Select Paramyxoviral V Proteins Inhibit IRF3 Activation by Acting as Alternative Substrates for Inhibitor of κB Kinase ε (IKKe)/TBK1

Abstract

V accessory proteins from Paramyxoviruses are important in viral evasion of the innate immune response. Here, using a cell survival assay that identifies both inhibitors and activators of interferon regulatory factor 3 (IRF3)-mediated gene induction, we identified select paramyxoviral V proteins that inhibited double-stranded RNA-mediated signaling; these are encoded by mumps virus (MuV), human parainfluenza virus 2 (hPIV2), and parainfluenza virus 5 (PIV5), all members of the genus Rubulavirus. We showed that interaction between V and the IRF3/7 kinases, TRAF family member-associated NFκB activator (TANK)-binding kinase 1 (TBK1)/inhibitor of κB kinase ε (IKKe), was essential for this inhibition. Indeed, V proteins were phosphorylated directly by TBK1/IKKe, and this, intriguingly, resulted in lowering of the cellular level of V. Thus, it appears that V mimics IRF3 in both its phosphorylation by TBK1/IKKe and its subsequent degradation. Finally, a PIV5 mutant encoding a V protein that could not inhibit IKKe was much more susceptible to the antiviral effects of double-stranded RNA than the wild-type virus. Because many innate immune response signaling pathways, including those initiated by TLR3, TLR4, RIG-I, MDA5, and DNA-dependent activator of IRFs (DAI), use TBK1/IKKe as the terminal kinases to activate IRFs, rubulaviral V proteins have the potential to inhibit all of them.

Innate immunity is stimulated by viruses in part via RNA. dsRNA2 specifically is present in several forms: viral genomes, single-stranded RNA virus replication intermediates, DNA virus symmetric transcription products, defective viral particles, and debris from lysed cells (1). Although extracellular dsRNA is sensed by Toll-like receptor 3 (TLR3), intracellular dsRNA is detected in part by the RNA helicases retinoic acid-inducible gene 1 (RIG-I) and melanoma differentiation-associated gene 5 (Mda-5). These receptors signal through Toll-IL-1R (TIR) domain containing adaptor-inducing IFNβ (TRIF) and IFNβ promoter stimulator 1 (IPS1), respectively, to activate the kinases TANK-binding kinase 1 (TBK1) and inhibitor of κB kinase ε (IKKe). They, in turn, phosphorylate IFN regulatory factor 3 (IRF3), promoting its nuclear translocation and subsequent IFN-stimulated regulatory element-mediated transcription of IFN-stimulated genes (ISG), such as ISG56, as well as IFN and other cytokines. IFN then, through Janus kinase (JAK)/STAT activation of IRF9, modulates microRNAs in addition to up-regulating itself and more ISGs to heighten the antiviral state and also to initiate the adaptive immune response by promoting dendritic cell maturation, memory T cell proliferation, and B cell differentiation (2–4).

To control this immune response, pathogens and hosts have developed methods of down-regulation and evasion at a variety of different points (5–7). At the level of sensing infection, NS1 from influenza virus binds to and sequesters dsRNA and also interacts with RIG-I (5, 6, 8). At the level of signal transduction, the NS3/4A protease from hepatitis C virus cleaves TRIF and IPS1 (9–11), and vaccinia virus A46R, which contains a TIR domain, inhibits TRIF-dependent activation of IRF3 (12, 13). At the level of IRF-mediated transcription, P proteins from Ebola (5, 6) and borna disease (14) viruses and human papilloma virus E16 (15) interfere with IRF3 activation. For a more global effect, M protein from vesicular stomatitis virus inhibits host machinery to prevent gene transcription (5, 6). Finally, at the level of IFN and cytokines binding to their receptors, human cytomegalovirus encodes a decoy for the chemokine RANTES (regulated on activation normal T cell expressed and secreted) to prevent further signaling (16).

