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ABSTRACT Viruses modulate the host immune system to evade host antiviral responses. The poxvirus proteins serine proteinase inhibitor 2 (SPI-2) and cytokine response modifier A (CrmA) are

involved in multiple poxvirus evasion strategies. SPI-2 and CrmA target caspase-1 to prevent apoptosis and cytokine activation. Here, we identified SPI-2 and CrmA as negative regulators of

virus-triggered induction of IFN-β. Ectopic expression of SPI-2 or CrmA inhibited virus-triggered induction of IFN-β and its downstream genes. Consistently, knockdown of SPI-2 by RNAi

potentiated VACV-induced transcription of antiviral genes. Further studies revealed that SPI-2 and CrmA associated with TBK1 and IKKε to disrupt the MITA-TBK1/IKKε-IRF3 complex. These

findings reveal a novel mechanism of SPI-2/CrmA-mediated poxvirus immune evasion. SIMILAR CONTENT BEING VIEWED BY OTHERS PBLD PROMOTES IRF3 MEDIATED THE TYPE I INTERFERON (IFN-I) RESPONSE

AND APOPTOSIS TO INHIBIT VIRAL REPLICATION Article Open access 03 October 2024 SARS-COV-2 NSP12 ATTENUATES TYPE I INTERFERON PRODUCTION BY INHIBITING IRF3 NUCLEAR TRANSLOCATION Article Open

access 26 February 2021 ISG15-DEPENDENT ACTIVATION OF THE SENSOR MDA5 IS ANTAGONIZED BY THE SARS-COV-2 PAPAIN-LIKE PROTEASE TO EVADE HOST INNATE IMMUNITY Article 16 March 2021 INTRODUCTION

Poxviruses comprise a large family of linear dsDNA viruses that replicate in the cytoplasm of host cells. The most widely studied genus, _Orthopoxviruse_ (OPXV), is pathogenic to humans,

cattle, and zoo animals. OPXVs include vaccinia virus (VACV), cowpox virus (CPXV), ectromelia virus (ECTV), and variola virus (VARV), the causative agent of smallpox. VACV, which was used in

the vaccination campaign for smallpox eradication, is now being developed as live vaccines against other infectious diseases and cancer1. CPXV infects a wide range of host species and

causes zoonosis, and the outbreaks of CPXV in recent years have caused a public health crisis2. OPXVs have evolved various mechanisms to evade and suppress host antiviral responses3. They

have large genomes with a variety of immunomodulatory genes and thus display a wide range of immune evasion strategies4, 5. Type I interferons (IFNs) are critical for both innate and

adaptive immune responses against viral infection6. Accordingly, the suppression of type I IFN signaling by viral immunomodulatory proteins is one of the key events in virus immune evasion.

Host germline-encoded pattern-recognition receptors (PRRs) recognize viral nucleic acids and trigger downstream signaling events7. Viral DNA can be recognized by cyclic GMP-AMP synthase

(cGAS)8, and viral RNA can be recognized by RIG-I-like receptors (RLRs)7. cGAS and RLRs activate the transcription factors interferon regulatory factor 3 (IRF3) and NF-κB though adaptor

proteins, mediator of IRF3 activation (MITA, also termed STING) and virus-induced signaling adaptor (VISA, also termed MAVS), respectively9,10,11,12,13. The signaling pathways of IRF3 and

NF-κB activation triggered by cGAS and RLRs converge at the level of TANK-binding kinase 1 (TBK1) and IκB kinases (IKKs). On the one hand, activation of cGAS or RLR leads to the recruitment

of kinases (TBK1 and IKKε) by MITA or VISA9,10,11,12. MITA or VISA is phosphorylated by the kinases and subsequently recruits IRF314. TBK1 and IKKε then phosphorylate IRF3, leading to the

dimerization and nuclear translocation of IRF314. On the other hand, inhibitor of NF-κB (IκB) is phosphorylated by the IKK complex (consisting of IKKα, IKKβ, and IKKγ) and then activated

NF-κB is released6. Activated IRF3 and NF-κB respectively bind to interferon-stimulated response element (ISRE) and the κB site, leading to the transcriptional induction of IFN-β7. Vaccinia

virus (VACV) strain Western Reserve (WR) immediate-early gene _B13R_ encodes a 38-kDa cytosolic protein SPI-2, which is highly homologous to its orthologue, CPXV cytokine response modifier A

(CrmA). SPI-2 and CrmA are the most extensively studied OPXV serine protease inhibitors. They are non-essential for virus replication and are involved in multiple immunomodulatory events.

