Severe Acute Respiratory Syndrome Coronavirus M Protein Inhibits Type I Interferon Production by Impeding the Formation of TRAF3·TANK·TBK1/IKKϵ Complex^*

Plasmids

pIFNβ-Luc and RIG-I expression vector (33) were kind gifts from Dr. Takashi Fujita (Kyoto University, Kyoto, Japan). TBK1 and IRF3 expression plasmids (34, 35) were provided by Dr. Genhong Cheng (University of California, Los Angeles, CA). IRF7 plasmid (36) was supplied by Dr. Luwen Zhang (University of Nebraska, Lincoln, NE). TRAF6 expression vector (37) was obtained from Dr. Tohru Ishitani (Kyushu University, Kyushu, Japan). TRAF3 cDNA (38) was kindly provided by Drs. Liusheng He and Peter Lipsky (NIAMS, National Institutes of Health, Bethesda, MD). pISRE-Luc was from Clontech. cDNA clones for IKKϵ, MDA5, VISA, and TANK were obtained from imaGenes GmbH (Berlin, Germany). TLR3 plasmid used in the construction of HEK293/TLR3 stable cell line has been described (39). pLTR-Luc reporter plasmid driven by the long terminal repeats of human T-cell leukemia virus type 1 and Tax expression vector pIEX have been described (40).

Viruses

The GZ50 strain of SARS coronavirus was propagated in Vero cells in a Biosafety Level 3 laboratory as described (31, 32). Sendai virus was obtained from American Type Culture Collection. HEK293/ACE2 cells infected with 5 multiplicity of infection of SARS coronavirus or Sendai virus were harvested to 1× SDS gel loading buffer (50 mm Tris-Cl, pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, and 0.02% bromphenol blue) for protein analysis or to cell lysis buffer (50 mm Tris·Cl, pH 8.0, 140 mm NaCl, 1.5 mm MgCl₂, 0.5% Nonidet P-40, and 1 mm dithiothreitol) for RNA analysis.

Antibodies

Anti-FLAG and anti-α-tubulin antibodies were purchased from Sigma. Anti-V5 was from Invitrogen. Anti-IFR3, anti-TRAF3, anti-IKKϵ, and anti-Myc were from Santa Cruz. Anti-phospho-IRF3 was from Cell Signaling. Anti-GM130 was from BD Transduction Laboratories. Anti-N was from Imgenex.

Protein Analysis and Reporter Assay

Immunoprecipitation, Western blotting, and dual luciferase assay were carried out as described (41, 42). Relative luciferase activity in arbitrary units was calculated by normalizing firefly luciferase activity to Renilla luciferase activity.

Confocal Microscopy

Confocal immunofluorescence microscopy was performed as described (43, 44). Cells were fixed in 1:1 (v:v) acetone/methanol for 10 min and imaged using LSM510 system (Carl Zeiss).

In Vitro Kinase Assay

TBK1/IKKϵ kinase activity was assayed in vitro as described (42, 43). Briefly, TBK1/IKKϵ-containing protein complex was immunoprecipitated from transfected HEK293 cells. The precipitates were suspended in kinase buffer (25 mm Tris-Cl, pH 7.5, 5 mm β-glycerophosphate, 2 mm dithiothreitol, 0.1 mm Na₃VO₄, 10 mm MgCl₂) and incubated with [γ-³²P]ATP and recombinant IκBα (Santa Cruz) or IRF3 (Abnova). Samples were analyzed by SDS-PAGE and autoradiography.

Inhibition of Type I Interferon Production by SARS Coronavirus M Protein

SARS coronavirus N protein has been shown to antagonize the production of type I interferons (7). In our study of the gene regulatory function of N protein (32), we included M protein as a control and found serendipitously that M protein was a more potent inhibitor of interferon β promoter when compared with N protein (Fig. 1). Considered together with the ability of M protein to interact with host factors and to modulate expression of cellular genes (25–29), we set out to characterize M protein-mediated inhibition of type I interferon production in detail.