The family Paramyxoviridae encompasses two subfamilies: Paramyxovirinae and Pneumovirinae. Viruses representing different genuses within the paramyxoviral subfamily are important pathogens for humans such as measles virus (MeV), a Morbillivirus, and mumps virus (MuV) and human parainfluenza virus 2 (hPIV2), both Rubulaviruses. For animals, examples include Hendra virus (HeV), a Henipavirus that infects flying foxes, Sendai virus, a Respirovirus that infects rodents, and parainfluenza virus 5 (PIV5), a Rubulavirus that infects dogs (17). Paramyxoviral V accessory proteins have been shown to be important in viral evasion of innate immunity. At the level of IFN signaling, V proteins act as ubiquitin-protein isopeptide ligases (E3) that target STATs for degradation or sequester them, preventing their nuclear translocation and subsequent transcriptional functions (18). At the level of sensing infection, V proteins bind to mda-5 and also to dsRNA itself to inhibit intracellular dsRNA signaling and protein kinase R activation, respectively (19–21). These functions highlight the importance of immune signaling in the viral life cycle. Indeed, VDC PIV5, a mutant PIV5 virus lacking the unique C terminus of V, is no longer able to inhibit IRF3 activation and causes a greater cytopathic effect in a variety of different cells (22, 23). Taken together, these lines of evidence indicate that V may inhibit dsRNA activation of IRF3 via TLR3.

Here we show that V proteins from hPIV2 (V_H), MuV (V_M), and PIV5 (V_P), but not HeV and MeV, inhibit TLR3 signaling. Analysis of the underlying mechanism revealed that the inhibitory V proteins interacted with the signaling kinases TBK1/IKKe and served as their substrates, thus preventing IRF3 phosphorylation. Our results indicated that the above interaction led to modifications of both partners and their degradation. Therefore, the V proteins from Rubulaviruses and the IRF3-activating kinases TBK1/IKKe are connected by a negative feedback loop.

EXPERIMENTAL PROCEDURES

Viruses, Cells, and Reagents—W3A strain WT and VDC PIV5 virus, gifts of B. He, were propagated in Vero (22, 24). HT1080-derived 2fTGH wild-type V protein-expressing cells, 293 and TLR3 293 have been described (25, 26). To make HT1080-derived 2fTGH cell lines permanently expressing mutant V_M, 2fTGH cells were co-transfected with respective pEF-V protein expression vectors and a puromycin-expressing vector for selection. To make TLR3 293 561-TK cells, TLR3 293 was transfected with 561-TK plasmid. This contains the ISG56 promoter (–654–+3) (27) driving the herpesvirus thymidine kinase (TK) (28), which was cloned via Sst1/SalI into pGL3B (Promega) to replace luciferase. Wild-type pEF-V protein expression vectors have been described (29, 30). MuV V mutants were constructed with QuikChange® II XL (Stratagene). PCR products from primers targeting residues were cloned with DpnI. PIV5 VDC mutant was constructed with the Expand Hi Fidelity PCR system (Roche Applied Science) using wild-type pEF-V protein as template and BamHI/BglII and NotI for cloning. pEF-Trif-FLAG (31) (gift of K. Fitzgerald), wild-type, and kinase inactive (K38A) IKKe (pCDNA3.1-Myc) (32) (gifts of U. Siebenlist), FLAG-IRF3 and IRF3 5D (33) (gifts of J. Hiscott), and HA-ubiquitin (gift of S. Fuchs) (34) vectors have been described. All transfections were carried out with FuGENE 6 (Roche Applied Science). Ganciclovir (GCV) (Invivogen), leptomycin B (LMB) (Sigma), MG132 (Sigma) in dimethyl sulfoxide (DMSO) (Sigma), and dsRNA (Amersham Biosciences) (27) were used in treatments at 10 μg/ml, 10 ng/ml, 10 μm, and 100 μg/ml, respectively, unless otherwise specified.

Cell Survival Assay—TLR3 293 561-TK cells were transfected with pEF-V and treated with dsRNA and GCV. Cells were maintained under selection 4 days to assay survival and create pools expressing V proteins.

Quantitative PCR—HT1080 V protein-expressing cells were treated with dsRNA for 6 h. Total RNA was isolated with RNA-Bee (Tel-Test). cDNA was synthesized with SuperScript III (Invitrogen). The quantitative-PCR reaction amplified bp 6–354 of ISG56 and control RPL32 (ribosomal protein L32) with SYBR Green (Applied Biosystems). ISG56 primers are as follows: sense, 5′-TCT CAG AGG AGC CTG GCT AAG-3′, and antisense, 5′-GTC ACC AGA CTC CTC ACA TTT GC-3′. RPL32 primers are as follows: sense, 5′-GCC AGA TCT TAT GCC CCA AC-3′, and antisense, 5′-CGT GCA CAT GAG CTG CCT AC-3′.