It has been reported that SPI-2 and CrmA inhibit extrinsic apoptosis and host inflammatory responses15,16,17,18,19,20. SPI-2 and CrmA target caspase-1 to protect virus-infected cells from

TNF- and Fas-mediated apoptosis as well as to prevent the proteolytic activation of interleukin-1β16,17,18,19. In the present study, we found that SPI-2 and CrmA acted as inhibitors of both

DNA- and RNA-virus-triggered induction of IFN-β. SPI-2 and CrmA functioned at the level of TBK1/IKKε to inhibit IRF3 but not NF-κB activation. SPI-2 and CrmA disrupted the

MITA-TBK1/IKKε-IRF3 complex by interacting with TBK1 and IKKε. Our findings suggest that SPI-2 and CrmA antagonize the type I IFN pathway by targeting TBK1/IKKε, thus representing a newly

identified mechanism of immune evasion of VACV and CPXV. RESULTS ROLES OF SPI-2 ORTHOLOGUES IN IFN-Β INDUCTION VACV strain Tian-Tan (TT), a widely-used smallpox vaccine strain in China, is

less virulent than strain WR21, 22. To evaluate the abilities of these VACV strains to stimulate type I IFN induction, THP-1 cells were infected with VACV strain WR and TT at an MOI of 1.

Quantitative real-time PCR results indicated that both VACV strains triggered IFN-β induction in THP-1 cells. However, VACV strain TT activated IFN-β more than strain WR (Fig. 1A). In VACV

strain WR and TT, several genes had polymorphic lengths, including _A39R_, _B13R_, _C4L_, _C14L_, and _C16R_ 22. Among them, C4L and C16R have been demonstrated to regulate type I IFN

induction23, 24. As type I IFN is critical in antiviral responses, we assumed that additional proteins target IFN induction signaling. SPI-2, encoded by the _B13R_ gene in VACV strain WR, is

split into two fragments (termed TB13R and TB14R) in VACV strain TT (Fig. 1B). As SPI-2 is highly homologous to CPXV CrmA, the well-known apoptosis inhibitor, we first constructed

expression clones encoding VACV SPI-2, TB13R, and TB14R and identified their roles in IFN-β induction. Luciferase reporter assays were utilized to identify their abilities to regulate the

activation of IFN-β promoter mediated by overexpression of cGAS and MITA in HEK293 cells. Ectopic expression of SPI-2 inhibited cGAS-and-MITA-mediated activation of IFN-β promoter and ISRE

reporter, but not NF-κB reporter, in a dose-dependent manner (Fig. 1C). However, ectopic expression of TB13R together with TB14R failed to inhibit cGAS-and-MITA-mediated activation of IFN-β

promoter and ISRE reporter (Fig. 1D). Although the sequences of TB13R and TB14R are highly homologous to SPI-2, these two proteins do not retain the function of the full-length protein,

which may be due to the conformational change resulting from splitting ORFs. These data indicated that full-length SPI-2 inhibits cGAS-and-MITA-triggered IFN-β induction. Next, we identified

the roles of SPI-2 orthologues in IFN-β induction. CPXV CrmA and ECTV C7 proteins have 92% and 94% amino acid identity with SPI-2 protein, respectively (Fig. 1B). Ectopic expression of CrmA

and C7 inhibited cGAS-and-MITA-mediated activation of IFN-β promoter and ISRE reporter, but not NF-κB reporter, in a dose-dependent manner (Fig. 1E and F). Thus, the SPI-2 orthologues, CrmA

and C7, inhibit cGAS-and-MITA-triggered IFN-β induction. Sendai virus (SeV) triggers induction of type I IFNs through RLR signaling. Consistent with their roles in regulation of

