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J Virol. Aug 2009; 83(16): 7828–7841.
Published online Jun 10, 2009. doi:  10.1128/JVI.02610-08
PMCID: PMC2715789

Nipah Virus Sequesters Inactive STAT1 in the Nucleus via a P Gene-Encoded Mechanism [down-pointing small open triangle]

Abstract

The Nipah virus (NiV) phosphoprotein (P) gene encodes the C, P, V, and W proteins. P, V, and W, have in common an amino-terminal domain sufficient to bind STAT1, inhibiting its interferon (IFN)-induced tyrosine phosphorylation. P is also essential for RNA-dependent RNA polymerase function. C is encoded by an alternate open reading frame (ORF) within the common amino-terminal domain. Mutations within residues 81 to 113 of P impaired its polymerase cofactor function, as assessed by a minireplicon assay, but these mutants retained STAT1 inhibitory function. Mutations within the residue 114 to 140 region were identified that abrogated interaction with and inhibition of STAT1 by P, V, and W without disrupting P polymerase cofactor function. Recombinant NiVs were then generated. A G121E mutation, which abrogated inhibition of STAT1, was introduced into a C protein knockout background (Cko) because the mutation would otherwise also alter the overlapping C ORF. In cell culture, relative to the wild-type virus, the Cko mutation proved attenuating but the G121E mutant virus replicated identically to the Cko virus. In cells infected with the wild-type and Cko viruses, STAT1 was nuclear despite the absence of tyrosine phosphorylation. This latter observation mirrors what has been seen in cells expressing NiV W. In the G121E mutant virus-infected cells, STAT1 was not phosphorylated and was cytoplasmic in the absence of IFN stimulation but became tyrosine phosphorylated and nuclear following IFN addition. These data demonstrate that the gene for NiV P encodes functions that sequester inactive STAT1 in the nucleus, preventing its activation and suggest that the W protein is the dominant inhibitor of STAT1 in NiV-infected cells.

Nipah virus (NiV) is a highly lethal member of the family Paramyxoviridae, genus Henipavirus. NiV was first recognized following a 1998-99 outbreak in Southern Malaysia and Singapore (7), and outbreaks have been recognized in India and almost annually in Bangladesh (5, 23). The large Malaysian outbreak was marked by severe, fatal encephalitis with 40% mortality, whereas the smaller, more recent Bangladeshi and Indian outbreaks displayed higher mortality rates (75 to 92%), potential human-to-human transmission, and an increased occurrence of severe respiratory disease (5, 17, 22). In addition to its high lethality, NiV is unique among paramyxoviruses in that it exhibits a relatively broad host range and is able to infect bats, pigs, humans, cats, dogs, and other species (7, 8, 12, 38).

Signal transducer and activator of transcription 1 (STAT1), a member of the STAT family of transcription factors, is a critical component of the JAK/STAT signaling pathways activated by alpha/beta interferon (IFN-α/β), IFN-γ, and other cytokines and growth factors (44). STAT protein activation involves tyrosine phosphorylation by JAK family kinases, resulting in STAT homo- or heterodimerization via SH2 domain-phosphotyrosine interactions (10, 28, 35). This directs the accumulation of STAT proteins in the nucleus, where they are able to modulate transcription. In the case of IFN-α/β signaling, STAT1-STAT2 heterodimers principally form, and these further complex with IFN regulatory factor 9 to create a transcription factor complex called ISGF-3 (10, 28). Presumably because IFNs are central to innate antiviral immunity, many viruses have evolved mechanisms to stop their production and to block STAT-dependent IFN signaling (16). Among nonsegmented negative-strand RNA viruses, including several paramyxoviruses, mechanisms have evolved to target STAT1 or STAT2 (1, 24, 29, 36, 37, 46). Among the best-characterized inhibitors of IFN production and STAT signaling are the V and W proteins of the paramyxoviruses (reviewed in references 14 and 21).

The NiV P gene encodes four proteins, C, P, V, and W (20, 26, 48). Faithful transcription of the P gene yields an mRNA that encodes the P protein (709 amino acids), an essential cofactor for the viral RNA polymerase which interacts with the viral nucleoprotein (N) and polymerase (L). The V and W proteins (456 and 450 amino acids, respectively) are encoded by edited transcripts where the viral polymerase adds nontemplated guanosine residues to the mRNA at a cis-acting editing site, causing a frameshift during translation (27, 45). As a result of this coding strategy, P, V, and W possess the same amino terminus but differ at their carboxy termini. The C protein is encoded by an internal alternate reading frame present in transcripts encoding P, V, or W (26, 48).