Immunoblotting and Immunoprecipitation—Immunoblotting was performed with antibodies HA Y-11 (Santa Cruz Biotechnology), actin (Sigma), p56 (35), c-Myc 9E10 (Santa Cruz Biotechnology), P396 IRF3 (Cell Signaling), total IRF3 (gift of M. David) (36), histone H1 AE4 (Santa Cruz Biotechnology), STAT2 C-20 (Santa Cruz Biotechnology), FLAG M2 (Sigma), and V5, a polyclonal antibody raised against a full-length PIV5 V·GST fusion protein at Cocalico Biologicals Inc.

To assay V interaction with IKKe, TLR3 293 cells were transfected with relevant expression plasmids. Immunoprecipitations were performed as described previously (30) with agarose-conjugated HA F7 (Santa Cruz Biotechnology), FLAG M2 (Sigma), or protein A/G PLUS-agarose (Santa Cruz Biotechnology) with c-Myc 9E10 at 4 °C. To assay for in vivo V phosphorylation, the same procedure was used to isolate V. The resulting samples were then treated with 100 units of calf intestine phosphatase (USB Corp.) 37 °C 3 h where indicated.

IRF3 Activation Assays—To assess Ser-396 phosphorylation and IRF3 nuclear localization, HT1080 V_M cells were treated with dsRNA for 3 h before collection of whole cell or nuclear lysates, respectively, described previously (27). To look at localization by immunofluorescence, the same cells were plated on coverslips and treated with dsRNA for 1 h and then LMB for 2 h. Cells were stained for IRF3 and 4′,6-diamidino-2-phenylindole (27). To determine IRF3 transcriptional activity, the same cells were treated with dsRNA for 1 h and then with LMB for 6 h. Whole cell extracts were prepared for immunoblotting.

In Vitro Kinase Assay—To purify FLAG-IRF3 and V_M, the respective vectors were transfected for 24 h into TLR3 293 cells, and lysates were prepared as described (37). FLAG-tagged proteins were immunoprecipitated with FLAG M2 agarose (Sigma), eluted with FLAG peptide (Sigma), and concentrated with Microcon filter devices YM-3 (Millipore). Myc-TBK1 was purified similarly with c-Myc 9E10 and Trueblot immunoprecipitation beads (eBioscience). For in vitro kinase assays, purified FLAG-IRF3 or V_M were incubated with GST·IKKe (Cell Signaling) or Myc-TBK1 in [γ-³²P]ATP (37). Quantitation for autoradiograph was performed by GE Healthcare PhosphorImager and for immunoblotting by Odyssey (Licor).

V_M degradation—To follow V_M degradation by IKKe, control empty or IKKe-expressing vectors were co-transfected with V-expressing vectors into TLR3 293 cells for 8 h, media were changed, and whole cell lysates were prepared 48 h later. To follow V_M degradation by dsRNA, the same cells were simultaneously transfected with pEF-V and dsRNA-treated with either MG132 or control DMSO for 16 h.

TLR3 Antiviral Activity and PIV5 Plaque Assay—293 or TLR3 293 cells were treated with dsRNA for 16 h. The cells were then infected with WT or VDC PIV5 at a multiplicity of infection of 0.1. Supernatants were collected 6 days after infection. Virus yields were determined by plaque assay on Vero (24).

RESULTS

Some, but Not All, Paramyxoviral V Proteins Inhibit TLR3 Signaling—We used a cell survival assay to examine the potentials of V proteins from different paramyxoviral subfamilies to inhibit TLR3 signaling. This assay uses a TLR3-expressing 293 cell line (TLR3 293) in which a selection gene has been introduced; this gene is driven by the promoter of ISG56 (27) and encodes the herpesvirus TK protein (28). Any signaling that activates transcription factors containing IRF proteins, such as TLR3 signaling or type I IFN signaling, induces TK production and causes cell death in the presence of GCV. If an inhibitor of signaling blocks TK production in a cell, that cell survives and proliferates even in the presence of GCV. Using this assay, we determined that V proteins from the Rubulaviruses hPIV2 (V_H), MuV (V_M), and PIV5 (V_P) inhibited TLR3 signaling (Fig. 1A, top panel, lanes 4–6). In contrast, V proteins from HeV and MeV were ineffective (Fig. 1A, top panel, lanes 2 and 3), although the different V proteins were expressed at similar levels in the transfected cells (Fig. 1B). Expression of the V proteins themselves, without any dsRNA treatment, did not affect cell survival at all (Fig. 1A, bottom panel). The above observations suggested that rubulaviral V proteins could block induction of cellular dsRNA-inducible genes as well, a conclusion that was confirmed by measuring the levels of ISG56 mRNA, using a quantitative reverse transcription-PCR assay in HT1080 cells permanently expressing different V proteins. The mRNA was induced strongly upon dsRNA treatment of cells (Fig. 1C, lane 5) and the induction was almost completely blocked by V_H, V_M, and V_P (Fig. 1C, lanes 6–8). Similar inhibitions of the induction of p56 protein were observed (Fig. 1D).

