Measles Virus C Protein Interferes with Beta ... - Journal of Virology

Measles Virus C Protein Interferes with Beta Interferon Transcription in the Nucleus Konstantin M. J. Sparrer, Christian K. Pfaller,* and Karl-Klaus Conzelmann Max von Pettenkofer-Institute and Gene Center, Ludwig-Maximilians-University Munich, Munich, Germany

Transcriptional induction of beta interferon (IFN-␤) through pattern recognition receptors is a key event in the host defense against invading viruses. Infection of cells by paramyxoviruses, like measles virus (MV) (genus Morbillivirus), is sensed predominantly by the ubiquitous cytoplasmic helicase RIG-I, recognizing viral 5=-triphosphate RNAs, and to some degree by MDA5. While MDA5 activation is effectively prevented by the MV V protein, the viral mechanisms for inhibition of MDA5-independent induction of IFN-␤ remained obscure. Here, we identify the 186-amino-acid MV C protein, which shuttles between the nucleus and the cytoplasm, as a major viral inhibitor of IFN-␤ transcription in human cells. Activation of the transcription factor IRF3 by upstream kinases and nuclear import of activated IRF3 were not affected in the presence of C protein, suggesting a nuclear target. Notably, C proteins of wild-type MV isolates, which are poor IFN-␤ inducers, were found to comprise a canonical nuclear localization signal (NLS), whereas the NLSs of all vaccine strains, irrespective of their origins, were mutated. Site-directed mutagenesis of the C proteins from an MV wild-type isolate and from the vaccine virus strain Schwarz confirmed a correlation of nuclear localization and inhibition of IFN-␤ transcription. A functional NLS and efficient nuclear accumulation are therefore critical for MV C to retain its potential to downregulate IFN-␤ induction. We suggest that a defect in efficient nuclear import of C protein contributes to attenuation of MV vaccine strains.

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ntiviral host defense relies on rapid recognition of invading viruses by pattern recognition receptors (PRRs), like the ubiquitous cytoplasmic RIG-I-like receptors (RLR), which include RIG-I, MDA5, and LGP-2, and a number of Toll-like receptors (TLR) operating mainly in specialized immune cells. Virus sensing by these receptors leads to activation of the transcription factors interferon regulatory factor 3 (IRF3) and IRF7 and nuclear factor kappa B (NF-␬B), which orchestrate transcriptional activation of type I interferons (alpha interferon [IFN-␣], IFN-␤, IFN-␭, and IFN-␻) and of proinflammatory cytokines (30, 43). Secreted early IFN-␤ alerts noninfected cells via binding to the IFN-␣ receptor (IFNAR) and activation of JAK/STAT signaling (50), which results in the induction of numerous IFN-stimulated genes (ISG). The established antiviral state limits further spread of the virus. As illustrated in the past decade, most viruses utilize multiple mechanisms to limit both the induction of and the response to type I IFN in order to be able to establish an infection (19, 55, 67). In the subfamily Paramyxovirinae, these functions are taken over by the diverse products of the phosphoprotein (P) gene (for a comprehensive review, see reference 22). Measles virus (MV) is a member of the genus Morbillivirus within the subfamily Paramyxovirinae and, in spite of the availability of safe live attenuated vaccines, remains a major cause of childhood mortality in developing countries (67a). MV infects primarily hematopoietic and epithelial cells and causes a severe transient immune suppression facilitating secondary infections that is due to interference with both innate and adaptive immune responses, including the type I IFN network (20, 23, 38, 39, 61). The MV P gene products, which comprise the P, V, and C proteins, have long been known as viral virulence factors (15, 35, 47, 52, 66), but only recently, more detailed knowledge of the multiplicity of their individual and cooperative roles in modulating innate immune responses is emerging. The MV P protein is the product of an mRNA exactly complementary to the viral P gene and has essential roles in viral RNA synthesis (24, 56). The nones-

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sential V protein is translated from an edited mRNA and shares a large 231-amino-acid (aa)-long N-terminal domain (NTD) with the P protein (VNTD) but has a V-specific C-terminal domain (CTD) of 68 amino acids (VCTD) (9) that proved to represent a hub module for binding and inhibiting the functions of a variety of cellular molecules (12, 44, 48, 53, 59). The nonessential 186-aalong C protein is expressed from an alternative open reading frame (ORF) in the P and V mRNAs (3) and, in contrast to the cytoplasmic P and V proteins, shuttles between the cytoplasm and nucleus (40). The MV C protein has been reported to downregulate viral transcription and replication (2, 57, 57) and to act as a viral release and infectivity factor (13). All of the P, V, and C proteins of MV appear to be involved in counteracting IFN-mediated JAK/STAT signaling, though V protein is considered the main player. The P and V proteins bind to STAT1 via their common NTD and interfere with its nuclear import (7, 14). V, in addition, binds to STAT2 and JAK1 via the VCTD (8, 41, 44, 54, 72). Also, C protein was implicated in preventing type I IFN-mediated expression of ISG, though less efficiently than V (16, 60, 70). As for IFN signaling, the MV V protein is a major factor counteracting specific IFN induction pathways, but P and C may contribute. V protein prevents MDA5-mediated IFN induction by

