Research Article

Inhibition of proinflammatory and innate immune signaling pathways by a cytomegalovirus RIP1-interacting protein

Claudia Mack, Albert Sickmann, David Lembo, and Wolfram Brune
PNAS February 26, 2008 105 (8) 3094-3099; https://doi.org/10.1073/pnas.0800168105

  1. Communicated by Thomas E. Shenk, Princeton University, Princeton, NJ, January 7, 2008 (received for review December 10, 2007)

Abstract

TNFα is an important cytokine in antimicrobial immunity and inflammation. The receptor-interacting protein RIP1 is an essential component of the TNF receptor 1 signaling pathway that mediates the activation of NF-κB, MAPKs, and programmed cell death. It also transduces signals derived from Toll-like receptors and intracellular sensors of DNA damage and double-stranded RNA. Here, we show that the murine CMV M45 protein binds to RIP1 and inhibits TNFα-induced activation of NF-κB, p38 MAPK, and caspase-independent cell death. M45 also inhibited NF-κB activation upon stimulation of Toll-like receptor 3 and ubiquitination of RIP1, which is required for NF-κB activation. Hence, M45 functions as a viral inhibitor of RIP1-mediated signaling. The results presented here reveal a mechanism of viral immune subversion and demonstrate how a viral protein can simultaneously block proinflammatory and innate immune signaling pathways by interacting with a central mediator molecule.

Antiviral innate immune responses are triggered by receptors and sensors that recognize pathogen-, damage-, or stress-associated molecular patterns (1). These receptors can be located at endosomal or cell surface membranes, as is the case for the Toll-like receptors (TLRs). Other receptors, such as the double-stranded RNA (dsRNA)-activated helicases RIG-I and Mda5, sense the presence of potentially dangerous molecular patterns inside of the cell. The receptors initiate specific signaling cascades that lead to the activation of transcription factors, (such as NF-κB and IFN regulatory factors) and MAPKs, or the initiation of programmed cell death (PCD). Very similar responses are triggered by proinflammatory cytokines such as TNFα, which also play important roles in controlling viral infections.

The receptor-interacting protein RIP1 (also called RIP) is located at the intersection of several signaling pathways [supporting information (SI) Fig. 7]. It integrates signals from membrane-bound receptors and intracellular stress sensors (reviewed in refs. 2 and 3). RIP1 has been investigated extensively because of its crucial role in the TNF receptor (TNFR)1 signaling pathway (4). Stimulation with TNFα initially induces the recruitment of RIP1, the TNFR-associated factor (TRAF)2, and the TNFR-associated death domain to the plasma membrane (5, 6). The subsequent ubiquitination of RIP1 by TRAF2 (7, 8) is required for activation of IκB kinase and NF-κB (9). RIP1 also activates the MAPKs p38 and ERK and participates in the activation of JNK (10, 11). In addition, RIP1 mediates NF-κB activation upon stimulation of TLR3 and TLR4 via the TIR domain-containing adaptor-inducing INF-β (TRIF) (12, 13). RIP1 also transmits signals derived from DNA-damage sensors (14) and from intracellular sensors of dsRNA (15, 16).

TNFR1 and other death receptors can trigger apoptosis by inducing the formation of a complex containing the Fas-associated death domain and procaspase-8, in which the latter is activated autocatalytically (5). However, when caspase-dependent cell death is blocked, an alternative, caspase-independent pathway to PCD can be activated, and this pathway depends on RIP1 (17, 18). The various downstream effects of RIP1, particularly the induction of cell death and activation of proinflammatory genes through NF-κB, can obviously be detrimental to viral replication and spread, and, therefore, they are potential targets for viral countermeasures.