FIGURE 1.

Select paramyxoviral V proteins inhibit TLR3-mediated gene induction. A, paramyxoviral V proteins were transiently expressed in TLR3 293 561-TK cells, which were then treated with dsRNA and GCV for 4 days (top panel) or GCV alone (bottom panel). Estimated cell survival is shown. Error bars represent standard error from two experiments. Lane 1, no V protein; lane 2, HeV protein; lane 3, MeV protein; lane 4, hPIV2 V protein/V_H (H); lane 5, MuV V protein/V_M (M); lane 6, PIV5 V protein/V_P (P). B, HA immunoblotting of extracts from the above cells was performed to determine the levels of HA-tagged V proteins. Lanes are as described above. Approximate molecular weights are marked. C, P56 mRNA levels were measured by quantitative reverse transcription-PCR in untreated (lanes 1–4) or dsRNA-treated (lanes 5–8) HT1080 cells stably expressing V proteins. Error bars represent standard error from triplicate samples. D, P56 protein levels were measured by immunoblotting in untreated (lanes 1–4) or dsRNA-treated (lanes 5–8) cells described in C. Immunoblotting for actin and FLAG-V served as controls.

V Proteins Block IRF3 Activation—Once we established that the three V proteins blocked TLR3 signaling, we investigated the underlying mechanism. Exogenous expression of signaling proteins downstream of TLR3 is known to activate IRF3 and induce synthesis of p56, as shown in Fig. 2A. Expression of TRIF, IKKe, and TBK1 induced p56 (lane 1), and the induction was blocked by all three V proteins. In contrast, p56 induction from expression of a constitutively active IRF3 5D mutant was not blocked (Fig. 2A, lanes 2–4). These results indicated that the V proteins blocked a step in signaling downstream of TLR3, TRIF, and the IRF3 kinases and upstream of events following activation of IRF3. Inactive IRF3 shuttles in and out of the nucleus, whereas activated IRF3 translocates to the nucleus and induces transcription of target genes, such as ISG56 (38). IRF3 was not localized to the nucleus in cells expressing V_M (Fig. 2B, lane 4), V_H, and V_P (data not shown). By using LMB, a drug that blocks nuclear export of proteins (39), we examined whether V_M blocked nuclear import of IRF3 or enhanced its export. As expected, in untreated cells, IRF3 was in the cytoplasm of both control and V-expressing cells (Fig. 2C, row 1). dsRNA treatment alone caused nuclear accumulation of IRF3 only in the absence of V protein (Fig. 2C, row 2). In both untreated and dsRNA-treated cells, LMB caused similar nuclear accumulation of IRF3 in the absence and the presence of V protein (Fig. 2C, rows 3 and 4). These results suggested that either V_M inhibited nuclear import of only activated IRF3 or V_M enhanced export of activated IRF3 through exportin 1, which is blocked by LMB (39). To address these possibilities, we examined whether the IRF3 sequestered within the nucleus by LMB was transcriptionally active in the presence of dsRNA. Nuclear IRF3 in LMB-treated cells could not induce p56 in control cells unless they were dsRNA-treated as well (Fig. 2D, lanes 1 and 3). In dsRNA-treated V-expressing cells, although LMB treatment caused nuclear translocation of IRF3, p56 was not induced (Fig. 2D, lanes 2 and 4). These results indicated that V protein inhibited import of activated IRF3 to the nucleus and did not enhance its nuclear export. Furthermore, these results indicated that nuclear localization, although necessary, was insufficient for IRF3 to induce genes; additional activation was required by post-translational modifications of the protein. We then asked whether the observed block of IRF3 activation was due to a block in its phosphorylation, which is known to activate it. Indeed, phosphorylation of Ser-396, a hallmark of IRF3 activation, was inhibited in the presence of V_M (Fig. 2E, lane 4). These results indicated that the major block caused by V_M was at the level of IRF3 phosphorylation.

FIGURE 2.