In transfection experiments, NiV P gene products suppress both the production of and signaling by IFN. V binds the cytoplasmic helicase mda-5 and inhibits activation of the IFN-β promoter, and both the V and W proteins block IFN regulatory factor 3-dependent gene expression (6, 41). The P, V, and W proteins all block the cellular response to IFN by binding to and preventing the tyrosine phosphorylation of STAT1 (39, 40, 42). Notably, following their individual expression, P and V are cytoplasmic and retain STAT1 in the cytoplasm; W, however, localizes to the nucleus and retains unphosphorylated STAT1 there. In one study, amino acids 50 to 150 from the amino terminus common to P, V, and W were sufficient to interact with STAT1 and to inhibit IFN-induced gene expression (42). In a separate study, residues 100 to 160 were sufficient to interact with STAT1 (39).

The ability of NiV P, V, and W to inhibit STAT1-dependent IFN signaling has thus far been demonstrated only in transfection experiments and not in NiV-infected cells. In the present study, mutations were identified that significantly impair STAT1 binding and IFN signaling inhibition by P, V, and W without abrogating P polymerase cofactor function. With these data and a newly established NiV reverse genetics system, recombinant NiVs were generated, including mutant viruses predicted to lack the STAT1 binding activity of P, V, and W. The NiV P gene was demonstrated to encode functions that regulate the trafficking and prevent the activation of STAT1 by sequestering it in the nucleus. These data suggest that the W protein is the dominant inhibitor of STAT1 in NiV-infected cells.

MATERIALS AND METHODS

Cells, antibodies, and expression plasmids.

For transfection experiments, HEK 293T and BSR-T7/5, a BHK-21 cell line stably expressing T7 RNA polymerase (2), cells were maintained in Dulbecco's modified Eagle medium (DMEM; Gibco, Carlsbad, CA) supplemented with fetal bovine serum to 10%. For generation of recombinant viruses, BSR-T7/5 cells were grown in Glasgow medium (Invitrogen, Carlsbad, CA) supplemented with 10% newborn calf serum, 29.5 g/liter tryptose phosphate broth (Eurobio, Paris, France), 2× nonessential amino acid mixture (Invitrogen), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine, and 0.5 mg/ml Geneticin (Gibco).

The anti-NiV M antibody was raised in rabbits against a peptide corresponding to amino acids 27 to 40 of the NiV M protein.

pSL1180 NiV GFP-CAT, a plasmid that produces a NiV minigenome RNA from a T7 promoter, was constructed by flanking a reporter gene encoding a green fluorescent protein (GFP)-chloramphenicol acetyltransferase (CAT) fusion protein with NiV genomic leader and trailer sequences. The leader and trailer sequences correspond to GenBank accession number AY029767 and were constructed by template-free PCR with overlapping deoxyoligonucleotides. The hepatitis delta virus ribozyme and T7 terminator sequences were placed adjacent to the leader sequence. A T7 promoter sequence was cloned adjacent to the NiV trailer sequence. The minigenome length was made to be divisible by six by adding nucleotides between the GFP-CAT gene and the L noncoding region. The three fragments were assembled into the pSL1180 vector.

The pCAGGS NiV P, V, and W constructs have been described previously (42). The P gene was hemagglutinin (HA) tagged at the amino terminus and subcloned into the pTM1 expression plasmid. All mutations were generated with the QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA). The pTM1-NiV N and L plasmids used for the minireplicon assay system were kindly provided by Paul Rota (Centers for Disease Control and Prevention, Atlanta, GA). The pCAGGS STAT1-GFP plasmid was described previously (36).

Minireplicon assay.

BSR-T7/5 cells were transfected with 3.5 μg pSL1180 NiV GFP-CAT minigenome; 0.05, 0.1, or 0.2 μg pTM1 HA NiV P; 0.75 μg pTM1 NiV N; 0.4 μg pTM1 NiV L; and 0.05 μg pTM1 firefly luciferase with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. At 24 h posttransfection (hpt), transfected cells were lysed in reporter lysis buffer (Promega, Madison, WI) and analyzed for CAT and luciferase expression. The CAT activity was quantified by PhosphorImager and normalized to the luciferase activity. The activity levels presented are relative to the activity of 50 ng of wild-type (WT) P, which was set to 100%.