CMVs, prototypes of the β-herpesviruses, are known to interfere with many innate defense pathways. In only a few cases, the responsible viral gene products have been identified. For instance, the murine CMV (MCMV) M27 protein and the human CMV (HCMV) IE1 protein block the induction of IFN-responsive effector genes by interacting with STAT1 and/or STAT2 (19, 20). Both viruses inhibit apoptosis upon stimulation of death receptors by a viral inhibitor of caspase-8 activation (vICA), encoded by the viral genes M36 and UL36, respectively (21, 22). Death receptor- as well as stress-induced PCD is also inhibited by the mitochondrial inhibitor of apoptosis encoded by the HCMV UL37x1 and the MCMV m38.5 gene (23, 24) (SI Fig. 7). It has also been shown that TNFα-induced NF-κB activation is blocked by CMVs (25–27), but the molecular mechanisms and the viral genes involved have remained unknown.

In this study, we show that the MCMV protein M45 binds to RIP1 and inhibits TNFα-induced activation of NF-κB and p38 MAPK, as well as the induction of caspase-independent cell death. Moreover, M45 also blocks the RIP1-mediated activation of NF-κB in response to TLR3 stimulation. These results show that M45 functions as a viral inhibitor of RIP1-mediated signaling.

Results

Inhibition of TNFα-Induced Cell Death by M45.

In previous work, we have shown that M45 is required for blocking PCD induced by MCMV infection itself and that endothelial cells and macrophages are particularly sensitive to virus-induced cell death (28). In the present study, we also observed that fibroblasts infected with an M45 deletion mutant (ΔM45) were highly sensitive to TNFα-induced cell death but resistant to staurosporine (STS)-induced apoptosis (Fig. 1 a). Similarly, ΔM45-infected cells were sensitive to Fas-mediated but resistant to actinomycin D-induced PCD (data not shown). By contrast, cells infected with wild-type (wt) MCMV or an M45 revertant virus (RM45) were protected from these cell death-inducing stimuli (Fig. 1 a). The finding that M45 is required to inhibit TNFR1- or Fas-induced cell death was surprising, because MCMV M36/vICA should inhibit caspase-8 activation upon death receptor stimulation (22). Fibroblasts infected with an M36 deletion mutant (ΔM36) were, indeed, sensitive to TNFα-induced apoptosis, and this was blocked by the caspase inhibitor z-VAD-fmk (Fig. 1 b). However, TNFα-induced cell death of ΔM45-infected cells was not blocked by z-VAD-fmk (Fig. 1 b), indicating that M45 is necessary to inhibit TNFα-induced caspase-independent cell death.

Fig. 1.
Fig. 1.

M45 is required to block TNFα-induced caspase-independent cell death. (a) 10.1 fibroblasts were infected with MCMVs as indicated and treated 6 h later with STS or TNFα plus CHX. Viability was measured 20 h later relative to cells treated with solvent only (for STS) or CHX only (for TNFα). Results that were similar to those with TNFα plus CHX were obtained with TNFα alone, with the exception of mock-infected cells, which did not die from TNFα alone (data not shown). (b) Cells were infected and treated with TNFα plus CHX and the caspase inhibitor z-VAD-fmk or DMSO. Cells infected with ΔM45 or ΔM36 were sensitive to TNFα-induced cell death, which was blocked by z-VAD-fmk only in the case of the ΔM36 virus.

M45 Interacts with RIP1.

To determine the molecular mechanism of action of M45, we tried to identify cellular and viral interaction partners of M45 by affinity purification. A recombinant MCMV was constructed that expresses an M45 protein with a tobacco etch virus (TEV) protease cleavage site and an HA tag at its C terminus (MCMV M45–TEV–HA). Lysates of cells infected with M45–TEV–HA or wt MCMV were applied to anti-HA affinity purification columns. M45 and proteins bound to it were specifically eluted by cleavage with TEV protease. Possible interaction partners were identified by mass spectrometry. By this approach, the TNFR-interacting protein RIP1 was identified as an interaction partner of M45 (SI Fig. 8). The M45–RIP1 interaction was confirmed by coimmunoprecipitation by using virus-infected cells (Fig. 2 a and b), as well as cells transfected with an M45 expression plasmid (Fig. 2 c). This showed that no other MCMV proteins are required for the binding of M45 to RIP1.

Fig. 2.
Fig. 2.

