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FEBS Letters 588 (2014) 28–34

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An anti-interferon activity shared by paramyxovirus C : Inhibition of Toll-like 7/9-dependent alpha interferon induction ⇑ Mayu Yamaguchi a, Yoshinori Kitagawa a, Min Zhou a, Masae Itoh b, Bin Gotoh a,

a Division of Microbiology and Infectious Diseases, Department of Pathology, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Shiga 520-2192, Japan b Department of Microbiology, Faculty of Bio-Science, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga 526-0829, Japan

article info abstract

Article history: Paramyxovirus C targets the host interferon (IFN) system for immune evasion. To Received 28 September 2013 identify its unknown anti-IFN activity, we examined the effect of Sendai virus C protein on Revised 31 October 2013 activation of the IFN-a promoter via various signaling pathways. This study uncovers a novel ability Accepted 6 November 2013 of C protein to block Toll-like receptor (TLR) 7- and TLR9-dependent IFN-a induction, which is spe- Available online 20 November 2013 cific to plasmacytoid dendritic cells. C protein interacts with a serine/threonine kinase IKKa and Edited by Hans-Dieter Klenk inhibits phosphorylation of IRF7. This anti-IFN activity of C protein is shared across genera of the Paramyxovirinae, and thus appears to play an important role in paramyxovirus immune evasion.

Keywords: Paramyxovirus Structured summary of protein interactions: C protein SeV C physically interacts with IKKalpha by anti tag coimmunoprecipitation (View interaction) TLR7 bPIV3 C physically interacts with IKKalpha by anti tag coimmunoprecipitation (1, 2) Interferon-a NiV C physically interacts with IKKalpha by anti tag coimmunoprecipitation (1, 2) pDC MeV C physically interacts with IKKalpha by anti tag coimmunoprecipitation (1, 2)

Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction protein is encoded by only members that belong to three genera; the Respirovirus, Morbillivirus, and Henipavirus. Virus strategies against host interferon (IFN) system are crucial IFN antagonism of C protein was primarily discovered from for efficient virus growth, and greatly affects virus pathogenesis. studies on a prototype paramyxovirus, Sendai virus (SeV) (mouse Such anti-IFN strategies have been found exactly in the subfamily PIV type 1) [3,4]. It was found that SeV C protein blocked the Paramyxovirinae including parainfluenza (PIVs), measles JAK-STAT pathway, which transmits signals from IFNs outside virus (MeV), and emerging deadly viruses such as Nipah virus the cell into promoters of the IFN-stimulated (ISGs) in (NiV) [1]. The Paramyxovirinae employ an accessory protein(s), V the nucleus [5,6]. Since ISGs include anti-viral proteins such as and/or C, expressed from viral P mRNAs, as an IFN antagonist double stranded (ds) RNA dependent protein kinase R (PKR), [2]. Whereas V protein is expressed by most paramyxoviruses, C the blockade of the JAK-STAT pathway results in prevention of establishment of the IFN-mediated anti-viral state. The anti-IFN activity uncovered next is to limit amounts of dsRNAs produced Abbreviations: IFN, interferon; PIVs, parainfluenza viruses; MeV, measles virus; during virus transcription and replication, thereby keeping NiV, Nipah virus; ORF, open reading frame; SeV, Sendai virus; ISGs, IFN-stimulated dsRNA dependent PKR inactive [7]. Since dsRNAs serve as path- genes; ds, double stranded; PKR, protein kinase R; PAMPs, pathogen-associated molecular patterns; IRF, interferon regulatory factor; RIG-I, retinoic acid-inducible ogen-associated molecular patterns (PAMPs) to stimulate IFN gene-I; MDA5, melanoma differentiation-associated gene 5; RLRs, RIG-I-like production, this ability also contributes to restriction of host receptors; TLR, Toll-like receptor; TRAF6, tumor necrosis factor receptor-associated IFN production. In contrast to these unequivocal anti-IFN 6; pDCs, plasmacytoid dendritic cells; IKKa,IjB kinase alpha; IRAK1, interleukin-1 activities, no conclusive evidence has been yet obtained for the receptor-associated kinase 1; IPS-1, IFN-b promoter stimulator-1; , TIR- ability of SeV C protein to block signaling pathways that activate domain-containing adapter-inducing interferon-b; N, nucleocapsid protein; P, phosphoprotein; bPIV3, bovine PIV type 3; MyD88, myeloid differentiation factor interferon regulatory factor 3 (IRF3) involved in IFN-b production 88; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody; SDS, [8–10]. Furthermore it remains to be determined whether C sodium dodecyl sulfate; pAb, polyclonal antibody protein blocks signaling pathways that activate IRF7 involved ⇑ Corresponding author. Fax: +81 77 548 2176. in IFN-a production. E-mail address: [email protected] (B. Gotoh).