Assay for IFN-induced gene expression.

293T cells (1.2 × 106) were transfected with 0.3 μg of plasmid encoding firefly luciferase under the control of the IFN-β-responsive ISG54 promoter, 0.05 μg of pRLTK encoding Renilla luciferase, and 2 μg of the indicated expression plasmids as described previously (37). At 24 hpt, 1,000 IU of IFN-β (PBL, Piscataway, NJ) was added to the medium. At 16 h posttreatment, cells were lysed and reporter gene expression was measured by dual-luciferase assay (Promega). Firefly luciferase values were normalized to Renilla luciferase values. Induction was calculated relative to that of an empty-vector-transfected, untreated control. Immunoblotting was performed with mouse monoclonal antibodies raised against the HA and β-tubulin epitopes (Sigma-Aldrich, St. Louis, MO).

Immunoblotting and immunoprecipitation.

To determine levels of STAT1 phosphorylation in 293T cells treated with IFN-β, cells were transfected with expression plasmids encoding the indicated NiV proteins and STAT1-GFP expression plasmids. Twenty-four hours later, the cells were serum starved for 3 h and then treated with medium containing 1,000 U IFN-β for 1 more h. The cells were then lysed in lysis buffer (50 mM Tris, pH 8.0, 280 mM NaCl, 0.5% NP-40, 0.2 mM EDTA, pH 8.0, 2 mM EGTA, 10% glycerol) supplemented with 1 mM dithiothreitol, 5 mM sodium orthovanadate, and protease inhibitor cocktail (Complete; Roche, Mannheim, Germany). Western blots of the cell lysates were probed with antibodies specific for STAT1 (BD Biosciences, San Jose, CA) or the tyrosine 701-phosphorylated form of STAT1 (Cell Signaling, Danvers, MA).

To detect the interaction of STAT1 with the WT and mutant NiV P proteins, 293T cells were transfected with the indicated expression plasmids. At 24 hpt, cells were lysed in lysis buffer as described above. Lysates were incubated with anti-HA antibody-conjugated resin (Sigma-Aldrich) at 4°C for 2 h with gentle agitation. After extensive washing, the precipitated proteins and respective whole-cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed with antibodies against STAT1 (BD Biosciences) and the FLAG, HA, or β-tubulin epitope (Sigma-Aldrich), as indicated.

Generation of recombinant NiVs.

The NiV genome was amplified, in fragments, from purified virus genomic RNA by reverse transcription-PCR. Specifically, the 18,246-nucleotide (nt) genome (corresponding to GenBank accession no. AY029767) was assembled into thirds from small PCR products. Each third (nt 1 to 6780, 6780 to 10404, and 10404 to 18246) was cloned so that each segment could be mutated individually and later assembled into a full-length cDNA clone in the pSL1180 cloning vector. T7 promoter and terminator sequences and hepatitis delta virus ribozyme sequences were appended via PCR (see Fig. Fig.7A),7A), and three NiV full-length clones (pFL-NiV WT, pFL-NiV CKO, and pFL-NiV CKO P G121E) were constructed. The C knockout (Cko) virus was generated by mutating the two initiating methionine codons in the C open reading frame (ORF) of the P, V, or W gene (nt 2406 to 4535 in the genome) from ATG to ACG (t2429c, t2432c). To further ensure the knockout of this ORF, a stop codon was also introduced into the C ORF (nt c2438a) without affecting the P, V, or W ORF. The G121E mutation (nt g2767a) in P, V, or W was engineered into the Cko backbone to generate the G121E mutant NiV.

FIG. 7.
Growth kinetics of WT and mutant NiVs. (A) Illustration of the recombinant NiV genome used to generate WT, Cko, and G121E mutant NiVs. Triangles indicate nucleotide changes designed to knock out the expression of the C ORF, and the star indicates the ...

BSR-T7/5 cells were cotransfected with plasmids encoding full-length NiV antigenomes (pFL-NiV-WT, pFL-NiV-CKO, and pFL-NiV-P-G121E) and pTM1 N, P, and L at the same ratios used for the minireplicon assay described above. At 72 hpt, virus-containing medium was harvested from transfected cells and passaged onto Vero E6 cells. To generate virus stocks, Vero E6 cells were inoculated with virus at a multiplicity of infection (MOI) of 0.01. The virus-containing medium was harvested at 48 h postinfection, clarified by low-speed centrifugation, aliquoted, and stored at −70°C. The presence of introduced mutations was confirmed by direct sequencing of reverse transcription-PCR fragments amplified from virus RNA isolated from virus stocks with an RNeasy kit (Qiagen, Valencia, CA). Virus stocks were also analyzed for the presence of NiV M protein by Western blotting with rabbit anti-NiV M polyclonal antiserum. Virus titers were determined after infection of Vero E6 cells by using serial dilutions and determining the 50% tissue culture infective dose.