Received 4 August 2011 Accepted 31 October 2011 Published ahead of print 9 November 2011 Address correspondence to Karl-Klaus Conzelmann, [email protected] -muenchen.de. * Present address: Department of Molecular, Cellular and Developmental Biology, University of California at Santa Barbara, Santa Barbara, California, USA. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.05899-11

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IFN-␤ Transcription Inhibition by MV C Protein

binding to the PRRs MDA5 and LGP2, but not RIG-I (10), and prevents TLR7/9-mediated induction of IFN-␣ by binding IKK␣ and IRF7 (48). Moreover, V binds the NF-␬B subunit p65 (RelA) to prevent NF-␬B activity, thereby contributing to interference with early IFN-␤ transcription (59). The MV P protein, in addition, was shown to induce transcription of the TLR inhibitor A20 in macrophage cell lines (71), which might enhance negative feedback to proinflammatory responses in these cells. A contribution of the C protein to the regulation of IFN induction was suggested recently. Specifically, MV mutants deficient for C protein production (Cko viruses) were better inducers of IFN-␤ than parental or Vko viruses (34, 36). The observed activation of PKR (65), which leads to enhancement of IFN induction via activation of ATF-2 and NF-␬B (33), suggested that in the presence of C the accumulation of viral double-stranded RNA (dsRNA) serving as a molecular pattern for RLR is downregulated (34). A direct effect of MV C protein on an IFN-activating signaling cascade, however, has not been shown. In particular, how MV counteracts IFN-␤ induction by RIG-I, which is the major sensor for MV (26, 51) and other negative-strand RNA viruses (25, 27, 49), remained unclear. Here, we assess the direct capacities of the P, V, and C proteins from MV to interfere with RIG-I-mediated IFN induction. Since wild-type (wt) MV isolates are apparently able to avoid or control IFN induction much better than laboratory-adapted and vaccine strains (28, 39, 63), proteins encoded by a wt MV isolate and by the MV vaccine strain Schwarz were included in the investigation. Both MV wt and MV Schwarz C proteins (CWT and CS, respectively) efficiently interfered with IFN-␤ promoter activity and with IFN-␤ mRNA transcription without preventing the activation and nuclear import of IRF3. However, CWT was superior to CS in inhibition. Comparison of the amino acid sequences of CWT and CS revealed a mutation in CS affecting the integrity of the nuclear localization signal (NLS). Indeed, the inhibitory capacity of virus-derived and in vitro-mutated C proteins was correlated with the presence of the authentic NLS from CWT, or an ectopic NLS from simian virus 40 (SV40), and with the degree of nuclear localization. Notably, the sequences of all vaccine strains comprise a mutated NLS, strongly suggesting a contribution to their attenuated phenotype. (This work represents part of the Ph.D. thesis of K.M.J.S.) MATERIALS AND METHODS Cell lines. HEK293T (293T), Vero, Vero-hSLAM (kindly provided by Y. Yanagi), and HeLa cells were propagated in Dulbecco’s minimal essential medium supplemented with 10% fetal bovine serum, 1⫻ L-glutamine, and penicillin/streptomycin (Invitrogen). Plasmids and transfection. The generation of expression vectors encoding individual P gene products of the MV Schwarz vaccine strain and their tagged versions and fragments was described recently (48, 59). Corresponding expression plasmids for the MV wt isolate (MVi/Berlin .DEU/04.08, genotype D5) were prepared after reverse transcription (RT)-PCR from infected cells with primers P-fwd-EcoRI (ATA GAA TTC GCC ACC ATG GCA GAA GAG CAG GCA) and P-rev-XhoI (ATA CTC GAG CTA CTT CAT TAT TAT CTT CAT). The plasmid encoding rabies virus (RV) P was described previously (6). The mutations in MVCWT⌬NLS (R44A and K45A) and MV-CSwtNLS (G44R) were introduced by site-directed mutagenesis. A sequence coding for the SV40 large T NLS (ACT GCT CCA AAG AAG AAG CGT AAG) was added to the N terminus of the MV CWT ORF by PCR amplification and cloned into pCR3 (BamHI/NotI). pcDNA3.1-fl-MV H was constructed from cDNA of MV

