SPI-2/CrmA inhibits IFN-β induction by targeting TBK1/IKKε

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.

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 cancer 1 . CPXV infects a wide range of host species and causes zoonosis, and the outbreaks of CPXV in recent years have caused a public health crisis 2 .
OPXVs have evolved various mechanisms to evade and suppress host antiviral responses 3 . They have large genomes with a variety of immunomodulatory genes and thus display a wide range of immune evasion strategies 4,5 . Type I interferons (IFNs) are critical for both innate and adaptive immune responses against viral infection 6 . 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 events 7 . 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), respectively [9][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 VISA [9][10][11][12] . MITA or VISA is phosphorylated by the kinases and subsequently recruits IRF3 14 . TBK1 and IKKε then phosphorylate IRF3, leading to the dimerization and nuclear translocation of IRF3 14 . 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 released 6 . Activated IRF3 and NF-κB respectively bind to interferon-stimulated response element (ISRE) and the κB site, leading to the transcriptional induction of IFN-β 7 .
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. Tan (TT), a widely-used smallpox vaccine strain in China, is less virulent than strain WR 21,22 . 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 induction 23,24 . 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.
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 studies 25,26 . 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 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 functions [15][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 evasion 3 . VACV encodes several immunomodulatory proteins targeting type I IFNs induction signaling, including E3 27, 28 , A46 29, 30 , C16 24 , K7 31 , C6 32 , and N2 33 . 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 DDX3 31 , and C6 interacts with TANK, NAP1, and SINTBAD, the scaffold proteins that associate with TBK1 and IKKε 32 . 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.
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.

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.