M45 binds to RIP1. (a) Lysates of cells infected with wt MCMV or an HA-tagged virus (M45HA) were used for immunoprecipitation with the indicated antibodies. RIP1 was detected by Western blot as a protein coprecipitating with M45. (b) HA-tagged M45 was coprecipitated with RIP1 in lysates of cells infected with MCMV–M45HA. (c) NIH 3T3 cells were transfected with plasmids expressing HA-tagged M45 or m143 (a different MCMV protein), respectively. RIP1 was coprecipitated with M45 but not with m143. (d) 3T3 fibroblasts derived from wt and knockout mice were infected with wt or M45HA-expressing MCMV. RIP1 was coprecipitated with M45HA in all but rip1 −/− knockout cells.

Upon TNFα stimulation, RIP1 and TRAF2 are recruited to TNFR1 to form a signaling complex (5). Even though TRAF2 and TNFR1 were not found in our affinity purification screen, we wanted to rule out the possibility that M45 binds to RIP1 only indirectly via TRAF2 or TNFR1. To do this, TNFR1- and TRAF2/5-deficient fibroblasts were infected with MCMV and used for coimmunoprecipitation experiments. As shown in Fig. 2 d, RIP1 was coprecipitated with M45 in the absence of TNFR1 or TRAF2 and -5, indicating that M45 binding to RIP1 does not require TNFR1 or TRAF2/5.

M45 Inhibits TNFα-Induced NF-κB Activation.

Because one of the main functions of RIP1 is signal transduction from TNFR1 to NF-κB activation, we analyzed whether M45 interferes with this pathway. Ubiquitination of RIP1 leads to the recruitment and activation of the IκB kinase (IKK) complex, which phosphorylates IκBα, the inhibitor of NF-κB (9). The subsequent degradation of IκBα leads to the release of NF-κB. We found that TNFα-induced IκBα degradation was blocked in cells infected with wt MCMV or the RM45 revertant virus but not in cells infected with the ΔM45 virus (Fig. 3 a).

Fig. 3.
Fig. 3.

M45 inhibits TNFα-induced NF-κB activation. (a) NIH 3T3 cells were infected with wt or mutant MCMV for 24 h and treated with TNFα for 15 min. M45-expressing viruses inhibited the degradation of IκBα. (b) rip1 −/− knockout fibroblasts were stably transduced with a RIP1-expressing or an empty MSCV retrovirus. Cells were also transduced with retroviral vectors expressing M45 or GFP (as control). TNFα-induced IκBα degradation was blocked in all RIP1-deficient cells and in RIP1-positive cells expressing M45. (c) HEK 293 cells were transfected with an NF-κB-dependent luciferase reporter plasmid and different expression plasmids. Luciferase activity was measured 12 h after TNFα stimulation and is shown as fold induction compared with transfected cells without TNFα. Luciferase induction was inhibited in cells expressing M45 or the cellular RIP1 inhibitor A20 but not in cells transfected with control plasmids expressing irrelevant proteins (m41 or GFP) or empty pcDNA3 vector.

To test whether M45 by itself can inhibit IκBα degradation, we expressed M45 or GFP in fibroblasts. RIP1-deficient cells and derivatives of these cells, in which the rip1 gene was reintroduced, were used to confirm that this process is RIP1-dependent. Fig. 3 b shows that M45 inhibited IκBα degradation in RIP1-expressing fibroblasts.

Although the analysis of IκBα degradation is an established assay for NF-κB activation, we used an independent test system to confirm the results. An NF-κB-dependent luciferase reporter plasmid was transfected together with M45-expressing or control plasmids into HEK 293 cells. Upon stimulation with TNFα, luciferase expression was induced in cells transfected with control plasmids but was blocked in cells expressing M45 or the cellular RIP1 inhibitor A20 (Fig. 3 c) (29).

M45 Inhibits Caspase-Independent Cell Death.