0014-5793/$36.00 Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2013.11.015 M. Yamaguchi et al. / FEBS Letters 588 (2014) 28–34 29

Production of IFN-a/b is induced following the detection of 2.2. Reporter assay PAMPs by pattern recognition receptors [11]. Cytoplasmic RNA helicase retinoic acid-inducible gene-I (RIG-I) and melanoma HEK293T or Vero cells (1.5 Â 105/well) in 24-well plates were differentiation-associated gene 5 (MDA5) known as RIG-I-like transfected with IFN-a6-promoter-driven reporter plasmid (80 ng/ receptors (RLRs), and endosomal Toll-like receptor 3 (TLR3) well), pRL-TK (10 ng/well) (Promega) and various combinations of sense nucleic acids of RNA viruses as PAMPs and activate plasmids using polyethyleneimine (Polysciences) [19,20]. The IRF3 and IRF7 [12]. Both transcription factors induce IFN-b, amount of plasmids used was 25 ng/well for HA-RIG-IN, HA- but production of IFN-a except for IFN-a4 subtype is regulated MDA5, and IPS-1, 5 ng/well for FLAG-TRIF, V5-myeloid differentia- by IRF7 but not IRF3 in the mouse [13]. Primary activation of tion factor 88 (MyD88), V5-TRAF6, V5-IKKa, 15 ng/well for the RLR- or TLR3-dependent pathway results in induction of myc-IRF7, and 50 ng/well for plasmids expressing viral protein. IFN-b via activation of endogenous IRF3, because most cells ex- The total mass of transfected DNA was held constant by the addi- press IRF3 but do not express a detectable level of IRF7. Once tion of the appropriate amount of pCA7 empty plasmid. At 20 h IFN-b is produced, however, secreted IFN-b stimulates the post-transfection, cells were assayed for luciferase activity as pre- JAK-STAT pathway and activates transcription of ISGs including viously described [25]. IRF7. IRF7 is in turn activated through the RLR- or TLR3-depen- dent pathway, resulting in IFN-a production. The RLR- and 2.3. Measurement of IFN-a TLR3-dependent pathways leading to activation of IRF7 are basically the same as those activating IRF3, but a recent study Levels of human IFN-a were determined by using the human revealed a difference in a portion of molecules participating IFN alpha enzyme-linked immunosorbent assay (ELISA) kit (PBL in the . Whereas tumor necrosis factor Interferon Source, Piscataway, NJ). receptor-associated 6 (TRAF6) is required for RLR-dependent activation of IRF7, it is dispensable for RLR-dependent activa- 2.4. Immunoprecipitation tion of IRF3 [14]. On the other hand, plasmacytoid dendritic cells (pDCs), which constitutively express a high level of IRF7, HEK293T cells (5.0 Â 105 cells/well) in a 6-well plate were have a particular signaling pathway mediated by endosomal transfected with various combinations of plasmids (400 ng/well TLR7 and TLR9 to produce a burst amount of IFN-a [15].Recog- each), using polyethyleneimine. At 24 h post-transfection, cells nition of PAMPs by TLR7/9 results in activation of IRF7 via were lysed in 400 ll of the lysis buffer [50 mM Tris–HCl pH 7.4, kinases IjB kinase alpha (IKKa) and/or interleukin-1 receptor- 150 mM NaCl, 1% TritonX-100, and protease inhibitor cocktail associated kinase 1 (IRAK1) [16]. (Roche)]. Cell lysates were then incubated with anti-FLAG mouse In the present study, we examined effect of SeV C protein on monoclonal antibody (mAb) (1E6: Wako) or anti-V5 mouse mAb various signaling pathways that activate IRF7 involved in IFN-a (SV5-Pk1: Invitrogen) together with Protein G Sepharose 4 Fast induction. Our study uncovered a novel inhibitory activity