V proteins from hPIV2, MuV, and PIV5 inhibit IRF3 activation. A, pools of TLR3 293 561-TK cells expressing V proteins were transfected with expression vectors for different components of the TLR3 pathway; the specific signaling protein expressed is shown on the left. Induced p56 levels were determined by immunoblotting. Actin served as a loading control. Lane 1, noV; lane 2, V_H (H); lane 3, V_M (M); lane 4, V_P (P). B, nuclear extracts from control cells (lanes 1 and 2) or cells stably expressing V_M (lanes 3 and 4), that were untreated (lanes 1 and 3) or treated with dsRNA (lanes 2 and 4) were immunoblotted for IRF3, with histone as loading control. C, control and V_M-expressing HT1080 cells were treated with dsRNA (rows 2 and 4) and LMB (rows 3 and 4). Subcellular location of IRF3 was determined by immunofluorescence. D, cell extracts from samples, as described for C, rows 3 and 4, were used for immunoblotting to detect p56, FLAG-V, and actin. E, IRF3 phosphorylation at serine 396 (P396) was detected by immunoblotting cell extracts with a phospho-IRF3-specific antiserum, with total IRF3 as a control. Whole cell extracts were prepared from control cells (lanes 1 and 3) or cells stably expressing V_M (lanes 2 and 4) that were untreated (lanes 1 and 2) or treated with dsRNA (lanes 3 and 4).

Interaction of V with IKKe Is Essential for Inhibiting TLR3 Signaling—To further analyze the mechanism of V-mediated blocking of TLR3 signaling, we examined possible interactions of the viral proteins with known components of the signaling pathways. V_H, V_M, and V_P did not interact with TLR3, TRIF, IRF3, Src, phosphatidylinositol 3-kinase, and IKK α, β, and γ as revealed by co-immunoprecipitation assays, although the same assay demonstrated their known interactions with STAT2 (Fig. 3, A and B). In contrast, all three viral proteins interacted with both IKKe (Fig. 3C) and TBK1 (data not shown). IKKe co-immunoprecipitated with V_H (lane 3), V_M (lane 5), and V_P (lane 7) (Fig. 3C, top panel). Note that the mobility of co-immunoprecipitated IKKe was slower than that of the protein in cell extracts. This was also the case for co-immunoprecipitated STAT2 (Fig. 3C, middle panel, lanes 3, 5, and 7). Since the V proteins have E3 ubiquitin ligase activity (18), the observed mobility differences could be due to V-mediated polyubiquitination of the co-immunoprecipitated proteins. Indeed, exogenously introduced ubiquitin was covalently bound to IKKe purified by immunoprecipitation, and this ubiquitination was V_M-dependent (Fig. 3D). The functional significance of the V-IKKe interaction was determined by testing mutant V_M. Among six mutant proteins tested (Fig. 3E), only the V_M mutant W174A/W178A/W188A (V_M-AAA) failed to bind to IKKe (lane 3, left top panel). This mutant protein could not block TLR3 signaling as revealed by the cell survival assay (data not shown) and in HT1080 cells permanently expressing mutant V_M-AAA (Fig. 3G, lane 8). In contrast, WT V_M and mutant V_C189A could inhibit signaling (Fig. 3G, lanes 4 and 6). Both bound to IKKe (Fig. 3E, lanes 7 and 8) and, as shown when electrophoresed longer, both shifted the mobility of co-immunoprecipitated IKKe, indicating its ubiquitination (Fig. 3F, lanes 2 and 4, compared with lanes 1 and 3). These results strongly indicated that the V proteins blocked TLR3 signaling by blocking the action of the signaling kinases.

FIGURE 3.