Stimulation of TNFR1 can activate caspase-8 and induce apoptosis (5) or lead to caspase-independent PCD mediated by RIP1 (17, 18). The latter pathway is activated when caspase-8-dependent apoptosis is blocked and has, therefore, been termed a “backup” pathway to cell death. During MCMV infection, caspase-8 activation is blocked by M36. Hence the virus needs M45 to inhibit TNFα-induced caspase-independent cell death (Fig. 1). Caspase-independent PCD can be induced by treatment of susceptible cells with TNFα and caspase inhibitors (17). Thus we tested whether M45 by itself can inhibit the caspase-independent cell death pathway. Fig. 4 a shows that SVEC4–10 endothelial cells died rapidly upon TNFα stimulation when the caspase-8-dependent pathway was blocked by a pan-caspase (z-VAD-fmk) or a caspase-8-specific inhibitor (z-IETD-fmk). By contrast, M45-expressing SVEC4–10 cells were protected. Similar results were obtained with L929 fibrosarcoma cells (Fig. 4 b), which have been used by other investigators for the analysis of RIP1-dependent PCD (17) and 10.1 fibroblasts (data not shown). We observed that different cell lines vary in their sensitivity to this cell death pathway, with SVEC4–10 cells being highly sensitive and NIH 3T3 cells rather insensitive.

Fig. 4.
Fig. 4.

M45 inhibits caspase-independent cell death and activation of p38 MAPK. (a) SVEC4–10 endothelial cells were transduced with retroviral vectors expressing M45, GFP, or nothing. Cells were treated with TNFα (without CHX) and z-VAD-fmk, z-IETD-fmk, or DMSO. Viability was measured after 24 h. M45 blocked the induction of caspase-independent cell death. (b) Similar results were obtained with L929 fibrosarcoma cells. (c) Fibroblasts were infected with wt or mutant MCMV and treated with TNFα for the indicated periods of time. Lower amounts of phosphorylated p38 MAPK (P-p38) were detected in cells infected with M45-expressing viruses (wt and RM45).

RIP1 is essential for TNFα signaling to p38 MAPK (10). To find out whether M45 inhibits activation of p38, cells were infected with wt and mutant MCMVs, and p38 phosphorylation was assayed at different times after TNFα addition. TNFα stimulation increased the amount of phosphorylated p38 in mock-infected cells and cells infected with ΔM45 but not in cells infected with the wt MCMV or RM45, both of which express M45 (Fig. 4 c). This shows that M45 inhibits p38 activation in infected cells. It is noteworthy that MCMV-infected cells showed a slightly increased baseline level of phospho-p38 (i.e., before TNFα stimulation) as compared with mock-infected cells.

The N-Terminal Domain of M45 Is Dispensable for Inhibition of RIP1.

M45 shows a high sequence similarity to the large subunit (R1) of ribonucleotide reductases (RNR) within its C-terminal part (28). However, a number of amino acid residues essential for RNR activity are not conserved in M45, and the protein is catalytically inactive (30). M45 also contains a large N-terminal domain of unknown function. To test whether parts of the protein are dispensable for its function, we constructed truncation mutants of M45 (Fig. 5 a). Surprisingly, almost the entire N-terminal domain could be deleted without losing the ability of the protein to interact with RIP1 (Fig. 5 b) and inhibit TNFα-induced NF-κB activation (Fig. 5 c). By contrast, a relatively moderate truncation at the C terminus abrogated both, binding to RIP1 and the ability to block NF-κB activation (Fig. 5 b and c). Thus, the R1 homology domain, but not the N-terminal domain, is essential for the function of M45 as inhibitor of RIP1-mediated signaling.

Fig. 5.
Fig. 5.

Large parts of the N-terminal, but not of the C-terminal, domain of M45 are dispensable for interaction with RIP1 and activation of NF-κB. (a) Schematic representation of M45 and truncation mutants. The unique N-terminal domain is shown as an open box, and the C-terminal HA tag is shown in black. (b) NIH 3T3 cells were transfected with plasmids expressing full-length or truncated M45, and HA-tagged proteins were immunoprecipitated. RIP1 was coprecipitated with Nt1, -2, and -3 but not with Ct. (c) Luciferase activity was measured in transfected HEK 293 cells as described in the legend of Fig. 3 c. The N-terminal, but not the C-terminal, truncation mutants inhibited TNFα-induced NF-κB activation.

M45 Inhibits TLR3-Mediated NF-κB Activation.