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Schwarz-infected cells. The IFN-␤ firefly luciferase (FL) reporter plasmid p125-luc, the IRF3 response element reporter plasmid p55c1b-luc, and plasmids encoding ⌬RIG-I and fl-IRF3 were kindly provided by T. Fujita, Kyoto, Japan, and J. Hiscott, Toronto, Canada. pRL-CMV encoding Renilla luciferase (RL) was purchased from Promega. For the reporter gene assays, 1 ⫻ 105 293T or Vero cells were seeded in 24-well microtiter plates. Sixteen hours after seeding, 100 ng of the reporter plasmid p125-Luc, together with the internal control pRL-CMV (10 ng; Promega) and the indicated amounts of viral plasmids or IFN agonists, was transfected using Lipofectamine 2000 (Invitrogen). For quantitative RT-PCRs (qRT-PCRs), 1 ⫻ 105 293T cells were seeded in 24-well microtiter plates. Sixteen hours after seeding, 400␮g of viral plasmids and 100 ng of pEF-Bos-⌬RIG-I were transfected using Lipofectamine 2000 (Invitrogen). Quantitative RT-PCR. 293T cells were harvested in RLT buffer, and the complete RNA was extracted using a Qiagen RNeasy Kit according to the manufacturer’s protocol. The DNA was digested using Fermentas RNase-free DNase I for 45 min at 37°C. After inactivation of the DNase by EDTA (Fermentas) at 65°C for 10 min, oligo(dT) primers (Invitrogen) were annealed at 65°C for 10 min. Reverse transcription of mRNA occurred at 55°C for 30 min using a Roche Transcriptor Transcriptase Kit. The cDNA was analyzed by real-time PCR with the Quantitect SYBR green PCR Kit (Qiagen) on a LightCycler 2.0 (Roche). Primers for human IFN-␤ (hIFNbfwd, 5=-TCC AAA TTG CTC TCC TGT TG-3=; hIFNbrev, 5=-GCA GTA TTC AAG CCT CCC AT-3=) and human GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (hGAPDHfwd, 5=-TGG TAT CGT GGA AGG ACT CA-3=; hGAPDHrev, 5=-CCA GTA GAG GCA GGG ATG AT-3=) were used. Values for relative quantification of single probes were calculated from a standard curve of serial cDNA dilutions, and IFN-␤ values were divided by GAPDH values. The results obtained, in arbitrary units, were recalculated by setting the mean value for the induction control to 100. The bars in the figures show mean values of duplicates, and the error bars represent standard deviations. For Western blotting, additional wells were transfected with the same mixture used for transfection of cells later subjected to real-time PCR analysis. Reporter gene assays. Cells transfected as indicated, and in some experiments stimulated by infection with Sendai virus (SeV) DI-H4 (kindly provided by D. Garcin) at 6 h posttransfection (p.t.), were harvested at 24 h p.t. in passive lysis buffer, and the lysates were analyzed using the Promega Dual Luciferase Assay system. Luciferase activity was determined using the Berthold Centro LB 960 luminometer according to the supplier’s instructions. The graphs show mean values and standard deviations of three independent experiments. For parallel Western blotting, reporter assay lysates were mixed 1:1 with SDS sample buffer (62.5 mM Tris, 2% SDS, 10% glycerol, 6 M urea, 5% ␤-mercaptoethanol, 0.01% bromophenol blue, 0.01% phenol red). Western blots and antibodies. SDS gel electrophoresis was performed as described previously (59). Anti-MV P/V (N terminus), anti-MV C, and anti-RV P antibodies were generated in rabbits injected with synthetic peptides (Metabion; MV C, aa 176 to 186; MV V/P, aa 2 to 15; RV P, aa 166 to 189). Anti-Flag-M2, anti-p386 IRF3, and anti-IRF3 were purchased from Sigma, Cell Signaling, and Santa Cruz, respectively. Native PAGE. For native gel analysis, 1 ⫻ 106 cells were seeded in 6-well microtiter plates and transfected with the stimulus and plasmids encoding viral proteins using polyethylenimine (PEI) (Sigma) 16 h postseeding. Cells were harvested 24 h p.t. and lysed under native conditions. The whole-cell lysates were subjected to gel electrophoresis using gels cast without detergents and blotted on polyvinylidene difluoride (PVDF) membranes as recently described (58). IRF3 phosphorylation assay. 293T cells (1 ⫻ 105) were seeded on 24-well microtiter plates, and pfl-IRF3 and plasmids encoding viral proteins were transfected 16 h after seeding of the cells. Whole-cell lysates in SDS sample buffer were analyzed using SDS gel electrophoresis. After blotting on PVDF membranes (Millipore), the proteins on the mem-

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branes were detected using anti-MV C, anti-RV P, anti-Flag, and antip386 IRF3 antibodies. Immunofluorescence microscopy. Approximately 5 ⫻ 104 HeLa cells grown on glass slides in 24-well microtiter plates were transfected with the indicated plasmids using PEI and were either fixed 24 h p.t. using 3% paraformaldehyde solution in phosphate-buffered saline (PBS) (Invitrogen) or stimulated with 200 ␮l of SeV DI-H4 6 h p.t. and fixed 18 h later. Free binding sites were blocked using 2.5% milk powder (Roth) in 0.1% Triton X-100 (Merck)–PBS (Invitrogen), and anti-IRF3 (1:50) or anti-MV C (C1242, 1:800; kindly provided by R. Cattaneo) primary antibody diluted in 0.1% Triton X-100 –PBS was incubated on the samples for 2 h at 4°C. The fluorescence-labeled secondary antibody dilution with goat ␣-rabbit Alexa Fluor 488 (1:200), ␣-mouse tetramethyl rhodamine (1:200) (both from Molecular Probes), or ToPRO3-iodide (Vector Laboratories; 1:2,000) for nuclear staining in 0.1% Triton X-100 –PBS was incubated with the fixed cells at 4°C for an additional 2 h. A drop of Vectashield solution (Vector Laboratories) was used to mount the glass slides on object plates, and nail polish was used for sealing. Confocal laser scanning microscopy was performed with a Zeiss LSM510 microscopy system. Excitation of Alexa Fluor 488, tetramethyl rhodamine, and ToPRO3-iodide occurred at wavelengths of 488 nm, 543 nm, and 633 nm, respectively. The images were edited with Zeiss LSM 5 Image Examiner and Microsoft PowerPoint 2010. Statistical analysis. Statistical analysis was done using the analysis of variance (ANOVA) one-way algorithm. The P values of the compared samples were determined as indicated in the figures. Significance tests were done according to the Bonferroni-Holm algorithm.