cGAS-and-MITA-mediated activation of IFN-β promoter, ectopic expression of SPI-2, CrmA, and C7, but not TB13R and TB14R, inhibited SeV-induced activation of IFN-β promoter and ISRE reporter

but not NF-κB reporter (Supplementary Fig. S1A to D). They also had no marked effect on IFN-γ-induced activation of IRF1 promoter (Supplementary Fig. S1E to H). These data suggest that SPI-2

and its homologues, CrmA and C7, inhibit SeV-induced activation of IFN-β promoter. SPI-2 AND CRMA INHIBIT VIRUS-TRIGGERED INDUCTION OF ENDOGENOUS IFN-Β To investigate the roles of SPI-2 and

CrmA in virus-triggered induction of endogenous IFN-β, THP-1 cells were stably transfected with Flag-tagged SPI-2 or CrmA (Fig. 2A and B). Quantitative real-time PCR analysis indicated that

ectopic expression of SPI-2 inhibited herpes simplex virus 1 (HSV-1)- and SeV-induced transcription of _IFNB1_ and its downstream genes _RANTES_ and _CXCL10_, but not _TNF_, downstream of

NF-κB signaling (Fig. 2C and D). Similarly, CrmA inhibited HSV-1- and SeV-induced transcription of _IFNB1_, _RANTES_, and _CXCL10_, but not _TNF_ (Fig. 2E and F). These data suggest that

SPI-2 and CrmA specifically inhibit virus-triggered induction of endogenous IFN-β. Since SPI-2 and CrmA inhibited virus-triggered induction of IFN-β, we examined their roles in cellular

antiviral responses. As shown in Supplementary Fig. S2, HSV-1 and Vesicular Stomatitis Virus (VSV) production was increased in THP-1 cells ectopically expressing SPI-2 or CrmA, consistent

with the role of SPI-2 and CrmA in negative regulation of both DNA- and RNA-virus-triggered IFN-β induction. These results suggest that SPI-2 and CrmA negatively regulate cellular antiviral

responses. KNOCKDOWN OF SPI-2 ENHANCES VACV-TRIGGERED INDUCTION OF IFN-Β GENE The role of endogenous SPI-2 in innate antiviral responses were next examined. RNAi knockdown strategy used in

our study has been successfully used in previous studies25, 26. Two SPI-2-RNAi plasmids were constructed that could inhibit the mRNA level of _B13R_ gene (Fig. 3A and B). In THP-1 cells

stably transfected with the SPI-2-RNAi plasmids, the mRNA levels of _B13R_ neighboring genes, _B12R_ and _B15R_, were not dramatically decreased (Fig. 3B). Quantitative real-time PCR

analysis indicated that knockdown of SPI-2 promoted VACV-induced transcription of _IFNB1_, _RANTES_ and _CXCL10_ (Fig. 3C), but it had no marked effect on HSV-1- or SeV-induced transcription

of _IFNB1_ (Fig. 3D and E). These results suggest that knockdown of SPI-2 potentiates VACV-triggered induction of IFN-β and its downstream genes. SPI-2 AND CRMA INHIBIT IRF3 ACTIVATION AT

THE LEVEL OF TBK1/IKKΕ To determine which step of IFN-β induction was targeted by SPI-2/CrmA, luciferase assays was utilized in which SPI-2 or CrmA was co-expressed with the components of

IFN-β induction signaling. Ectopic expression of SPI-2 inhibited the activation of IFN-β promoter and ISRE reporter mediated by overexpression of cGAS and MITA but not their downstream TBK1,

IKKε, and IRF3 (Fig. 4A and B). IKKβ and p65 act upstream of NF-κB activation. Consistent with the role of SPI-2 in NF-κB activation (Figs 1C, 2C and D), SPI-2 did not markedly affect IKKβ-

or p65-mediated activation of IFN-β promoter (Fig. 4C). Similarly, CrmA inhibited cGAS-, MITA-, but not TBK1-, IKKε- and IRF3-mediated activation of IFN-β promoter and ISRE reporter (Fig. 