Interaction of V with IKKe is essential for inhibition of TLR3 signaling. A, TRIF and V proteins were transiently expressed in TLR3 293 cells. V proteins were immunoprecipitated (IP), and co-immunoprecipitated proteins were immunoblotted to detect TRIF (indicated by arrow), HA-tagged V, stably expressed FLAG-tagged TLR3 and endogenous STAT2, phosphatidylinositol 3-kinase p85 (PI3K P85), c-Src (indicated by arrow), and IRF3 (indicated by arrow). Cell extracts (W) served as controls for expression levels. B, the same as A except that immunoblotting to detect endogenous IKK α, β, and γ was used in place of other TLR3 signaling mediators. C, IKKe and V proteins were transiently expressed in TLR3 293 cells. V proteins were immunoprecipitated, and co-immunoprecipitated proteins were immunoblotted to detect Myc-tagged IKKe, STAT2, and HA-tagged V; whole cell extracts served as controls for expression levels. Lanes 1 and 2, noV; lanes 3 and 4, V_H (H); lanes 5 and 6, V_M (M); lanes 7 and 8, V_P (P). D, IKKe, transiently expressed with tagged ubiquitin (Ub) and V_M or empty vector control, was immunoprecipitated. Samples were immunoblotted to detect HA-tagged ubiquitin (top panel, IKKe^Ub). Membranes were stripped and reprobed to detect Myc-tagged IKKe (bottom panel, IKKe) at the same molecular weight. E, FLAG-tagged WT and V_M mutants M-AAA (W174A/W178A/W188A), E95D, E95R, C189A, C214A, and C217A were immunoprecipitated (top two panels), and co-immunoprecipitated proteins were immunoblotted to detect Myc-tagged IKKe and FLAG-tagged V. The bottom two panels show corresponding analyses of whole cell extracts. F, FLAG-tagged WT and V_M C189A mutant were immunoprecipitated (lanes 2 and 4) as in D. Samples were then electrophoresed alongside whole cell extracts to show shifted as compared with unshifted mobilities of Myc-tagged IKKe. G, P56 protein levels were measured by immunoblotting in untreated (lanes 1, 3, 5, and 7) or dsRNA-treated (lanes 2, 4, 6, and 8) HT1080 cells stably expressing WT or V_M mutants C189A or M-AAA. Immunoblotting for actin and FLAG-V served as controls.

V Proteins Are Substrates for IKKe and TBK1—Because the V proteins bound to the kinases, in the next experiments, we tested whether they were phosphorylated as a consequence. Indeed, both V_M (Fig. 4A) and V_P (Fig. 4B) were phosphorylated by IKKe in vivo. As expected, the phosphorylated V proteins migrated more slowly (Fig. 4, A and B, lane 2), and upon phosphatase treatment, they co-migrated with the unphosphorylated proteins (Fig. 4, A and B, lane 3). Similar results were seen for TBK1 (data not shown). Expression of enzymatically inactive IKKe did not cause phosphorylation of the V proteins (Fig. 4, A and B, lane 4), suggesting that IKKe directly phosphorylated V. To test this suggestion, purified V_M and IKKe were added to an in vitro protein kinase reaction in the presence of [γ-³²P]ATP, and radiolabeling of V_M was measured. As a positive control, purified IRF3, a known substrate of IKKe, was used. Both IRF3 and V_M were phosphorylated by IKKe (Fig. 4C, lanes 1 and 2). Control reactions showed that the addition of both IKKe and V_M was needed to observe the radiolabeled protein (Fig. 4C, lanes 3 and 4). Quantification of the incorporated phosphates demonstrated that, on a molar basis, V_M was a slightly better substrate than IRF3 (114 versus 100). Similar in vitro reactions demonstrated the ability of purified TBK1 to phosphorylate V_M (130 versus 100) (Fig. 4D, lane 3). These results established the V proteins as authentic substrates for IKKe/TBK1.

FIGURE 4.

MuV and PIV5 V proteins are phosphorylated by IKKe and TBK1. A, V_M was expressed by itself (lane 1) or co-expressed with kinase active (lanes 2–3) or kinase inactive (KI)(lanes 4–5) IKKe, immunoprecipitated, and treated with calf intestine phosphate (CIP)(lanes 3 and 5). HA immunoblotting was used to detect V. B, the same as A except that V_P was used in place of V_M. C, in vitro [γ-³²P]ATP kinase assays were conducted with GST·IKKe and V protein or IRF3. Radiolabeled proteins were visualized by autoradiography (top two panels) and immunoblotted (bottom two panels) to determine total amounts of IRF3 or V in the loaded samples. Lane 1, V_M + IKKe; lane 2, IRF3 + IKKe; lane 3, IKKe; lane 4, V_M. D, in vitro [γ-³²P]ATP kinase assays were performed using purified Myc-TBK1. The top two panels are autoradiographs, and the bottom two panels are immunoblots. Lane 1, TBK1; lane 2, V_M; lane 3, TBK1 + V_M; lane 4, IRF3; lane 5, TBK1 + IRF3.