As a first step, we expressed M protein transiently in HEK293 and HeLa cells. The M protein-expressing cells did not show any growth defect, and no signs of apoptosis were observed (Fig. 1A). M protein was abundantly found in transfected cells (Fig. 1B). Consistent with a predominant Golgi localization shown in previous reports (21, 23), M protein was found to co-localize substantially with GM130 (Fig. 1A), a marker of the Golgi apparatus (45). Expression of M protein did not suppress transcription driven by the long terminal repeats of human T-cell leukemia virus type 1, which was stimulated by viral transactivator Tax (Fig. 1C). Hence, M protein is unlikely a general inhibitor of RNA polymerase II-dependent transcription. Next, we assessed the influence of M protein expression on the induction of interferon β gene transcription using pIFNβ-Luc, a luciferase reporter construct driven by interferon β promoter (33). Notably, expression of M protein led to significant and dose-dependent suppression of dsRNA-induced activation of interferon β promoter (Fig. 1D). This suppression by M protein was more pronounced than the effect of two previously reported viral antagonists of interferon production, SARS coronavirus N protein and influenza A virus NS1 protein (7, 39). In contrast, E or ORF7a protein of SARS coronavirus did not modulate the activation of interferon β promoter by dsRNA (Fig. 1D). When we repeated the luciferase assay with a reporter construct driven by canonical ISRE enhancer elements, which were bound to IRF3 and IRF7 (36, 46, 47), the same pattern of inhibitory activity mediated by M, N, and influenza A virus NS1 proteins was observed (Fig. 1E). These results suggested that SARS coronavirus M protein is a potent inhibitor of the activity of IRF-responsive elements in the interferon β promoter.

SARS Coronavirus M Protein Inhibits Interferon-inducing Activity of TBK1/IKKϵ

We next addressed at which step of the signaling pathway M protein might inhibit dsRNA-induced production of type I interferons. Various transducer proteins that are previously known to stimulate interferon production (13–17) were used to activate ISRE-dependent expression of luciferase reporter (Fig. 2). These include cytoplasmic dsRNA sensors RIG-I (Fig. 2A) and MDA5 (Fig. 2B), adaptor protein VISA/IPS1/MAVS/Cardif (Fig. 2E), and protein kinases TBK1 (Fig. 2C) and IKKϵ (Fig. 2D) as well as ISRE binding transcription factors IRF3 (Fig. 2F) and IRF7 (Fig. 2G). All of these transducer proteins tested strongly activated the enhancer activity of ISRE (Fig. 2).

Interestingly, expression of M protein suppressed ISRE-driven transcriptional activity induced by RIG-I, MDA5, VISA, TBK1, and IKKϵ but not by IRF3 or IRF7 (Fig. 2). In these assays, SARS coronavirus N, E, or ORF7a protein was unable to counteract any ISRE activator. Thus, although both N and M proteins inhibited interferon production (7), they plausibly acted through different mechanisms. Consistent with previous findings (48–50), influenza A virus NS1 protein inhibited the stimulatory activity of RIG-I, MDA5, TBK1, and IKKϵ (Fig. 2, A ∼ D). However, unlike SARS coronavirus M protein, NS1 had no influence on the activity of VISA (Fig. 2E). These results suggested that M protein suppressed interferon production in a manner distinct from that of NS1.

Association of SARS Coronavirus M Protein with TBK1/IKKϵ

M protein inhibited TBK1/IKKϵ-dependent activation of interferon production but was unable to counteract the action of transcription factors IRF3 and IRF7 (Fig. 2). This pattern of inhibitory activity suggested that M protein might function at a step before nuclear activation of IRF3 and IRF7. In light of this, we next investigated whether M protein physically interacted with the cytoplasmic transducer proteins. We overexpressed M and individual transducer proteins RIG-I, MDA5, TBK1, IKKϵ, TANK, TRAF3, and TRAF6 in HEK293 cells and performed reciprocal co-immunoprecipitation to assess their interactions (Fig. 3). Although both M protein and the transducer proteins were expressed abundantly in HEK293 cells (Fig. 3A), M protein was found to co-precipitate with RIG-I, TBK1, IKKϵ, and TRAF3 but not with MDA5, TANK, or TRAF6 (Fig. 3, B and C). Notably, precipitations with two different antibodies yielded similar results (Fig. 3B compared with Fig. 3C), suggesting that M protein consistently formed a complex with RIG-I, TBK1, IKKϵ, and TRAF3. In keeping with this notion, M protein was found to co-localize substantially with IKKϵ and TRAF3 in HeLa cells to discrete cytoplasmic subdomains that were compatible with the Golgi complex (Fig. 4). Particularly, IKKϵ was sporadically found in multiple cytoplasmic dots in the absence of M protein (Fig. 4, panels 1–3). In contrast, IKKϵ was more concentrated to the M protein-containing region in cells expressing M protein (Fig. 4, panels 7–9).