4D and E). Additionally, CrmA had no marked effect on IKKβ- or p65-mediated activation of IFN-β promoter (Fig. 4F). SPI-2 and CrmA also inhibited the activation of IFN-β promoter and ISRE

reporter mediated by the RLR adaptor VISA, which is upstream of TBK1/IKKε (Supplementary Fig. S3). These data indicate that SPI-2 and CrmA inhibit IRF3 activation at the level of TBK1/IKKε.

SPI-2 AND CRMA DISRUPT THE MITA-TBK1/IKKΕ-IRF3 COMPLEX BY INTERACTING WITH TBK1/IKKΕ Since SPI-2 and CrmA function at the level of TBK1/IKKε, we examined whether SPI-2 and CrmA interacted

with TBK1 and IKKε. Transient transfection and co-immunoprecipitation experiment indicated that SPI-2 and CrmA were associated with TBK1 and IKKε in HEK293 cells (Fig. 5A and B). To

investigate the mechanism by which SPI-2 and CrmA regulated IRF3 activation, we examined whether SPI-2 and CrmA affected the interaction between components of the MITA-TBK1/IKKε-IRF3 complex

as well as the stability of the components. Ectopic expression of SPI-2 and CrmA attenuated the interaction of TBK1/IKKε with MITA and IRF3, and SPI-2 and CrmA did not markedly affect the

protein levels of MITA, TBK1, IKKε, and IRF3 (Fig. 5C and D). These results suggested that SPI-2 and CrmA disrupted the MITA-TBK1/IKKε-IRF3 complex through interacting with TBK1 and IKKε.

Thus, SPI-2/CrmA inhibits IFN-β induction by targeting TBK1/IKKε. DISCUSSION Previous studies have demonstrated that SPI-2 and CrmA have anti-apoptotic and anti-inflammatory

functions15,16,17,18,19,20. Here, we identified SPI-2 and CrmA as negative regulators of virus-triggered induction of IFN-β. Our findings reveal a novel mechanism of SPI-2/CrmA mediated OPXV

immune evasion. SPI-2 and its orthologue CrmA inhibited the induction of IFN-β triggered by both DNA and RNA viruses, and SPI-2 and CrmA inhibited the activation of IFN-β promoter triggered

by cGAS and RLR signaling. SPI-2 and CrmA specifically inhibited virus-triggered induction of IFN-β by inhibiting of IRF3, but not NF-κB, activation. Luciferase reporter assays suggested

that SPI-2 and CrmA inhibited the activation of IFN-β promoter and ISRE reporter at the level of TBK1 and IKKε, consistent with the roles of TBK1 and IKKε in the activation of IRF3 but not

NF-κB. Further experiments suggested that SPI-2 and CrmA were associated with TBK1 and IKKε. Additionally, TBK1 and IKKε disrupted the MITA-TBK1/IKKε-IRF3 complex. Based on our findings, we

developed a working model of how SPI-2 and CrmA negatively regulate virus-triggered type I IFN induction (Fig. 6). The recognition of cytosolic viral DNA by PRRs triggers host antiviral

responses. SPI-2 and CrmA inhibit the induction of type I IFNs by targeting TBK1 and IKKε. These findings have not only verified a novel immunomodulatory function of SPI-2/CrmA, but they

also provide an example of how viruses escape host immune attack using distinct mechanisms mediated by one immunomodulator. Consistent with the ability of SPI-2 to inhibit IFN-β induction,

knockdown of SPI-2 by RNAi enhanced VACV-induced transcriptional activation of IFN-β gene in THP-1 cells. We observed that knockdown of _B13R_ gene by SPI-2-RNAi plasmids slightly inhibited

the mRNA levels of its neighboring genes, _B12R_ and _B15R_, likely because the elevation of IFN-β production inhibited the replication of VACV in SPI-2 knockdown cells. Multiple viral

proteins may target a same cellular pathway to ensure successful immune evasion3. VACV encodes several immunomodulatory proteins targeting type I IFNs induction signaling, including E327,