As a consequence of phosphorylation, V appeared to be destined for faster degradation (Fig. 5A). Co-expression of V with kinase active (lane 2), but not kinase inactive (lane 3) IKKe, caused major diminution of the cellular level of V, as compared with control (lane 1). Furthermore, this was not caused just by overexpression of IKKe because dsRNA-mediated activation of the endogenous kinase caused a dramatic lowering of the level of V as well (Fig. 5B, lane 2), and this diminution could be inhibited by the proteasome inhibitor MG132 (Fig. 5B, lane 3). Finally, interaction between V and IKKe was required for degradation; V_M-AAA, a mutant that did not co-immunoprecipitate with IKKe (Fig. 3E, lane 3), was not degraded (Fig. 5C).

FIGURE 5.

Phosphorylated MuV V protein is degraded. A, V_M was expressed alone (lane 1), or along with WT kinase active (lane 2) or kinase inactive (KI) (lane 3) IKKe. Levels of HA-tagged V and Myc-tagged IKKe were determined by immunoblotting of cell extracts. B, TLR3 293 cells transiently expressing V_M were treated with DMSO as a control (lane 1), dsRNA and DMSO (lane 2), or dsRNA and MG132 (lane 3). FLAG immunoblotting was used to detect FLAG-tagged V with actin as control. C, V_M WT (lanes 1 and 2) and mutant M-AAA (lanes 3 and 4) were expressed with WT kinase active (lanes 1 and 3) or kinase inactive (lanes 2 and 4) IKKe. Levels of FLAG-tagged V and Myc-tagged IKKe were determined by immunoblotting of cell extracts.

V Protein Determines the Efficacy of Virus Replication in TLR3-activated Cells—To evaluate the biological significance of our observations, we chose to use the PIV5 mutant virus VDC, which encodes a C-terminally truncated V protein (22). VDC was not phosphorylated in vivo by IKKe as seen in WT PIV5 V protein (Fig. 6A). Furthermore, we determined that the truncated V protein could not block signaling induced by exogenous expression of IKKe (data not shown). Results showed that WT virus replicated better as compared with the mutant virus in both TLR3-expressing and non-expressing cells (Fig. 6B). Since the mda-5/RIG-I pathway is still intact in these cells, this difference may be due to the lack of ability of the truncated V protein to inhibit that signaling pathway (19). In cells not expressing TLR3, treatment with dsRNA did not have any effect on the replication of either virus. In contrast, in TLR3-expressing cells, dsRNA treatment strongly inhibited the replication of the mutant virus (5 logs), whereas the effect on WT virus was minor. These results demonstrated that inhibition of TLR3 signaling by V protein was relevant for effective virus replication.

FIGURE 6.

Effects of dsRNA signaling on WT and mutant PIV5 replication. A, PIV5 WT (lane 1) or mutant C-terminally truncated (VDC) (lane 2) V proteins were co-expressed with IKKe and immunoprecipitated. V5 immunoblotting was used to detect V and Myc to detect IKKe. B, virus yields 6 days after infection were measured for WT or VDC PIV5 replication in 293 cells with and without TLR3 and pre- or mock-treated with dsRNA. Bars represent standard error derived from two independent experiments. PFU, plaque-forming units.

DISCUSSION

The results presented above revealed several new features of the equilibrium established in an infected cell between the virus and the host (Fig. 7). Induction of viral stress-inducible genes, including IFNs, is blocked by rubulaviral V proteins by inhibiting IRF3 phosphorylation, which is required for its activation because the V proteins can themselves be phosphorylated by TBK1/IKKe. A similar observation has been made by Unterstab et al. (14) for borna disease virus P protein. It is not yet clear exactly how V proteins can inhibit IRF3 phosphorylation by TBK1/IKKe. Because they themselves are substrates of the same kinases, one possibility is that V simply competes out IRF3 as a substrate. In the in vitro kinase assays, quantitation of phosphorylation showed that IRF3 and V_M were equally phosphorylated by IKKe, when present in equimolar amounts either singly or in combination (data not shown), indicating that the two proteins are equally competent substrates of the kinases. Hence, to compete out IRF3, V has to be present at a high molar excess, a possibility not unlikely because V proteins are produced in large quantities in Paramyxovirus-infected cells (17). Alternatively, V proteins can have stronger affinity for TBK1/IKKe, thus not allowing access to IRF3. The fact that V and IKKe were co-immunoprecipitated efficiently indicated that the two proteins could interact strongly, thus providing credence to the second scenario. Although our study was designed to determine whether different paramyxoviral V proteins could inhibit TLR3 signaling, our observations are equally relevant for other signaling pathways that converge on TBK1/IKKe. Thus, we can expect rubulaviral V proteins to block signaling initiated by the cytoplasmic receptors RIG-I, mda-5, and DNA-dependent activator of IRFs (DAI) or the membrane receptor TLR4, which are known to be triggered by RNA, DNA, or lipopolysaccharide (3). Indeed, we observed that RIG-I/mda-5 signaling to IRF3 triggered by transfected dsRNA was inhibited by V_H, V_M, and V_P (data not shown).