All experiments involving full-length genomes and infectious NiV were carried out in the Jean Merieux P4 Center biosafety level 4 (BSL4) laboratory in Lyon, France.

Virus growth kinetics.

293T or Vero E6 cells were inoculated with equal amounts of the WT, Cko, and G121E mutant viruses as determined by Western blot analysis with anti-M antibody. This amount for the WT virus corresponded to an MOI of 0.05. Both the Cko and G121E mutant viruses were determined to be approximately 200 times less infectious than the WT virus. Culture supernatants were collected at days 1 to 3 and analyzed by 50% tissue culture infective dose titration on Vero E6 cells.

STAT1 localization in NiV-infected cells.

To monitor the effect of infection on STAT1-GFP localization, Vero E6 cells were transfected with pCAGGS STAT1-GFP and infected at 10 hpt with recombinant NiVs (WT, Cko, or G121E) at an MOI of 0.2. At 17 h postinfection, the cells were washed and starved in serum-free medium supplemented with 0.3% bovine serum albumin (BSA) for 1 h at 37°C. Cells were treated with 1,000 U/ml human IFN-β (PBL) in serum-free medium for 40 min. STAT1-GFP localization was observed in live cells under BSL4 containment by fluorescence microscopy.

To monitor the effect of infection on endogenous STAT1, Vero E6 cells grown on glass coverslips in a 12-well plate were mock infected or infected with recombinant NiVs (WT, Cko, or G121E) at an MOI of 0.2. At 1 h postinfection, the virus inoculum was replaced with DMEM containing 2.5% fetal bovine serum and the cells were incubated at 37°C for 12 h. At 12 h postinfection, the cells were washed with serum-free medium and then treated with 1,000 U/ml human IFN-β (PBL) in serum-free DMEM supplemented with 0.3% BSA for 40 min at 37°C. For untreated controls, infected cells were incubated with IFN-free medium. Cells were washed twice with cold phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde-PBS solution for 20 min at room temperature, and subsequently treated with 0.1 M glycine for 10 min. Cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min and washed with a blocking solution (2% BSA and 0.2% Tween 20 in PBS). Immunofluorescence staining was performed with mouse anti-phospho-Stat1 (pY701) antibody or rabbit anti-NiV M antibody. To visualize the nuclei, the cells were stained with 0.1 μg of 4′,6-diamidino-2-phenylindole hydrochloride (DAPI).

RESULTS

The amino terminus of P is essential for its function in a minireplicon assay.

Amino acids 50 to 150 were previously shown to be sufficient for STAT1 to interact with the common amino-terminal domain shared by the NiV P, V, and W proteins (39, 40, 42). To determine whether this region is essential for the function of P in viral RNA synthesis, a NiV minireplicon system was developed. In the minireplicon assay, BSR-T7 cells, which constitutively express T7 RNA polymerase, are transfected with plasmids that express from T7 promoters a replica minigenome viral RNA encoding a GFP-CAT fusion protein and the NiV N, P, and L proteins, which reconstitute the viral RNA polymerase complex. The negative-sense minigenome RNA is encapsidated by the nucleocapsid (N) protein and transcribed and replicated by the reconstituted viral RNA polymerase, P, and L. GFP-CAT reporter expression is indicative of the efficiency of the polymerase function, and CAT activity was used for the quantitative measurement of polymerase activity. The system was optimized by systematically adjusting the ratio of N, P, and L plasmids transfected by using as a starting point the ratios used by Halpin et al. (19). As has been seen in similar minigenome systems for NiV and other nonsegmented negative-strand RNA viruses (15, 25, 34), the NiV system proved to be sensitive to variations in the expression of the P protein; specifically, increasing amounts of transfected P plasmid from 50 to 200 ng resulted in decreasing levels of CAT activity (Fig. (Fig.1).1). Decrease of the transfected amount of P plasmid to 25 and 12.5 ng also decreased polymerase activity (data not shown), indicating that 50 ng approximates the optimal amount of WT P expression plasmid for this assay.