RESULTS

Inhibition of IFN induction by MV C protein. To compare the IFN-inhibitory capacities of the P, V, and C proteins from wt MV and vaccine strains, cDNA was cloned from mRNAs of VerohSLAM cells (42) infected with a clinical MV isolate of the D5 genotype (MVi/Berlin.DEU/04.08) provided by A. Mankertz (RKI Berlin). pCR3 vectors expressing individual P and V proteins were constructed by silent mutations disrupting the C initiation codons and by introducing a nearby stop codon as previously described for the respective MV Schwarz constructs (48). Dual-luciferase assays were used to determine the abilities of the expressed proteins to interfere with the expression of IFN-␤ promoter-controlled FL from transfected p125-luc after stimulation of 293T cells with a preparation of SeV defective interfering particles (DI-H4) (62). In mock-transfected control cells, FL activity was strongly induced by DI-H4 infection. RV P, which counteracts activation of IRF3 by TBK-1 (6, 58), was used as a positive control in these assays and showed the strongest inhibitory effect. While expression of P of the MV wt isolate (PWT) had only a marginal effect on the activation of the IFN-␤ promoter, expression of V and C proteins (VWT and CWT, respectively) substantially and dose-dependently diminished induction (Fig. 1B). Since DI-H4 infection activates both MDA5 and RIG-I (74) and MV V is known to prevent MDA5 activation and NF-␬B activity, an inhibitory effect of VWT was not unexpected. Notably, however, CWT protein revealed a similar strong inhibitory effect, suggesting that the protein can interfere with RLR-mediated IFN-␤ induction. To determine whether CWT is able to prevent IFN induction upon stimulation of cells with factors downstream of RLR, cells were stimulated by expression of the kinase fl-TBK-1. Indeed, the presence of CWT and VWT again resulted in significant and dosedependent inhibition (Fig. 1C). Together, these results identify the C protein of wt MV as bona fide inhibitor of the IFN-␤ induction pathway.

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FIG 1 (A) Schematic representation of the MV genome and P gene expression. P protein is expressed from an exact transcript of the P gene; the V mRNA is generated by cotranscriptional editing, resulting in insertion of a nontemplated single G residue. The P and V proteins share their 231 N-terminal amino acids but differ in their C-terminal domains. The C protein is translated from an alternative ORF on both mRNAs. (B) Inhibition of IFN-␤ promoter activity by P gene products from the wild-type MV isolate MVi/Berlin.DEU/04.08. Dual-luciferase assays were performed on 293T cells transfected with plasmids encoding either the PWT, VWT, or CWT protein or RV P as a positive control and 6 h later infected with SeV DI-H4. The lysates were harvested 18 h postinfection (p.i.) for luciferase assays and Western blotting with specific antisera (bottom). (C) Cells were stimulated by cotransfection of fl-TBK-1 and were assayed 18 h p.t. FL was normalized with RL coexpressed from transfected pRL-CMV, and the values from mock-transfected cells were set to 1. Standard deviations of triplicates are displayed. Statistical differences according to oneway ANOVA calculation are indicated (ⴱⴱ, P ⬍ 0.001; ⴱⴱⴱ, P ⬍ 0.0001; n.s., nonsignificant).

Differential inhibition of IFN induction by wild-type and vaccine MV C proteins. To reveal whether the C protein of the widely used MV Schwarz vaccine strain (CS) has retained the ability to inhibit IFN-␤ induction, CWT- and CS-encoding plasmids were transfected in parallel into 293T cells. Indeed, expression of CS reduced IFN-␤ promoter activity after stimulation with DI-H4 or fl-TBK-1 (Fig. 2). Notably, however, inhibition by CS was consistently less pronounced than that by CWT. While after infection with DI-H4 only marginal differences were detectable (Fig. 2A),