against Flow (GE healthcare Bio-Science) at 4 °C for 2 h. After washing TLR7/9-dependent IFN-a induction. SeV C protein binds to a serine/ beads, proteins binding to the beads were subjected to immuno- threonine kinase IKKa and inhibits phosphorylation of IRF7. These blot analysis. features of C protein are shared across genera of the Paramyxovir- inae, suggesting that the novel activity plays an important role in 2.5. Analysis of phosphorylated IRF7 paramyxovirus immune evasion. HEK293T cells in a 24-well plate were transfected with various combinations of plasmids. The amounts and the ratios of plasmids 2. Materials and methods were the same as those used in the reporter assay. At 20 h post- transfection, cells were lysed in Laemmli sample buffer [50 mM 2.1. Cells and plasmids Tris–HCl pH 6.8, 2% sodium dodecyl sulfate (SDS), 0.1% bromophe- nol blue, 10% glycerol and 5% 2-mercaptoethanol] and then HEK293T and Vero cells were maintained in Dulbecco’s subjected to immunoblot analysis. modified Eagle’s medium containing 2 mM L-glutamine, penicillin (100 IU/ml), streptomycin (100 lg/ml), and 10% fetal bovine serum 2.6. Immunoblot analysis [6,17]. pEFneo-IFN-b promoter stimulator-1 (IPS-1), pEFneo-FLAG- TIR-domain-containing adapter-inducing interferon-b (TRIF), pEF- Samples were resolved by SDS-(10–12%) polyacrylamide gel neo-HA-MDA5, and pEFneo-HA-RIG-IN [N-terminal domain (aa electrophoresis, and then electroblotted onto PVDF membranes 1–229) of RIG-I] were a gift from T. Komatsu [8,9]. pCAGGs-puro/ (Immobilon-P; Millipore). Subsequent procedures were performed N-FLAG-NS3/4A and pCAGGs-SeV nucleocapsid protein (N) were as described previously [20]. Anti-FLAG mAb, anti-V5 mAb, anti- gifts from T. Abe [18] and T. Sakaguchi, respectively. IFN-a6 pro- phospho-IRF7 (Ser471/472) rabbit polyclonal antibody (pAb) moter-driven luciferase reporter plasmid was created previously (anti-pIRF7; Cell signaling technology), anti-SeV N rabbit pAb, or by insertion of the mouse IFN-a6 promoter into the NheI/BglII site anti-SeV C rabbit pAb, were used as the first antibody. of PGV-B2 reporter plasmid (Wako) [19,20]. The cDNA fragments encoding other cellular or viral proteins with N- or C-terminal myc, V5, or FLAG tag were created by using mouse intracellular 3. Results mRNAs, pCP721 containing the NiV phosphoprotein (P) gene (kindly provided by L.-F. Wang), and viral genomic from 3.1. A novel ability of SeV C protein to block TLR7/9-dependent IFN-a SeV Z strain, bovine PIV type 3 (bPIV3) 910 N strain and MeV induction Edmonston strain, as a template, and then subcloned into a multi-cloning site downstream of the cytomegalovirus enhancer We previously employed a reconstitution system in HEK293T chicken b-actin hybrid promoter [21] of pCA7 [22] or pCAGGs. C cells to examine effect of paramyxovirus V proteins on TLR7/9- mutant proteins with F170S, K151A/E153A/R154A, or R173A/ dependent pathway [19,20,26], since pDCs are less amenable to biochemical analysis. In this system, IRF7 in addition to a set of E175A/E176A substitutions (CF170S, Cm5, and Cm8) [23,24] were created as described previously [9]. upstream signaling molecules (MyD88, TRAF6, and IKKa) was 30 M. Yamaguchi et al. / FEBS Letters 588 (2014) 28–34 transfected into HEK293T cells together with IFN-a6-driven repor- inhibitory effect was observed for SeV C protein. This inhibition ter plasmid and an internal control pRL-TK. A similar reconstitu- was further confirmed by ELISAs for IFN-a