FIG. 1.
Residues 81 to 120 of NiV P are important for function in viral RNA synthesis. BSR-T7 cells were transfected with the NiV GFP-CAT minigenome plasmid along with T7 expression plasmids encoding NiV N, L, and the indicated HA-tagged NiV P constructs (wedges ...

Initial gross deletion of the amino-terminal 50, 100, or 150 amino acids of P resulted in mutants lacking any detectable function in the minireplicon assay (data not shown), suggesting that the P amino terminus is required for viral RNA synthesis. Therefore, a series of internal-deletion mutants was generated by targeting the amino acid 50 to 150 region in 10-amino-acid increments, and these were assayed in the minireplicon system (Fig. (Fig.1).1). To control for any variation in GFP-CAT reporter induction due to differential expression of the various P mutant constructs, three different amounts (50, 100, and 200 ng) of P plasmid were transfected. Figure Figure11 shows that WT P supports the minireplicon and that the mutants with deletions between amino acids 51 and 80 and amino acids 121 and 150 function comparably to the WT. Interestingly, although the Δ51-60 and Δ61-70 mutants displayed WT-like activity at the lowest concentration of P plasmid transfected, the intermediate (100 ng) transfection yielded decreased activity (Fig. (Fig.1A).1A). This may reflect the modestly higher expression levels of mutant P relative to that seen in the corresponding transfection with the WT P plasmid (Fig. (Fig.1A).1A). In contrast, the deletions within the amino acid 81 to 120 region (Δ81-90, Δ91-100, Δ101-110, and Δ111-120) caused a substantial decrease in reporter expression, indicating that this region plays a critical role in polymerase function (Fig. 1A and B).

Amino acids 111 to 140 of P are required for inhibition of IFN-α/β signaling.

We next sought to determine whether those regions of the P amino terminus critical for polymerase function are also essential for P-mediated inhibition of IFN signaling. The 10 deletion mutants were transfected into 293T cells along with an IFN-inducible ISG54 promoter-firefly luciferase (ISG54-luciferase) reporter construct and a constitutively expressed Renilla luciferase plasmid to control for transfection efficiency. Luciferase levels were measured at 16 h after IFN-β treatment (Fig. (Fig.2).2). Mutants with deletions between amino acids 51 and 110 and amino acids 141 and 150 efficiently inhibit induction comparably to WT P. The three deletion mutants that fail to antagonize IFN signaling span residues 111 to 140 (Δ111-120, Δ121-130, and Δ131-140) (Fig. (Fig.2).2). These data suggest that this 30-amino-acid region is required for inhibition of IFN signaling.

FIG. 2.
NiV P residues 111 to 140 are critical for efficient inhibition of IFN signaling. NiV P WT and mutant constructs were cotransfected with an IFN-inducible, ISG54-driven firefly luciferase plasmid and a Renilla luciferase plasmid into 293T cells. NiV P ...

IFN signaling mutants fail to bind and inhibit STAT1.

Previous reports have correlated the ability of P to bind and sequester STAT1 in the cytoplasm with its ability to inhibit IFN signaling (42). We therefore investigated by coimmunoprecipitation the capacity of the P mutants to interact with STAT1 (Fig. (Fig.3A).3A). In this experiment, a WT NiV W expression plasmid was included as an additional control. 293T cells were transfected with the HA-tagged WT or mutant P construct, and the P proteins were immunoprecipitated with an antibody against the HA tag. Western blotting of the immunoprecipitates with anti-STAT1 antibody indicated that the mutants that are capable of inhibiting IFN signaling (Δ51-110 and Δ141-150) retained the ability to bind endogenous STAT1 (Fig. (Fig.3A).3A). On the other hand, those three mutations that abrogated IFN signaling inhibition cause loss of detectable STAT1 binding activity (Fig. (Fig.3A).3A). Deletion of amino acids in the region of positions 111 to 140 also abolished the inhibition of STAT1 tyrosine phosphorylation in response to IFN-β treatment (Fig. (Fig.3B).3B). These mutations cause a loss of interaction with STAT1 in the V and W proteins as well (Fig. (Fig.3C),3C), indicating that this domain is critical for all three NiV IFN signaling antagonists. In combination with the ISG54 reporter data (Fig. (Fig.2),2), these data further correlate loss of STAT1 binding with a loss of IFN signaling inhibition and define amino acids 111 to 140 as critical for inhibition of IFN signaling.