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FIG 2 Differential inhibition of IFN-␤ promoter activity by C proteins from wild-type and vaccine strain MV. (A and B) Increasing amounts of plasmids encoding C protein from CWT or CS (125, 250, 500, and 750 ng) were transfected in 293T cells as indicated. RV P- and fl-MV H-encoding plasmids (500 and 750 ng) were transfected as positive and negative controls, respectively. Cells were stimulated by infection with SeV DI-H4 at 6 h p.t (A) or by cotransfection of pfl-TBK-1 (B) and processed for dual-luciferase assay and Western blotting at 24 h p.t. as described in the legend to Fig. 1. (C) Vero cells transfected with plasmids encoding the indicated viral proteins and the constitutive active ⌬RIG-I were analyzed for FL activity and Western blotting as before. FL was normalized with coexpressed RL, and the values of mock-transfected (nonstimulated) cells were set to 1. Standard deviations of triplicates are indicated. Statistical analyses were performed using one-way ANOVA (ⴱⴱ, P ⬍ 0.001; ⴱⴱⴱ, P ⬍ 0.0001).

the dissimilarity became more apparent after more downstream stimulation by overexpression of fl-TBK-1 (Fig. 2B). As confirmed by Western blot experiments of the lysates used in the reporter gene assays, the weaker performance of CS was not due to generally lower expression levels. MV C protein was reported to interfere with JAK/STAT signaling (37, 60), which could differentially affect the induction of pro-


teins involved in the IFN induction pathways. Experiments were therefore also performed in Vero cells lacking type I IFN genes, which were stimulated by expression of ⌬RIG-I, a constitutively active fragment of RIG-I. In this case, the superior inhibition by CWT became even more evident (Fig. 2C), with low levels of CWT causing greater inhibition of ⌬RIG-I-induced IFN-␤ promoter activity than higher levels of CS. In summary, the C proteins of a wt MV isolate and the Schwarz vaccine strain differ in their abilities to prevent IFN induction by RIG-I. C proteins of MV vaccine strains have a mutation in the NLS. To address the reason for the better performance of CWT, we compared the amino acid sequences of CWT and CS. Notably, one of the few exchanges concerns the previously described NLS of wildtype MV (40). While the sequence of CWT comprised the canonical NLS motif (41-PPARKRRQ-48), a mutation of R44 (underlined) to G (R44G) in CS disrupted the basic cluster of the NLS predicted to be required for proper function of the NLS (11, 29) (Fig. 3A). Most intriguingly, an inspection of the sequences of a variety of vaccine strains revealed the same R44G mutation in all strains irrespective of their origins (1), while all wt MV isolates of different genotypes and origins have retained the original NLS sequence, strongly suggesting functional importance. Nuclear localization of C proteins correlates with inhibition of IFN induction. Although the MV C protein is small enough for diffusion through nuclear pores, the observed R44G mutation in the NLSs of CS and of C proteins from other vaccine strains should decrease active nuclear import by importins. To experimentally address the effect of the NLS on nuclear accumulation, CWT and CS were expressed in HeLa cells, which were stained for confocal fluorescence microscopy with MV C-specific antibodies. Indeed, whereas CWT almost exclusively localized to the nuclei of transfected cells, CS was distributed equally in the cytoplasm and nucleus (Fig. 4B and C, respectively), indicating a defect of the mutated NLS. To further confirm the role of the NLS, mutants were generated in which the NLS of CWT was destroyed by replacing R44 and K45 with alanine residues (CWT⌬NLS) or in which the NLS of CS was repaired by the exchange of G44 for arginine (CSwtNLS). CWT⌬NLS, which should be incompetent for active transport, was located in both the cytoplasm and nuclei of the cells (Fig. 4E), similar to CS. As its cytoplasmic localization seemed to be even more pronounced than that of CS, residual active transport of CS protein resulting from a weak NLS cannot be excluded. Like CWT, CSwtNLS was almost exclusively located in the nucleus, verifying that the observed altered subcellular localization of CS is due to the R44G mutation in its NLS (Fig. 4D). CWT⌬NLS was further modified to contain the NLS of SV40 large T at its N terminus. This mutant again accumulated well in the nucleus, like CWT, demonstrating that an ectopic NLS can bring about the same cellular distribution of C as its authentic internal NLS (Fig. 4F). Nuclear localization of C proteins correlates with inhibition of IFN-␤ transcription. To test whether the distinct subcellular locations of the C constructs affect their ability to inhibit IFN-␤ induction, expression of FL from p125-luc was determined after stimulation of cells by fl-TBK-1 overexpression. As before, CWT was superior to CS in inhibiting the reporter gene activity (Fig. 5A). In striking contrast, CWT⌬NLS had almost completely lost the inhibitory activity, whereas CS carrying a functional NLS (CSwtNLS) had regained function so that it prevented luciferase expression as efficiently as CWT. The R44G mutation in CS is therefore entirely responsible for the weaker inhibition by the vaccine

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FIG 3 Alignment of the primary sequences of C proteins from genetically divergent MV wild-type isolates and vaccine strains (vac) of different origins. Genotype and GenBank accession numbers are indicated. Deviations from the consensus sequence are shown in black. A canonical NLS (boxed) is present only in the proteins of wild-type isolates. Vaccine strains possess a common R44G exchange (boldface).