SARS Coronavirus M Protein Impedes TRAF3·TANK·TBK1/IKKϵ Complex Formation and IRF3 Phosphorylation

The association of M protein with TBK1/IKKϵ (Fig. 3) raised two possibilities that might explain the inhibition of interferon production. First, M protein might inhibit catalytic activity of TBK1/IKKϵ to phosphorylate IRF3/IRF7. Second, M protein might interfere with the formation of TRAF3·TANK·TBK1/IKKϵ complex required for phosphorylation of IRF3/IRF7 in the cytoplasm. To test these possibilities, we assessed the impact of M protein on the kinase activity of TBK1/IKKϵ both in vitro and in vivo. We expressed M protein and TBK1/IKKϵ in HEK293 cells (Fig. 5A) and immunoprecipitated TBK1/IKKϵ-containing protein complex (Fig. 5B). The precipitates were then incubated with recombinant IκBα or recombinant IRF3. Phosphorylated IκBα and IRF3 were analyzed by autoradiography (Fig. 5, C and D). Notably, the TBK1/IKKϵ complex purified from M protein-expressing cells contained M protein (Fig. 3C). However, the TBK1/IKKϵ complexes containing or not containing M protein showed similar activity to phosphorylate IκBα or IRF3 (Fig. 5, C and D, lane 3 compared with lane 2 and lane 6 compared with lane 5). Thus, the expression of M protein did not modulate the catalytic activity of TBK1/IKKϵ kinase.

On the other hand, phosphorylated IRF3 was detected in cultured HEK293 cells expressing TBK1/IKKϵ alone but not in cells simultaneously expressing TBK1/IKKϵ and M protein, whereas comparable amounts of IRF3 were found in all different groups of cells (Fig. 6, lane 2 compared with lane 3 and lane 4 compared with lane 5). These results were consistent with the notion that M protein inhibits in vivo phosphorylation of IRF3 by TBK1/IKKϵ.

To verify the relevance of M protein-mediated inhibitory effects to SARS coronavirus infection, we also examined phosphorylation of IRF3 and production of interferon β transcript in cells infected with SARS coronavirus. Both phosphorylated IRF3 and elevated expression of interferon β mRNA were detected in cells infected with Sendai virus (Fig. 7, A and C). In contrast, although IRF3 was abundantly found in cells infected with SARS coronavirus, neither phosphorylation of IRF3 nor induced expression of interferon β transcript was observed. These results were consistent with previous findings (4–12).

The formation of TRAF3·TANK·TBK1/IKKϵ complex in the cytoplasm is an essential step in the activation of IRF3/IRF7 (15–17, 34). M protein physically interacted with TBK1/IKKϵ and TRAF3 (Fig. 3). In addition, they also colocalized to the cytoplasmic subdomains that were likely the Golgi apparatus (Fig. 4). Plausibly, M protein might impede the formation of a functional TRAF3·TANK·TBK1/IKKϵ complex by masking the interaction domains or retaining some of the subunits in the Golgi complex. Indeed, when we co-expressed TBK1, TRAF3, and M in HEK293 cells, the interaction between TBK1 and TRAF3, which was evident in cells expressing TBK1 and TRAF3, was not detected (Fig. 8A, lane 2 compared with lane 1). Likewise, the interaction between IKKϵ and TRAF3 was not observed in M protein-expressing cells (Fig. 8A, lane 4 compared with lane 3). Furthermore, the formation of TRAF3-TANK complex was also blocked in the presence of M protein (Fig. 8A, lane 6 compared with lane 5). Hence, M protein impeded the formation of a functional TRAF3·TANK·TBK1/IKKϵ complex. Importantly, the formation of this complex was also impaired in SARS coronavirus-infected cells (Fig. 8B). As such, the interaction of TRAF3 with TBK1, IKKϵ, and TANK was impeded in infected cells where N protein was expressed (Fig. 8B, lanes 2, 4, and 6 compared with lanes 1, 3, and 5, respectively). Although the roles of other viral proteins could not be excluded, the same pattern for the impeded formation of TRAF3·TANK·TBK1/IKKϵ complex in cells expressing M protein alone and cells infected with SARS coronavirus supported the notion that M protein contributes significantly to the inhibition of type I interferon production during viral infection.