FIG. 3.
NiV P, V, and W IFN signaling mutants fail to bind and inhibit STAT1. (A) 293T cells were transfected with HA-tagged NiV P WT or internal deletion constructs spanning residues 51 to 150. At 24 hpt, immunoprecipitation (IP) was performed with anti-HA antibody ...

Fine mapping of the amino acid 111 to 120 region of NiV P.

Our deletion mutagenesis indicated that loss of residues 111 to 120 abolished the function of P in both the minireplicon and IFN signaling assays. In order to determine if this region is important for both functions, we generated a series of alanine scanning mutants across this region in three-amino-acid increments. These constructs were then studied in the minireplicon (Fig. (Fig.4A)4A) and IFN signaling assays (Fig. (Fig.4B).4B). The substitution of alanine for amino acids 111 to 113 markedly reduced P function in the minireplicon system, whereas substitutions between amino acids 114 and 122 had no effect (Fig. (Fig.4A).4A). The 111-to-113 mutant inhibited IFN signaling comparably to WT P and WT W, which was included as an additional control, indicating that these residues are not required for IFN signaling inhibition (Fig. (Fig.4B).4B). The substitutions between amino acids 114 and 122 did, however, impair IFN inhibition (Fig. (Fig.4B).4B). These data implicate the 81-to-113 region of P in its polymerase cofactor function and residues 114 to 122 in its IFN-inhibitory function. Therefore, these two functions of P are separable and suggest that within the P amino terminus there are adjacent but discrete domains required for RNA synthesis and STAT1 binding.

FIG. 4.
Separation of NiV P functional domains in minireplicon and IFN signaling assays. (A) BSR-T7 cells were transfected as described for Fig. Fig.1,1, with plasmids encoding the NiV minigenome and N, L, and P WT and alanine scanning mutants. Induction ...

Mutation of G121, G125, G127, G13, or Y116 impairs inhibition of IFN signaling but does not affect P polymerase cofactor function.

We next sought to define individual residues that are important for interaction with STAT1 and inhibition of IFN signaling. Hagmaier et al. reported that a NiV V point mutant in which glycine 125 was replaced with glutamic acid was unable to inhibit IFN signaling (18). As this mutation lies in the common amino terminus of P, V, and W and within the 114-to-140 putative STAT1-binding domain, we investigated in our assays the importance of this and other glycines in the vicinity of residue 125 for replication function and for inhibition of IFN signaling. Specifically, glycines 120, 121, 127, and 135, in addition to glycine 125, were mutated to glutamic acid in our NiV P expression plasmid. We tested these mutants for their anti-IFN signaling properties, and results are shown in Fig. Fig.5.5. As was seen when the mutation was present in NiV V, the G125E P mutant was unable to inhibit IFN-induced transcription from the ISG54 promoter (Fig. (Fig.5A).5A). Inhibition of IFN signaling was also abrogated by substitution of P residues G121 and G127, and to a lesser extent G135, whereas the G120E mutant protein functioned as well as WT P. Western blotting indicated that all mutant P proteins were expressed to comparable levels. The status of interaction with STAT1 was also determined for these mutant proteins (Fig. (Fig.5B).5B). Those mutations that caused loss of signaling inhibition also caused loss of detectable STAT1 binding. Interestingly, the G135E mutant protein does not detectably interact with STAT1 but retains partial inhibition of signaling, as observed by reporter gene assay. That not all glycine substitutions disrupt inhibition of the IFN signaling pathway provides evidence that these residues (G121, G125, and G127) contribute specifically to STAT1 binding and signaling inhibition.

FIG. 5.
Single amino acid substitutions decrease NiV P's inhibition of IFN signaling and STAT1 binding. HA NiV P constructs harboring the indicated point mutations were assayed for polymerase cofactor activity, IFN signaling inhibition, and STAT1 binding as described ...