virus C protein. Importantly, the CWT construct in which the authentic NLS was destroyed and replaced by an N-terminal SV40 NLS showed the same inhibition as CWT (Fig. 5B). The sequence of the NLS motif of the C proteins is therefore not involved in the mechanism of IFN inhibition but merely determines the degree of

nuclear import, which determines inhibition of IFN-␤ promoter transcription. To verify that the presence of nuclear MV C constructs is able to prevent transcription of the endogenous IFN-␤ mRNA, 293T cells were stimulated with ⌬RIG-I in the presence of CWT,

FIG 4 A functional NLS is a prerequisite for nuclear accumulation of MV C. The intracellular localization of the MV CWT and CS proteins and of the indicated mutants was analyzed by confocal laser scanning microscopy in plasmid-transfected HeLa cells 24 h p.t. C proteins were stained with the C-specific peptide/rabbit antiserum C1242 and visualized with anti-rabbit–Alexa 488 secondary antibody (green). Nuclei were stained with ToPRO3 iodine (white). Mutated amino acids are shown in bold.

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CWT⌬NLS had lost most of the inhibitory activity. Moreover, while CS was inefficient in reducing IFN-␤ mRNA levels, the CS protein with the repaired NLS (CSwtNLS) was highly effective (Fig. 5C). Inhibition of IRF3-dependent transcription by MV CWT protein in spite of unimpaired IRF3 activation and nuclear import. Transcriptional induction of the IFN-␤ promoter critically requires activation of the latent cytoplasmic transcription factor IRF3. As revealed by dual-luciferase experiments, MV CWT, but not MV CS protein, was able to dose-dependently inhibit the IRF3-dependent expression of luciferase from p55c1b-luc upon stimulation by overexpressed fl-IRF3 (Fig. 6A). This suggests that inhibition of IRF3-dependent transcription is responsible for the observed inhibition of the IFN-␤ promoter. IRF3 activation involves phosphorylation, dimerization, and import into the nucleus (31, 73). To address whether MV C protein might interfere with these processes, cells expressing CWT and fl-IRF3 were infected with SeV DI-H4 to activate IRF3. As revealed by Western blot experiments with phosphospecific IRF3 antibodies, CWT was not able to prevent the critical phosphorylation of the IRF3 S386 residue, in contrast to RV P (6), which was included as a positive control (Fig. 6B). In fact, no substantial difference from mocktransfected cells or cells transfected with an fl-MV H proteinencoding plasmid was observed, indicating that C is completely inactive in this respect. Similarly, and in contrast to RV P, neither CWT nor CS prevented dimerization of endogenous IRF3 upon transfection of cells with TBK-1, as indicated by native PAGE (Fig. 6C). To study whether MV CWT might interfere with nuclear accumulation of IRF3, the localization of endogenous IRF3 was determined by confocal fluorescence microscopy. Infection of cells with SeV DI-H4 led to conspicuous redistribution of cytoplasmic IRF3 to the nucleus (Fig. 6D, compare upper two and lower two rows). This behavior was not notably changed in cells expressing CWT (Fig. 6D, bottom row), indicating that activated IRF3 is still able to accumulate in the nucleus in the presence of MV C and that inhibition occurs at a downstream step within the nucleus. DISCUSSION FIG 5 Nuclear localization of MV C is required for efficient inhibition of IFN-␤ promoter activity. (A and B) Dual-luciferase assays were used to determine activation of the IFN-␤ promoter. Plasmids encoding the indicated protein constructs (300 and 600 ng for C, 200 ng for fl-TBK-1, and 600 ng for RV P or fl-MV H) were transfected into293Tcells,andinductionofp125-luc-directedFLactivitywasdetermined24hp.t. All assays were normalized with coexpressed RL, and mock values were set to 1. Relative induction is shown, and standard deviations of triplicates are displayed. (A) The inhibitoryactivityofCWT iscompletelylostbyafterdisruptionoftheNLS,andCS gains full activity after repair of the NLS R44G mutation. (B) An ectopic heterologous SV40 large T antigen NLS can substitute for the authentic internal C protein NLS in SV40NLS-CWT⌬NLS. (C) Quantitative RT-PCR of 293T cells stimulated by expression of ⌬RIG-I (100 ng) in the presence of the indicted viral protein constructs (400 ng). IFN-␤ mRNA was normalized with GAPDH mRNA. The bars show average values from two (C) or three (A and B) experiments, and the error bars represent standard deviations. Statistical calculations were done using one-way ANOVA (ⴱ, P ⬍ 0.01; ⴱⴱ, P ⬍ 0.001; ⴱⴱⴱ, P ⬍ 0.0001; n.s., nonsignificant).