Recent studies have demonstrated that TLR3 and TLR4 can activate NF-κB by signaling through the adaptor proteins TRIF and RIP1 (12, 13). Because TLR4 (but not TLR3) can activate NF-κB also independently of RIP1 via MyD88 (13), we tested whether M45 was also able to inhibit TLR3-induced NF-κB activation. Macrophages, which naturally express TLR3, are notoriously difficult to transfect. Therefore, we transfected HEK 293 cells with a TLR3 expression plasmid and an NF-κB-dependent luciferase reporter plasmid and stimulated the cells with poly(I:C) as performed by others previously (31). In 293 cells, NF-κB activation upon poly(I:C) addition depends on TLR3, because these cells do not respond to poly(I:C) in the absence of TLR3 (ref. 31 and unpublished results). As shown in Fig. 6 a, TLR3 stimulation induced luciferase expression in cells cotransfected with control plasmids expressing an inactive truncation mutant of M45 or an irrelevant MCMV protein. By contrast, M45 and the cellular RIP1 inhibitor A20 inhibited luciferase induction upon the addition of poly(I:C) (Fig. 6 a), indicating that M45 also inhibits RIP1-mediated TLR3 signaling.

Fig. 6.
Fig. 6.

M45 inhibits TLR3-induced NF-κB activation and ubiquitination of RIP1. (a) HEK 293 cells were transfected with an NF-κB-dependent luciferase reporter plasmid and expression plasmids for TLR3 and the indicated proteins. Luciferase activity was measured 6 h after poly(I:C) addition and is shown as a fold induction as compared with transfected cells without poly(I:C). Induction of luciferase activity was inhibited by M45 and A20 but not by an irrelevant MCMV protein (m41) or a truncated M45 (Ct). (b) (Upper) Cells were transfected with plasmids as indicated. The total amount of plasmid for each transfection was normalized with empty pcDNA3 vector. MycRIP was precipitated with an anti-Myc antibody, and polyubiquitinated RIP1 was detected as a high-molecular-weight ladder by using an anti-HA antibody (lane 2). Ubiquitination was reduced in cells expressing A20 or M45. (Lower) The same blot after stripping and incubation with anti-M45 and anti-Flag antibodies. Comparing the two blots reveals that the anti-HA antibody also detects coprecipitated M45 and A20, indicating that A20 and M45 are also ubiquitinated. (c) Control Western blots show MycRIP, Flag-A20, and M45 in the cell lysates used for immunoprecipitation. (d) Overexpression of MycRIP in HEK 293 cells activates NF-κB, as measured with a luciferase reporter assay. NF-κB activation is inhibited by M45 and A20.

M45 Inhibits Ubiquitination of RIP1.

Upon TNFα stimulation, RIP1 is ubiquitinated and activated by TRAF2 (7, 8, 32). Lysine 63 (K63)-linked ubiquitination of RIP1 is required for recruitment of IKKγ/NEMO and activation of IκB kinase, whereas K48-linked ubiquitination causes proteasomal degradation of RIP1 (3, 9, 33). Overexpression of RIP1 leads to its ubiquitination and activation of NF-κB, and this process is inhibited by the cellular protein A20. A20 counteracts RIP1 activation by removing K63-linked and attaching K48-linked ubiquitin molecules (33). M45 shows no obvious sequence similarity to A20 or other deubiquitinating enzymes, but we speculated that the strong binding of M45 to RIP1 could block its ubiquitination. To test this hypothesis, we overexpressed Myc-tagged RIP1 (MycRIP) in HEK 293 cells by transient transfection. An A20 expression vector, an empty vector plasmid, or increasing amounts of a plasmid expressing M45, as well as an expression vector for HA-tagged ubiquitin (HA–Ub), were cotransfected. As shown in Fig. 6 b and c, A20 and M45 inhibited the ubiquitination of MycRIP. HEK 293 cells were also transfected with similar sets of plasmids, but an NF-κB-dependent luciferase reporter plasmid was used instead of HA–Ub. Consistent with the results in Fig. 6 b, MycRIP overexpression activated NF-κB, and A20, as well as M45, markedly decreased its activation (Fig. 6 d). These results show that M45 inhibits ubiquitination of RIP1.