NiV P possesses a tyrosine residue at position 116 which is present within a hexapeptide sequence (114VVYHDH119). The context of this NiV tyrosine is similar to that of tyrosine 110 of the measles virus P protein (within the hexapeptide 109YYVYDH114), which is defined as critical for its inhibition of STAT1 phosphorylation and activation (4, 11, 13, 32). To test the importance of this tyrosine residue for inhibition of IFN signaling by NiV P, we generated constructs where Y116 was replaced with alanine or phenylalanine. Because of the potential for phosphorylation, we also replaced Y116 with the phosphomimetic glutamic acid residue. Replacing Y116 with alanine resulted in a P protein with a reduced ability to inhibit IFN signaling (Fig. (Fig.5D).5D). Similar results were obtained with the Y116E substitution, indicating that the phosphomimetic residue cannot replace the tyrosine residue at this position. However, replacement with phenylalanine allowed the mutant to function comparably to the WT, suggesting that an aromatic residue at position 116 is important and that phosphorylation at position 116 is not necessary for function. (Fig. (Fig.5D).5D). Figure Figure5E5E demonstrates the ability of the tyrosine mutant proteins to interact with STAT1. As expected, the Y116A and Y116E mutant proteins lacked detectable interaction with STAT1 but the Y116F mutant protein was efficiently coprecipitated with STAT1 (Fig. (Fig.5E5E).

The glycine and tyrosine point mutants were separately assayed for function in the minireplicon assay. As with the other mutant P constructs, various concentrations of P plasmid were cotransfected with constant amounts of the minigenome, N, and L plasmids. Figure 5C and F demonstrate that the point mutants all yield levels of reporter gene expression comparable to that seen with WT P, indicating that the amino acid substitutions have little or no effect on P polymerase cofactor function. Taken together, these data identify specific residues within the 114-to-140 region that are important for IFN signaling inhibition and further demonstrate that the STAT1 binding and polymerase cofactor functions of P can be separated.

Point mutations abolish the interaction of NiV V and W with STAT1.

Mutations in the amino-terminal half of the P gene will also be present in the V and W proteins. We investigated the dependence of V and W on the glycine residues defined above as critical for P-STAT1 interaction. NiV V and W constructs harboring glycine-to-glutamic-acid substitutions were expressed in 293T cells and immunoprecipitated. As we and others have previously demonstrated, STAT1 coprecipitated with WT V and W; however, glutamic acid substitutions at glycines 121, 125, 127, and 135 disrupt this interaction (Fig. (Fig.6A).6A). Under normal conditions, STAT1 is phosphorylated at Y701 in response to IFN-β treatment, and this activation of STAT1 is blocked in 293T cells expressing WT P, V, and W. In contrast, a representative point mutation, G121E, that caused loss of the P-, V-, or W-STAT1 interaction, causes the P, V, and W proteins to lose the ability to block STAT1 phosphorylation following IFN treatment (Fig. (Fig.6B).6B). As previously indicated (42), the STAT1-inhibitory activity of the V and W proteins appears to be stronger than that of P.

FIG. 6.
Point mutant forms of NiV V and W do not interact with or inhibit STAT1. (A) Plasmids encoding the HA-tagged NiV V and W WT or mutant proteins were transfected in 293T cells and immunoprecipitated (IP) as described in the legend to Fig. Fig.3. ...

A P gene-encoded function regulates STAT1 localization and activation during NiV infection.

Taking this information and using a newly developed reverse genetics system, we generated recombinant WT NiV or NiVs possessing either an intact or a defective P, V, or W STAT1-binding domain (as illustrated in Fig. Fig.7A).7A). The region required for STAT1 binding in P, V, and W (amino acids 114 to 140) is overlapped by the ORF that encodes the C protein. Thus, including STAT1 binding mutations in the P gene would result in mutation of the C protein. Therefore, mutations to the P gene were engineered in a Cko background where C expression was blocked by mutation to ACG of the first two AUG codons of the C ORF and by replacement of the fourth codon with a stop codon (indicated by triangles in Fig. Fig.7A).7A). All of these mutations are silent for the P, V, and W proteins. The P, V, or W STAT1-binding region was mutated by introducing the G121E mutation, which causes a loss of STAT1 binding, as demonstrated in Fig. Fig.55 and and66 (indicated by the star in Fig. Fig.7A).7A). The growth of the resulting recombinant viruses was compared in 293T and Vero E6 cells (Fig. 7B and C, respectively). Interestingly, the disruption of the C ORF attenuates NiV growth in both cell lines compared to WT virus growth. However, the G121E mutant does not display further attenuation and replicates with kinetics identical to those of the Cko virus.