secreted from transfec- tion system was applied to studies on RLR- and TLR3-dependent ted HEK293T cells (Fig. 1F). These results suggest that SeV C pro- IFN-a induction [8,9]. To stimulate RLR-dependent pathway, RIG- tein inhibits TLR7/9-dependent IFN-a induction. Stimulation of IN (constitutive active form of RIG-I), MDA5, and a mitochondrial TLR7/9-dependent pathway induces IFN-a, which in turn activates adaptor molecule IPS-1 were used (Fig. 1A–C). On the other hand, the IRF7 gene via the JAK-STAT pathway. Since the JAK-STAT path- a cytoplasmic adaptor molecule TRIF was utilized to stimulate way is blocked by SeV C protein, this blockade may affect total TLR3-dependent pathway (Fig. 1D). amount of IRF7, thereby lowering activation of the IFN-a promoter. Expression of RIG-IN, which activates endogenous IRF3, re- To exclude this possibility, Vero cells, which never produce IFNs, sulted in no activation of the IFN-a6 promoter, unless IRF7 was were used instead. Even in Vero cells, similar results were obtained co-expressed (Fig. 1A). Similar results were obtained for MDA5, (Fig. 2A). Moreover C mutant proteins, CF170S, Cm5, and Cm8, which IPS-1 and TRIF (Fig. 1B–D), indicating no response of the IFN-a6 lack the ability to block the JAK-STAT pathway [3,24], were found promoter to endogenous IRF3 in HEK293T cells. Expression of to have the inhibitory activity comparable to wild type C (Fig. 2B). IRF7 alone resulted in slight activation of the IFN-a6 promoter. These results demonstrate that the novel inhibitory activity is Whereas co-expression of RIG-IN, MDA5, IPS-1, and TRIF strikingly independent of the ability to block the JAK-STAT pathway. enhanced the IFN-a6 promoter activation, this enhancement ex- cept for a case of TRIF was suppressed by HCV NS3/4A, which 3.2. SeV C protein binds to IKKa and inhibits phosphorylation of IRF7 cleaves and inactivates IPS-1 (Fig. 1A–D) [27,28]. Previous studies demonstrated that paramyxovirus V protein targeted MDA5 for To identify a molecular target of SeV C protein, co-immunopre- inhibition of MDA5-dependent IFN-b induction [29]. As expected, cipitation experiments were performed. V5-tagged MyD88, TRAF6, MDA5-mediated IFN-a6 promoter activation was suppressed by IKKa, or IRF7 was transfected into HEK293T cells together with SeV V protein (Fig. 1B). On the other hand, no suppression of FLAG-tagged SeV C, and transfected cells were subjected to RIG-IN-, MDA5-, IPS-1-, and TRIF-induced IFN-a activation was ob- immunoprecipitation. As shown in Fig. 3A, FLAG-tagged C was served for SeV C protein as well as SeV N and P proteins (Fig. 1A– co-immunoprecipitated with anti-V5 antibody in cells transfected D), consistent with previous observations [8]. with V5-tagged IKKa, but not V5-tagged MyD88, TRAF6 and IRF7. We next examined effect on TLR7/9-dependent IFN-a induc- Conversely, not only V5-tagged IKKa (Fig. 3B) but also endogenous tion. A previous study uncovered an anti-IFN activity of SeV P IKKa (Fig. 3C) was co-immunoprecipitated with anti-FLAG anti- and V proteins against TLR7/9-dependent IFN-a induction [19]. body. These results suggest that IKKa is a candidate target of SeV Indeed expression of the P and V proteins significantly inhibited C protein. IFN-a6 promoter activation induced by transfection with MyD88, Since IKKa is involved in phosphorylation of IRF7 [30],we TRAF6, and IKKa together with IRF7 (Fig. 1E). Comparable examined effect of SeV C protein on phosphorylation of IRF7. An