FIGURE 7.

A negative feedback loop between rubulaviral V proteins and IKKe/TBK1. Although rubulaviral V proteins block TBK1/IKKe kinase activity by acting as an alternative substrate to IRF3/7, the resulting phosphorylated V protein (V^PO4) is degraded. V proteins also mediate ubiquitination of TBK1/IKKe (Ub), leading to their degradation.

An additional consequence of the action of V proteins as alternative substrates of IKKe could be inhibition of phosphorylation of STAT1. Because STAT1 phosphorylation by IKKe is essential for its ability to induce a subset of ISGs (40), in addition to causing STAT1 degradation (18), rubulaviral V proteins may block actions of IFN using this mechanism.

The interaction with V caused ubiquitination of IKKe as indicated by the observed slower mobility of co-immunoprecipitated IKKe and its V-dependent ubiquitination when co-transfected with tagged ubiquitin. A similar change in the mobility of IKKe was observed when C189A mutant of V_M was used; this was unexpected because the mutant cannot bind DDB1 (data not shown), a component of the ubiquitin ligase complex (18). This indicates the existence of alternative pathways for the ubiquitin ligase activity of V_M. Here, polyubiquitination of IKKe leads to its proteasome-mediated degradation. Thus, V not only blocks IRF3 phosphorylation but destroys the activating kinase as well.

What was clear from our results was that the host could fight back by degrading V proteins. Just as phosphorylation of IRF3 leads to its degradation, phosphorylation of V protein was accompanied by its destruction (Fig. 5). This is consistent with the degradation of V_M observed over the course of a viral infection (41). Because V was needed for the optimum replication of viruses, even in the absence of dsRNA signaling (Fig. 6), its destruction should be a potent antiviral mechanism in that context as well. Phosphorylation and degradation of V provide an opportunity for temporal regulation of IRF3 activation; when the level of V is high, IRF3 phosphorylation is blocked by competition. As more and more V is phosphorylated and degraded, the relative concentration of IRF3 will increase, leading to its phosphorylation, activation, and degradation. Thus, two negative feedback loops, one between V and IRF3 and the other between IKKe/TBK1 and V, regulate the equilibrium between the virus and the cell.

It is known that IKKe interacts with STAT1 and that STAT1 can interact with V (30, 40). However, the interaction between IKKe and V reported here is not mediated by STAT1 because V_M could block TLR3 signaling in U3B cells (42), which lack functional STAT1 (data not shown). The tryptophan residues of V that were required for IKKe binding are located in the C-terminal region of the protein (43). Because this region is not shared between viral V and P proteins, whose mRNAs are produced by alternative transcription of the same open reading frame, P proteins are not expected to bind to IKKe. This prediction is consistent with previous findings that V, but not P, blocks dsRNA-mediated IRF3 activation (22).

The experiments with LMB (Fig. 2, C and D) illuminated subtle, but important, aspects concerning IRF3 activation. According to the current paradigm of the activation process, IRF3 is phosphorylated at multiple serine residues, and this leads to its nuclear translocation, a process that is necessary for its action as a transcription factor (38). We found that forcing nuclear localization of IRF3 by LMB treatment of cells was not sufficient for its ability to induce genes. Therefore, there are consequences to IRF3 phosphorylation in addition to nuclear translocation that are necessary for activation.

It is interesting to note that among the V proteins from paramyxoviral subfamilies tested, all inhibit IFN signaling, but only members of the genus Rubulavirus were able to inhibit dsRNA-TLR3-mediated IRF3 activation (17). These observations seem to correlate with what seems to be a more important role for V in the life cycle of Rubulaviruses as compared with others. Rubulaviral V proteins, in contrast to P in other Paramyxoviruses, represent the default open reading frame transcribed at the P/V locus, resulting in higher levels of basal expression. Correspondingly, virions from Rubulaviruses contain a much higher level of V as compared with others and are conveniently present during the initial stages of infection and signaling (17). This importance of rubulaviral V proteins may be in part compensation for the lack of W or C accessory proteins, which Respiroviruses and Henipaviruses encode to counteract host defense (5, 17, 44).