CWT⌬NLS, CS, and the repaired protein CSwtNLS, and the levels of IFN-␤ mRNA were determined by qRT-PCR. As indicated previously in the reporter gene assays, CWT was able to strongly inhibit transcription of the endogenous IFN-␤ mRNA, whereas


Here, we have identified the MV C proteins of both wild-type and vaccine strain MV as autonomous inhibitors of IFN-␤ induction. MV C protein interferes with the induction of IFN-␤ after infection with a heterologous virus (SeV DI-H4), expression of a constitutive active RLR (⌬RIG-I), or overexpression of the IRF3 kinase TBK-1 or of IRF3 itself. The differentially pronounced activities of CWT and CS were independent of the previously described interference of MV C with type I IFN signaling (60), as was clarified by experiments in Vero cells, which lack type I IFN genes and therefore cannot support positive feedback to IFN induction pathways (Fig. 2C). Using the CWT protein, we could demonstrate that activation of IRF3 by phosphorylation and nuclear accumulation of IRF3 was not disturbed in the presence of C (Fig. 6), revealing that a nuclear function of C is responsible for interference with IFN-␤ transcription. This was corroborated by comparison of the subcellular localizations of CWT and CS and of diverse C mutants, which revealed a clear correlation of nuclear localization and inhibition of IFN-␤ induction (Fig. 5). The reduced nuclear accumulation of the C protein of MV Schwarz, a member of the Edmonston lineage (1, 46), was due to a mutation in the NLS, but

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FIG 6 MV C interferes with IRF3-dependent transcription (A) without inhibiting IRF3 phosphorylation (B), dimerization (C), or nuclear accumulation (D). (A) Dual-luciferase assays were performed with p55c1b comprising the IRF3 response elements from the IFN-␤ promoter. Plasmids encoding the indicated protein constructs (125 to 750 ng for C; 200 ng for fl-IRF-3) were transfected into 293T cells, and induction of p55c1b-luc-directed FL activity was determined 24 h p.t. The assays were normalized with coexpressed RL, and mock values were set to 1. Standard deviations of triplicates are displayed. Statistical calculations were done using one-way ANOVA (ⴱⴱ, P ⬍ 0.001; ⴱⴱⴱ, P ⬍ 0.0001). (B) 293T cells expressing MV C, RV P, and fl-MV H, along with Flag-tagged IRF3 (fl-IRF3), were infected with DI-H4 as indicated to stimulated activation of IRF3. Phosphorylation of the IRF3 S386 residue was analyzed in Western blots of whole-cell lysates harvested 12 h posttransfection. (C) Native PAGE of whole lysates collected from 293T cells expressing the indicated proteins 24 h posttransfection of fl-TBK-1. IRF3 dimerization was analyzed by staining with anti-IRF3 antibodies. (D) HeLa cells were mock infected or infected with SeV DI-H4. At 18 h p.i., endogenous IRF3 was visualized by staining with anti-IRF3 antibody and Alexa 488 (green), and fl-CWT was expressed from transfected plasmids by anti-Flag and Alexa 555 (red). Nuclei were stained with ToPRO3 (white). Representative examples are shown.

upon repair of the NLS, this protein was as effective in IFN-␤ inhibition as the wt protein. The inhibition by MV C of a step downstream of IRF3 activation fills a gap in the knowledge of viral control of IFN-inducing pathways, since the MV V protein is so far considered to merely prevent activation of MDA5, but not of RIG-I (10, 45), which is the major sensor of MV infection (26, 51). However, it is noteworthy that here we observed significant inhibition of TBK-1stimulated IFN induction by the VWT protein, as well (Fig. 1C). Whether this can be entirely attributed to the recently observed inhibition of NF-␬B activity (59), which plays a role in early IFN-␤ induction in human cells (21), or whether V has additional targets downstream of MDA5 and TBK-1, as was reported for V proteins of rubulaviruses and of SeV (32, 69), remains to be clarified. In any case, it is obvious that both V and C proteins of MV do contribute to inhibition of MDA5-independent IFN-␤ induction. While V is predicted to act in the cytoplasm, one target step of C is in the nucleus, as determined here. A comparison of the protein sequences of CWT and CS, and of C proteins from other measles viruses, readily indicated the reason behind the differential IFN-␤ inhibitory capacities of the proteins. As reported previously for another wt MV isolate, CWT contains a typical NLS, which mediates active import into the cell nucleus, and a Crm1-dependent nuclear export signal (NES)-like sequence (40). Of the 7 amino acid exchanges between the CWT and CS proteins, 1 affected the NLS, while the NES remained unchanged (Fig. 3), suggesting different subcellular locations of the proteins.

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Because the MV C protein is small enough to diffuse through nuclear pores, the loss of the NLS in CS was not expected to result in complete exclusion from the nucleus, but rather, in a more pronounced cytoplasmic localization, whereas CWT actively accumulated in the nucleus. This was confirmed by (i) repairing the CS NLS, resulting in nuclear accumulation; (ii) destruction of the NLS of CWT, resulting in a more cytoplasmic location; and (iii) the rescue of nuclear localization of the latter mutant by an ectopic SV40 NLS (Fig. 4). The last experiment also revealed that the primary basic amino acid sequence of the NLS is not involved in the inhibitory mechanism in the nucleus but is only required to accumulate C in the nucleus. The mechanism of inhibition in the nucleus remains to be revealed. Though formally not excluded, a specific influence on IFN-␤ mRNA stability or nuclear export by C is very unlikely, as indicated by corresponding results from reporter gene assays and qRT-PCR of the endogenous IFN-␤ mRNAs. A more likely target is transcription from the IFN-␤ promoter, which is a highly ordered and tightly controlled process involving the assembly of an enhanceosome comprising AP1, IRF3/7, and NF-␬B at the IFN-␤ enhancer, which probably requires interchromosomal interactions, followed by histone modifications, and recruitment of the transcriptional coactivator CBP/p300 and the Pol II complex (for a review, see reference 17). Although CS protein can moderately interfere with NF-␬B activity (59), this activity alone is most likely insufficient for mediating the observed strong inhibition of IFN-␤ production. Physical association of C with IRF3, or degradation of