Fig. 1. Effect of SeV C protein on IFN-a6 promoter activation via RLR-, TLR3-, or TLR7/9-dependent pathway. HEK293T cells were transfected with indicated plasmids together with (A–E) or without (F) IFN-a6-promoter-driven reporter plasmid and pRL-TK. At 20 h post-transfection, relative luciferase activity of cell lysates (A–E) or the level of IFN-a of the culture media (F) was determined. M. Yamaguchi et al. / FEBS Letters 588 (2014) 28–34 31

Fig. 2. Effect of SeV C or mutant C proteins on IFN-a6 promoter activation via TLR7/9-dependent pathway in Vero or HEK293T cells. Vero (A) or HEK293T (B) cells were transfected with indicated plasmids together with IFN-a6-promoter-driven reporter plasmid and pRL-TK. At 20 h post-transfection, relative luciferase activity was determined.

Fig. 3. Interaction of SeV C protein with IKKa. HEK293T cells were transfected with indicated plasmids. At 24 h post-transfection, cells were lysed in the lysis buffer. N- terminal V5-tagged (A) or FLAG-tagged proteins (B and C) were immunoprecipitated (IP), and then subjected to immunoblot analysis (IB). An asterisk indicates position of the antibody heavy chain.

anti-phospho-IRF7 antibody was used for detection of phosphory- Y2 with IKKa (Fig. 5A and B), indicating that the N-terminal region lated form of IRF7. IRF7 was present predominantly in an of C is not required for binding to IKKa. Nevertheless, Y1 and Y2 unphosphorylated form in cells transfected with IRF7 alone showed less ability to inhibit TLR7/9-dependent IFN-a induction, (Fig. 4). Co-expression of MyD88, TRAF6 and IKKa enhanced the le- compared with full size C (Fig. 5C). In accordance with magnitude vel of phosphorylated IRF7, whereas this enhancement was strik- of their inhibitory activity, IRF7 phosphorylation was less inhibited ingly suppressed by co-expression of C, suggesting that SeV C (Fig. 5D). These results indicates that the N-terminal region of C is protein inhibits IKKa-mediated phosphorylation of IRF7. Elevation not required for binding to IKKa, but partly involved in the ability of the IRF7 expression level was reproducibly observed in cells co- to block TLR7/9-dependent IFN-a induction. transfected with the upstream signaling molecules regardless of the presence or absence of viral protein, although the reason is 3.4. The novel activity of C protein is shared across genera unknown. The subfamily Paramyxovirinae consists of seven distinct gen- 3.3. Y1 and Y2 bind to IKKa but exhibit less ability to inhibit TLR7/9- era, but only members of the Respirovirus, Morbillivirus, and Heni- dependent IFN-a induction pavirus genera have acquired the C open reading frame (ORF) in the P gene during evolution. To determine whether the novel Not only full size C (aa 1–204) but also Y1 (aa 23–204) and Y2 inhibitory activity is shared across genera, we examined effect of (aa 29–204), N-terminally deleted forms of C, are expressed in SeV- the C proteins of bPIV3 of the same Respirovirus, MeV of the Morbil- infected cells [31]. Co-immunoprecipitation experiments using livirus, and NiV of the Henipavirus. As shown in Fig. 6A, not only cells transfected with Y1 or Y2 revealed the interaction of Y1 and bPIV3 C but also MeV and NiV C exhibited a similar inhibitory 32 M. Yamaguchi et al. / FEBS Letters 588 (2014) 28–34