28, A4629, 30, C1624, K731, C632, and N233. Among them, K7 and C6 have been reported to inhibit IRF3 activation by targeting the kinase complex containing TBK1 and IKKε. However, each

protein targets distinct components of this complex. K7 inhibits IRF3 phosphorylation by targeting DDX331, and C6 interacts with TANK, NAP1, and SINTBAD, the scaffold proteins that associate

with TBK1 and IKKε32. Our findings suggest that SPI-2 inhibits the activation of IRF3 in a different manner than K7 and C6. SPI-2 directly targets the kinases TBK1 and IKKε, which are

responsible for the phosphorylation of both MITA and IRF3. SPI-2 may work in coordination with these immunomodulatory proteins to inhibit the activation of IRF3 triggered by VACV infection.

The mechanism study of immune evasion mediated by SPI-2 and CrmA not only reveals new insights into the virus-host interaction but also has important implications for rational vaccine design

and antiviral drug development against OPXV infection. MATERIALS AND METHODS REAGENTS AND ANTIBODIES Recombinant IFN-γ (R&D Systems) and mouse monoclonal antibodies against FLAG,

Flag-HRP, β-actin (Sigma), Myc (CST), and HA (Covance) were purchased from the indicated manufacturers. HEK293 and THP-1 cells, SeV, HSV-1, and VSV have been previously described26,

34,35,36. CONSTRUCTS IFN-β, ISRE, NF-κB and IRF1 luciferase reporter plasmids, mammalian expression plasmids for Flag- or HA-tagged cGAS, MITA, TBK1, IKKε, IRF3, and β-actin have been

previously described26, 34. Mammalian expression plasmids for human Flag-, HA- or Myc-tagged SPI-2 and CrmA were constructed using standard molecular biology techniques. RNAI Double-stranded

oligonucleotides corresponding to the target sequences were cloned into the pSuper.Retro-RNAi plasmid (Oligoengine). The following sequences were targeted for SPI-2 mRNA: #1

5′-GGAGAACATGGATAAGGTT-3′, #2 5′-GCGATATTCTGCCGTGTTT-3′. The sequence targeted by the control RNAi plasmid is 5′-GGAAGATGTATGGAGACATGG-3′. TRANSFECTION AND REPORTER ASSAYS HEK293 cells were

seeded and transfected the following day using the standard calcium phosphate precipitation method or FuGENE (Roche), according to the procedures recommended by the manufacturer. Empty

control plasmids were added to ensure that each transfection received the same amount of total DNA. To normalize for transfection efficiency, pRL-TK Renilla luciferase reporter plasmids were

added to each transfection. Luciferase assays were performed using a dual-specific luciferase assay kit (Promega). Firefly luciferase activities were normalized based on Renilla luciferase

activities. QUANTITATIVE REAL-TIME PCR Total RNA was isolated from THP-1 cells (1 × 106) using TRIzol reagent (Takara) according to the manufacturer’s instructions. cDNA was synthesized from

2 μg of purified total RNA using reverse transcriptase (Invitrogen). Quantitative real-time PCR was performed using Ssoadvanced Universal SYBR Green Supermix (Bio-Rad) to measure the

expression of mRNA. The related fold difference in expression of the target gen between the cells were analyzed with 2∆∆CT method. Data represent the relative abundance of the indicated mRNA

normalized to that of GAPDH. Gene-specific primer sequences were as described34 or as follows: B13R: (forward, 5′-CTCCGACGGAAATGGTAGAT-3′; reverse, 5′-TGCCGAATGATTCCTTTACA-3′). CrmA:

(forward, 5′-GTGTCGACGCTATGATCCAC-3′; reverse, 5′-GATGAACGGATGATCTGCAC-3′). B12R: (forward, 5′-ACAACTGGACACGGGAACG-3′; reverse, 5′-CCTTTGGGGCGAATACTCTT-3′). B15R: (forward,