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IRF3, as was observed for several positive-strand RNA and DNA viruses (67), was not apparent in preliminary experiments (data not shown). Further biochemical analyses should reveal the target structures of nuclear MV C. Notably, the significance of nuclear inhibition of IFN-␤ by MV C is supported by the recent observation that the C protein of rinderpest virus (RPV), which is closely related to MV (18), also inhibits the induction of IFN without preventing the activation of IRF3 (5). A potential NLS is also present in the RPV C protein sequence, though it is not conserved and does not correspond to that of MV C (5). Although the role of nuclear localization of RPV C in IFN-␤ inhibition was not revealed in this work, a similar role of C in this animal morbillivirus can be assumed. Most intriguingly, all MV vaccine strains analyzed in this study possessed the R44G mutation in their NLSs, clearly distinguishing them from wt isolates, which have all retained a functional NLS. In the P and V proteins, this mutation merely causes a conservative exchange from arginine to lysine, which seems to be acceptable, as no differences between vaccine- and wt-derived V and P proteins in terms of IFN-␤ (unpublished data) or NF-␬B (59) inhibition were apparent. Changing other amino acids of the NLS might result in more dramatic changes, thereby favoring the R44 mutation. The four vaccine strains included in Fig. 3 as examples were generated from wt isolates of different origins, mostly by multiple passaging on interferon-negative cells (1). It is therefore suggested that in vivo there is a selective pressure on the conservation of the NLS in wt isolates, which is lost in cell culture, where other selection criteria, such as speed of replication, might be more pertinent. While on one hand the reduction of nuclear C protein in MV vaccine strains should result in less powerful inhibition of IFN-␤ induction in the nucleus, on the other hand, the gain in cytoplasmic C protein may have advantageous effects. MV C protein is obviously involved in supporting viral transcription and replication in the cytoplasm (1, 2, 57), probably by interacting with the viral polymerase L (57) or with viral RNA (36, 37). Indeed, deletion of C results in viruses severely compromised in replication and production of infectious virions (13, 65). In addition, a cytoplasmic location of C is required for association with activated STAT1 (70), which should contribute to attenuation of IFN signaling (60). Importantly, in the viral context, cytoplasmic C protein was shown to contribute to the control of IFN-␤ production by restraining activation of PKR and ATF-2 (34, 65), probably by limiting the generation of viral dsRNA, as observed for the C protein of human parainfluenza virus type 1 (4). This is a task that presumably cannot be performed by nuclear C. Intriguingly, in this respect, we observed only minor differences between CWT and CS in counteracting IFN-␤ promoter activity in cells infected with SeV DI-H4, which strongly activates PKR (64). In contrast, CS revealed major defects after stimulation of cells with the downstream TBK-1. This prompts the speculation that cytoplasmic MV C can prevent dsRNA-dependent PKR activation in the course of a heterologous virus infection, or even independent of virus infection. Evidently, the nuclear localization of C, as well as the capacity to shuttle, is important in the course of the viral life cycle. In early stages of wt MV infection, C was found to reside in the nucleus (3, 40), whereas in later stages of infection, it colocalizes with the RNPs (13). Therefore, when comparing IFN-␤ induction of wt,


vaccine strain, and C-deficient viruses (28, 36), the fundamentally different growth characteristics of these viruses have to be considered, as well as differential expression of replication products, which might act as ligands for RLR, and the location-dependent functions of C. In particular, wt MV seems to control viral gene expression and growth much more restrictively than vaccine strain or C-deficient viruses, and therefore, the different control mechanisms may apply differently. To our knowledge, however, apart from the well-known mutations leading to differential receptor use during adaptation of MV to cell culture and to its attenuation (68), the R44 mutation is the first marker common to all vaccine strains. In other words, the intact NLS seems to define all MV wild-type isolates. We conclude that the R44 mutation in vaccine strains and the altered intracellular localization contribute to attenuation. However, whether this is primarily due to a reduced capacity to counteract IFN in the nucleus or to other functions of C that are affected through differential localization remains to be determined. ACKNOWLEDGMENTS We thank Nadin Hagendorf for perfect technical assistance. The MV isolate MVi/Berlin.DEU/04.08 of the D5 genotype was kindly provided by A. Mankertz (RKI Berlin), the Vero-SLAM cell line by Y. Yanagi, and SeV DI-H4 by D. Garcin. Antibodies were provided by R. Cattaneo, and plasmids by T. Fujita and J. Hiscott. We thank Kerstin Schuhmann for constructive feedback on the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) through GraKo 1202, SFB 455, and SFB 870.

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