4. Discussion

Paramyxovirus C proteins are expressed from mRNAs of the P gene through the use of an alternative initiation codon, with the C ORF being in the +1 reading frame relative to the P ORF [1]. Although their coding strategy is shared across genera, amino acid sequences of C protein are extremely diverse and there is at most 20% amino acid sequence identity between C proteins of any two genera [31]. It is thus unclear whether C protein is evolved from a common ancestral origin across genera. The present study uncov- ered a novel inhibitory activity of SeV C protein against TLR7/9- dependent IFN-a induction, and further revealed that the novel anti-IFN activity of C protein was shared across genera. In addition, all the C proteins belonging to a separate genus interacted with IKKa and inhibited phosphorylation of IRF7, suggesting that they share a common underlying mechanism. These findings suggest that there is a common ancestral C origin of the Respirovirus, Mor- billivirus, and Henipavirus, and the novel anti-IFN activity is con- served during evolution because of its important role in virus Fig. 4. Effect of SeV C protein on phosphorylation of IRF7 via TLR7/9-dependent immune evasion. Functional kinship of the C proteins between pathway. HEK293T cells were transfected with indicated plasmids. At 20 h post- transfection, cells were lysed in Laemmli sample buffer and then subjected to genera was also observed for another anti-IFN ability to limit immunoblot analysis (IB) with specific antibodies including anti-phospho-IRF7 dsRNA production [32,33]. In contrast, the ability to block the antibody. JAK-STAT pathway may be genus-specific [34–38], suggesting that it might have been independently acquired after the common ancestral C origin split into three genera. activity against TLR7/9-dependent IFN-a induction. Co-immuno- The present study does not provide sufficient evidence for a precipitation experiments and immunoblot analysis showed that molecular target of C protein. It is possible that signaling molecules all the C proteins were capable of binding to IKKa (Fig. 6B and other than IKKa is involved in the underlying mechanism. To ob- C), and inhibited phosphorylation of IRF7 (Fig. 6D). These results tain convincing evidence, we will need to create several C mutant suggest that the novel anti-IFN activity and its underlying mecha- proteins that lack the binding capacity for the target molecule. nism are conserved during evolution. More important question is why particular paramyxoviruses have

Fig. 5. Effect of Y1 and Y2 on IFN-a6 promoter activation via TLR7/9-dependent pathway. HEK293T cells were transfected with indicated plasmids together with (C) or without (A, B and D) IFN-a6-promoter-driven reporter plasmid and pRL-TK. (A and B) At 24 h post-transfection, cells were lysed in the lysis buffer. N-terminal FLAG-tagged proteins were immunoprecipitated (IP) and then subjected to immunoblot analysis (IB). (C) At 20 h post-transfection, relative luciferase activity of cell lysates was determined. (D) At 20 h post-transfection, cells were lysed in Laemmli sample buffer, and then subjected to IB with specific antibodies including anti-phospho-IRF7 antibody. An asterisk indicates position of the antibody light chain. M. Yamaguchi et al. / FEBS Letters 588 (2014) 28–34 33

Fig. 6. Effect of bPIV3, MeV, and NiV C proteins on IFN-a6 promoter activation via TLR7/9-dependent pathway. HEK293T cells were transfected with indicated plasmids together with (A) or without (B–D) IFN-a6-promoter-driven reporter plasmid and pRL-TK. (A) At 20 h post-transfection, relative luciferase activity of cell lysates was determined. (B and C) At 24 h post-transfection, cells were lysed in the lysis buffer. C-terminal FLAG- or V5-tagged proteins were immunoprecipitated (IP) and then subjected to immunoblot analysis (IB). (D) At 20 h post-transfection, cells were lysed in Laemmli sample buffer, and then subjected to IB with specific antibodies including anti- phospho-IRF7 antibody. An asterisk indicates position of the antibody light chain.

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