Discussion

Numerous virus recognition systems and cytokine signaling pathways activate a limited number of effector systems. The cellular kinase RIP1 is at the converging point of several pathways (2, 3). Its central role should make it an ideal target for inhibition by a virus, because several signaling pathways could be blocked at the same time (SI Fig. 7). In this study, we demonstrate that the MCMV M45 protein binds to RIP1, inhibits its activation by ubiquitination, and blocks the TNFα-induced activation of NF-κB, p38 MAPK, and caspase-independent PCD. We also show that M45 inhibits TLR3-induced NF-κB activation.

Previously, we have reported that M45 is required to inhibit PCD induced by the virus itself (28). The molecular mechanism of action of M45 described here offers an explanation for the ability of M45 to block cell death induced by both external and internal stimuli. RIP1 transduces signals from intracellular sensors of DNA damage (14) and dsRNA (15, 16), and it is known that CMV infection can trigger dsRNA-activated pathways (34) and induce a DNA-damage response (35). Hence, virus replication could induce cell death via internal sensors that signal through RIP1. The apparent differences in sensitivity to RIP1-mediated cell death described here correlate with our previous observation that SVEC4–10 endothelial cells are much more sensitive to cell death induced by infection with an M45 mutant virus than NIH 3T3 fibroblasts (28). A recent report has suggested that the sensitivity of cells to caspase-independent cell death may depend on the level of RIP1 expression and the ability of cells to secrete TNFα (36). Like murine cells, human cells also appear to differ in their sensitivity to caspase-independent PCD (17, 18).

TLR3 and TLR4 are pattern-recognition receptors activated by dsRNA or LPS, respectively. The role of TLR3 in MCMV infection is under debate (37, 38), but the fact that RNA has been detected in CMV particles (39) and that dsRNA-binding proteins are incorporated into the tegument (40) suggests that dsRNA may stimulate TLR3 or cytoplasmic dsRNA sensors. M45 is also part of the viral tegument (30) and could inhibit RIP1-mediated signaling immediately if delivered in sufficient amounts to the infected cell. Whether TLR4 plays a role during MCMV infection is questionable, because a previous study has shown that TLR2, but not TLR4, is stimulated by HCMV (41).

Considering the central role of RIP1 in different pathways, one would expect that an M45 mutant MCMV should be severely attenuated in its natural host. In a previous study, we have shown that M45 mutants are completely avirulent in SCID mice, even though these mice are highly susceptible to MCMV infection (30). Unfortunately, it is not possible to test whether RIP1 deficiency can reverse the attenuated phenotype of an M45-deficient virus, because rip1 knockout mice die within the first 3 days of life (4).

Stimulation of death receptors can induce apoptosis by activation of caspase-8 (5). To inhibit this pathway, many viruses, including CMVs, γ-herpesviruses, and poxviruses, express caspase-8 inhibitors (21, 22, 42, 43). Our results show that the mere inhibition of caspase-8 can render infected cells sensitive to TNFα-induced caspase-independent PCD and that an additional inhibitor is required to block this backup pathway to cell death. Hence, it is likely that other viruses that block caspase-8 also inhibit this RIP1-dependent pathway, possibly in a similar way like M45.

The ability of M45 to inhibit both NF-κB activation and caspase-independent cell death may seem paradoxical, because NF-κB can induce the expression of antiapoptotic proteins (5). However, a recent study has shown that caspase-independent PCD is not affected by NF-κB activation (44), indicating that the function of M45 is not as conflicting as it appears.

Unlike α- and γ-herpesviruses, β-herpesviruses seem to have abandoned the strategy of supplying enzymes required for the biosynthesis of DNA precursors. Genes for a thymidine kinase, a thymidylate synthase, and for the small RNR subunit are absent, and those for the large RNR subunit and dUTPase encode catalytically inactive proteins. The M45 gene became a paradigm of the latter case. The ability of MCMV to induce the cellular RNR allowed M45 to mutate and lose a direct involvement in ribonucleotide reduction. M45 apparently maintained or gained a second function that is indispensable for viral replication in certain cells and dissemination in vivo (28, 30). This study reveals the molecular mechanism of the function of M45 and demonstrates how a viral protein can simultaneously block innate immune and proinflammatory signaling pathways by interacting with a central mediator molecule.