5′-TTAACGCGCCTGAATGTATCG-3′; reverse, 5′-CAGTAGGTTTCGTTCGTGGTAATG-3′). RNAI-TRANSDUCED STABLE THP-1 CELLS HEK293 cells were transfected with two packaging plasmids (pGAG-Pol and pVSV-G)

together with control or SPI-2-RNAi retroviral plasmids respectively by calcium phosphate precipitation. Twenty-four hours after transfection, the cells were incubated with new medium

without antibiotics for another twenty-four hours. The recombinant virus-containing medium was filtered with a 0.22-μm filter (Millex) and then added to cultured THP-1 cells in the presence

of polybrene (4 μg/mL). The infected cells were selected with puromycin (0.5 μg/mL) for at least seven days before performing additional experiments. COIMMUNOPRECIPITATION AND IMMUNOBLOT

ANALYSIS HEK293 cells (5 × 106) were lysed in 1 mL IP lysis buffer (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 1 mM

phenylmethylsulphonyl fluoride) on wet ice. Coimmunoprecipitation and immunoblot analysis were performed as previously described10, 26, 34,35,36. DATA AVAILABILITY All data generated or

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adaptor STING. _Nature immunology_ 17, 1057–1066, doi:10.1038/ni.3510 (2016). Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Yu-Zhi Fu, Dan Li, Tian-Tian

Liu, Tian Xia, and Ze-Min Song for technical assistance and stimulating discussions; we thank Xue-Wu Zhang, Ai-Ping Mao, Cao-Qi Lei, and Yu Zhang for critically reading the manuscript; and

we thank Yan-Yi Wang (Wuhan Institute of Virology, Chinese Academy of Sciences), Zhi-Gao Bu and Xi-Jun Wang (Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences)

for reagents. This work was supported by the National Natural Science Foundation of China (31600696, 81470727, and 81501750), the National Key Research and Development Program

(2016YFC1000505), the Fundamental Research Funds for the Central Universities (413000006), and the Open Research Fund Program of Hubei-MOST KLOS & KLOBME (201501). AUTHOR INFORMATION

AUTHORS AND AFFILIATIONS * The State Key Laboratory Breeding Base of Basic Science of Stomatology, Hubei Province and Key Laboratory of Oral Biomedicine, Ministry of Education (Hubei-MOST

KLOS & KLOBME), School and Hospital of Stomatology, Wuhan University, Wuhan, 430079, China Yue Qin, Sheng-Long Zhou, Wei Yin & Zhuan Bian * State Key Laboratory of Virology, College

of Life Sciences, Wuhan University, Wuhan, 430072, China Mi Li & Hong-Bing Shu * Medical Research Institute, Collaborative Innovation Center for Viral Immunology, School of Medicine,

Wuhan University, Wuhan, 430071, China Hong-Bing Shu Authors * Yue Qin View author publications You can also search for this author inPubMed Google Scholar * Mi Li View author publications

You can also search for this author inPubMed Google Scholar * Sheng-Long Zhou View author publications You can also search for this author inPubMed Google Scholar * Wei Yin View author

publications You can also search for this author inPubMed Google Scholar * Zhuan Bian View author publications You can also search for this author inPubMed Google Scholar * Hong-Bing Shu

View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Conceived and designed the experiments: Y.Q., M.L., H.B.S. Performed the experiments: Y.Q.,

M.L. Analyzed the data: Y.Q., S.L.Z., W.Y., Z.B., H.B.S. Wrote the paper: Y.Q. CORRESPONDING AUTHOR Correspondence to Yue Qin. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare

that they have no competing interests. ADDITIONAL INFORMATION ACCESSION NUMBER: UniProtKB/Swiss-Prot accession numbers (parentheses) are indicated for the proteins mentioned in text: SPI-2

(P15059), CrmA (P07385), TBK1 (Q9UHD2), IKKε (Q14164), MITA (Q86WV6), IRF3 (Q14653). PUBLISHER'S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published

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http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Qin, Y., Li, M., Zhou, SL. _et al._ SPI-2/CrmA inhibits IFN-β induction by

targeting TBK1/IKKε. _Sci Rep_ 7, 10495 (2017). https://doi.org/10.1038/s41598-017-11016-3 Download citation * Received: 15 May 2017 * Accepted: 17 August 2017 * Published: 05 September 2017

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