine fever virus NS5A regulates viral RNA replication through binding to NS5B 520 and 3'-UTR

Influence of NS5A protein of classical 522 swine fever virus (CSFV) on CSFV internal ribosome entry site-dependent translation

Classical 525 swine fever virus NS5B protein suppresses the inhibitory effect of NS5A on viral translation 526 by binding to NS5A

Characterization of the C-terminal sequence of NS5A necessary for the assembly and 529 production of classical swine fever virus infectious particles

Annexin A2 is 531 involved in the production of classical swine fever virus infectious particles

Rab1A is 534 required for assembly of classical swine fever virus particle

Rab18 binds to classical 536 swine fever virus NS5A and mediates viral replication and assembly in swine umbilical vein 537 endothelial cells

The eukaryotic 539 translation initiation factor 3 subunit E binds to classical swine fever virus NS5A and 540 facilitates viral replication

Cellular Hsp27 interacts with classical swine fever virus NS5A protein and negatively 543 regulates viral replication by the NF-κB signaling pathway

eEF1A interacts with the NS5A protein and inhibits the growth of classical swine fever virus

Viperin inhibits classical swine fever virus replication by 549 interacting with viral nonstructural 5A protein

Heat 551 shock protein 70 is associated with CSFV NS5A protein and enhances viral RNA replication

FKBP8 interact with classical swine fever virus NS5A protein and promote virus RNA 555 replication

Classical swine fever NS5A protein changed inflammatory 557 cytokine secretion in porcine alveolar macrophages by inhibiting the NF-kB signaling 558 pathway

Molecular basis of NF-κB signaling

Sensing of Lys 63-linked 562 28 polyubiquitination by NEMO is a key event in NF-kappaB activation

Activation of IKK by TNFalpha requires 565 site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO

Israë l A. 568 1998. Complementation cloning of NEMO, a component of the IκB kinase complex essential 569 for NF-κB activation

Regulation of adaptive immunity by the innate immune system

NF-κB and innate immunity

Viral interference with innate immunity by preventing NF-kB activity

C-type lectin receptor-induced NF-κB activation in innate immune 577 and inflammatory responses

Selective inhibition 579 of NF-kappaB activation by a peptide that blocks the interaction of NEMO with the IkappaB 580 kinase complex

A role for NF-kappaB essential

modifier/IkappaB kinase-gamma (NEMO/IKKgamma) ubiquitination in the activation of the 583

IkappaB kinase complex by tumor necrosis factor-alpha

The zinc finger of 585 NEMO is a functional ubiquitin-binding domain

NF-κB and innate immunity

Classical swine fever virus failed to activate nuclear factor -kappa b signaling pathway 590 both in vitro and in vivo

Israë l A. 592 1998. Complementation cloning of NEMO, a component of the IκB kinase complex essential 593 for NF-κB activation

Selective inhibition 595 of NF-kappaB activation by a peptide that blocks the interaction of NEMO with the IkappaB 596 kinase complex

Development of novel NEMO-binding domain mimetics 600 for inhibiting IKK /NF-κB activation

Novel non-peptide small molecules preventing 604

IKKβ/NEMO association inhibit NF-κB activation in LPS-stimulated J774 macrophages

Foot-and-mouth disease virus 3C protease cleaves NEMO to impair 608 innate immune signaling

Epidemic Diarrhea Virus 3C-Like Protease Regulates Its Interferon Antagonism by Cleaving 611 NEMO

Porcine reproductive 613 and respiratory syndrome virus nonstructural protein 4 antagonizes beta interferon expression 614 by targeting the NF-κB essential modulator

Molluscum Contagiosum Virus

Protein MC005 Inhibits NF-κB Activation by Targeting NEMO-Regulated IκB Kinase

NF-κB essential modulator (NEMO) interaction 620 with linear and lys-63 ubiquitin chains contributes to NF-κB activation

Innate immune-directed NF-κB signaling requires site-specific NEMO ubiquitination

Molluscum contagiosum virus MC159 abrogates cIAP1-NEMO 626 interactions and inhibits NEMO polyubiquitination

SARS-CoV-2 ORF9b inhibits RIG-I-MAVS 630

antiviral signaling by interrupting K63-linked ubiquitination of NEMO

This work was supported by the National Nature Science Fundation of China (Grant 502No.31972677). 503