Materials and Methods

Cells.

NIH 3T3 (ATCC CRL-1658) and 10.1 cells are immortalized mouse embryonic fibroblasts. L929 (ATCC CCL-1) and SVEC4–10 (CRL-2181) are murine fibrosarcoma and endothelial cell lines. 3T3-like fibroblasts derived from rip1, tnfr1, and traf2/traf5 knockout mice (4, 32) were a gift from M. Kelliher (University of Massachusetts, Boston, MA). Human embryonic kidney (HEK) 293 cells were purchased from Invitrogen.

Plasmids and Transfections.

The following expression plasmids were used: pCAGGS-FlagA20 (LMBP plasmid collection, University of Ghent), pFlagCMV1-huTLR3 (Addgene), pRK5-MycRIP (a gift from Z. G. Liu, National Institutes of Health, Bethesda, MD), pHA–Ub (provided by M. Nevels, University of Regensburg, Germany), pRSV-βGal (Promega), pTranslucent NF-κB (Panomics), pcDNA-CrmA (43), pcDNA-m143HA (34), and pcDNA-m41 (45). The pcDNA-M45HA and pcDNA-M45 plasmids were obtained by inserting the PCR-amplified M45 (GenBank accession no. DQ978788) via KpnI and XbaI into pcDNA3 (Invitrogen). For the truncation mutant Nt1, nucleotides 162 to 559 of M45 were amplified by PCR and inserted between the KpnI and BamHI sites of pcDNA-M45HA. Nt2 and Nt3 were generated by digesting this plasmid with KpnI and HindIII or EcoRI, respectively, blunting, and religation. For the Ct truncation mutant, pcDNA-M45HA was digested with XhoI and XbaI, and a synthetic linker encoding an HA tag was inserted. Transient transfections were performed by calcium phosphate precipitation or with Polyfect (Qiagen) according to the recommendations of the manufacturer.

Retroviral Transduction.

The murine rip1 cDNA (IMAGE clone 5721177) was inserted into pMSCVpuro (Clontech). The M45HA sequence was inserted into the PmlI site of pRetroEBNA to generate pRetroM45. The pRetroEBNA and pRetroGFP plasmids were obtained from Tom Shenk (Princeton University, Princeton, NJ). Production of retroviruses by using Phoenix A cells and transduction of target cells was performed as described (45).

CMVs and Infection.

MCMV–GFP and the ΔM45 deletion mutant have been described (28, 45). The ΔM36 mutant was constructed essentially as described (22), with the exception that a zeocin resistance gene was used. For the RM45 revertant virus, the M45HA gene was used to replace the nonessential genes m02 to m06 in ΔM45 by using the pReplacer plasmid as described (46). The M45–TEV–HA mutant was generated by tagging of the M45 sequence at the 3′ end with a TEV protease cleavage site and an HA tag as described (45). Viruses were grown on fibroblasts according to standard procedures. Unless stated otherwise, cells were infected at a multiplicity of infection (moi) of three median tissue culture infective doses (TCID50) per cell.

Affinity Purification.

Cells (8 × 107 10.1) were infected at an moi of one TCID50 per cell with MCMV M45–TEV–HA or control virus. After 48 h, cells were lysed (50 mM Tris, pH 7.5/150 mM NaCl/2.5% Nonidet P-40, complete protease inhibitor mixture; Roche) and centrifuged for 1 h at 20,000 × g. Supernatants were loaded onto anti-HA 3F10 affinity columns (Roche). After washing (20 mM Tris, pH 7.5/0.1 M NaCl/0.1 M EDTA/0.05% Tween-20), M45 and associated proteins were eluted by digestion with 100 units of AcTEV protease (Invitrogen) for 1 h at room temperature. Eluted proteins were concentrated and separated by SDS/PAGE. Silver-stained bands were excised and analyzed by mass spectrometry as described (47).

Western Blots and Immunoprecipitation.

Monoclonal mouse antibodies against RIP1 (Clone 38, BD Biosciences), Flag (M2, Sigma), Myc (4A6, Upstate), and β-actin (Ac-74, Sigma) were purchased as indicated. Polyclonal rabbit antibodies against RIP1, IκBα, and p38 MAPK were from Santa Cruz Biotechnology; anti-phospho-p38 was from Cell Signaling; and anti-HA was from Sigma. The anti-M45 antibody has been described (30). A mouse monoclonal antibody against MCMV m41 was generated in our laboratory by Maren Syta. HRP-coupled secondary antibodies were obtained from Cell Signaling or Dako. For immunoprecipitation, 6 × 106 cells were lysed in lysis buffer with 1% Nonidet P-40. The proteins of interest were precipitated overnight with 2.5 μg of antibody and protein A Sepharose at 4°C. Precipitates were washed five times, eluted with sample buffer, and separated by SDS/PAGE. For the analysis of RIP1 ubiquitination, cells lysates were harvested 26 h after transfection by using radioimmunoprecipitation assay buffer [20 mM Tris·HCl (pH 7.5), 300 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS] and homogenized with QIAshredder columns (Qiagen). MycRIP was precipitated with an anti-Myc antibody and protein G Sepharose.

For the analysis of IκBα degradation, infected or transduced cells were stimulated for 15 min with 20 ng/ml recombinant murine TNFα (Promokine). For p38 MAPK activation, cells were stimulated with 7.5 ng/ml TNFα. Proteins were harvested with boiling SDS/PAGE sample buffer, sonicated, and analyzed by Western blot.

Luciferase Assay.

HEK 293 cells were transfected with pTranslucent–NF-κB luciferase reporter vector, pRSV-βGal control vector, pFlagCMV1-huTLR3 (for analysis of TLR3 signaling), or pRK5-MycRIP (for RIP overexpression) and expression vectors for the proteins to be analyzed. After 24 h, cells were stimulated with 20 ng/ml TNFα for 12 h or with 100 μg/ml poly(I:C) for 6 h. Luciferase and β-galactosidase activities were analyzed with a Dual-Light reporter assay kit (Applied Biosystems). Experiments were done in triplicate, and results are shown as fold induction of luciferase activity in induced samples in comparison to noninduced samples with standard deviation.

Cell Viability Assay.

Cell death was induced with 150 nM STS or 20 ng/ml TNFα with or without 250 ng/ml cycloheximide (CHX). MCMV-infected cells were treated 6 h after infection at an moi of 3 TCID50 per cell. Where indicated, TNFα was added to the cells together with z-VAD-fmk, z-IETD-fmk (MBL International), or DMSO. Cell viability was measured by using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay as described (46). The results are shown as percentages of viability in comparison with control cells with SD of four to eight parallel samples.

Acknowledgments

We thank C. Hasselberg-Christoph and C. Winkler for technical assistance; M. Kelliher for providing knockout cells; and A. Loewendorf, C. Benedict, I. Mohr, and S. Voigt for a critical reading of the manuscript. This work was supported by Deutsche Forschungsgemeinschaft Grant SFB421, Project B14 (to W.B.) and by grants from Ministero dell'Istruzione, dell'Universitá e della Ricerca Grants ex 60% and PRIN 2005 (to D.L.).

Footnotes

  • §To whom correspondence should be addressed. E-mail: BruneW{at}rki.de

  • Author contributions: C.M. and W.B. designed research; C.M. and A.S. performed research; D.L. contributed new reagents/analytic tools; C.M., A.S., and W.B. analyzed data; and C.M., D.L., and W.B. wrote the paper.

  • The authors declare no conflict of interest.

  • This article contains supporting information online at www.pnas.org/cgi/content/full/0800168105/DC1.

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Inhibition of proinflammatory and innate immune signaling pathways by a cytomegalovirus RIP1-interacting protein
Claudia Mack, Albert Sickmann, David Lembo, Wolfram Brune
Proceedings of the National Academy of Sciences Feb 2008, 105 (8) 3094-3099; DOI: 10.1073/pnas.0800168105
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