To determine the phosphorylation status of STAT1 in infected cells, WT, Cko, and G121E mutant virus-infected Vero cells were treated with IFN-β. Forty minutes after IFN-β addition, an indirect immunofluorescence assay was performed to detect endogenous, tyrosine-phosphorylated STAT1. Staining was also performed for the NiV M protein as a marker of infection (Fig. (Fig.8).8). Little to no tyrosine-phosphorylated STAT1 was detectable in the nuclei of cells infected with the WT and Cko viruses, which possess intact P, V, and W STAT1-binding domains, whereas adjacent, uninfected cells exhibited strong nuclear phospho-STAT1 staining (Fig. (Fig.8).8). In contrast, a strong tyrosine-phosphorylated STAT1 signal was present in G121E mutant virus-infected cells following addition of IFN (Fig. (Fig.8,8, right column). Consistent with this, when nuclear phospho-STAT1 was observed in Cko virus-infected cells, the signal was of substantially lower intensity than that seen in G121E mutant virus-infected cells (Fig. (Fig.8).8). Upon examination, 91% (68 out of 75) of the G121E mutant virus-infected, IFN-treated cells contain phosphorylated STAT1 in their nuclei, in contrast to only 33% of Cko virus-infected cells (27 out of 82).

FIG. 8.
Impact of WT and mutant NiVs on the localization and phosphorylation of endogenous STAT1. Vero E6 cells were infected with the indicated viruses and treated with IFN-β at 12 h postinfection. The phospho-Y701 form of endogenous STAT1 (P-STAT1, ...

Next, to investigate the localization of STAT1 during infection, Vero E6 cells expressing STAT1-GFP were mock infected or infected with the WT, Cko, or G121E mutant virus. In mock-infected cells, STAT1-GFP is localized in the cytoplasm and translocates to the nucleus upon IFN treatment (Fig. (Fig.9,9, left column). At 17 h postinfection, a time when infection has progressed such that syncytia are clearly evident, the WT and Cko virus-infected cells exhibited a striking phenotype. STAT1-GFP was exclusively localized to the nucleus (seen as clustered nuclei in syncytia) before or 40 min after the addition of 1,000 U of IFN-β, and STAT1-GFP remained nuclear even 24 h after IFN was added (Fig. (Fig.9,9, middle column). This pattern was identical in both WT and Cko NiV-infected cells and is reminiscent of the pattern seen in cells expressing the NiV W protein (42). In contrast, in G121E mutant virus-infected cells (Fig. (Fig.9,9, right column), STAT1-GFP was exclusively cytoplasmic prior to IFN-β addition. Forty minutes after IFN-β addition, the STAT1-GFP subcellular localization was mostly nuclear and 24 h after IFN addition, STAT1-GFP had regained its cytoplasmic distribution (Fig. (Fig.9,9, left column). This pattern mirrors what was seen in mock-infected Vero cells and suggests that the G121E mutation renders the virus unable to disrupt normal STAT1 trafficking. These data indicate that the P gene encodes a function that directs STAT1 to the nucleus such that it is unable to be tyrosine phosphorylated. Therefore, although NiVs possessing disrupted P, V, and W STAT1-binding domains are replication competent, they are unable to sequester STAT1 in the nucleus to prevent its activation by IFNs.

FIG. 9.
WT and Cko viruses direct STAT1 to the nucleus, but the G121E mutant virus fails to control STAT1 localization. STAT1-GFP localization following NiV infection and subsequent IFN treatment. Vero E6 cells were transfected with STAT1-GFP plasmid, and at ...

DISCUSSION

This study has identified regions of NiV P that, when mutated, abrogate its function in viral RNA synthesis but do not impair its ability to inhibit STAT1 activation. Conversely, it has also identified regions of the P protein that, when mutated, have little impact on viral RNA synthesis but greatly impair P inhibition of STAT1 activation. Importantly, these latter mutations, when introduced into the V or W protein, also impair their STAT1-inhibitory function. In addition to providing insight into how the NiV P protein encodes both a polymerase cofactor and a STAT1-inhibitory function, this work suggested strategies that allowed the generation of recombinant NiVs lacking the ability to inhibit STAT1 function. Analysis of these recombinant viruses revealed a striking and unique phenotype: viruses expressing WT P, V, and W fully sequester STAT1 in the nucleus in a non-tyrosine-phosphorylated state. In contrast, the G121E mutant virus further demonstrates that the NiV P gene encodes functions that direct unphosphorylated STAT1 to the nucleus to prevent its activation.

Our investigation demonstrates that the two functions previously ascribed to the NiV P protein, polymerase cofactor and inhibitor of IFN signaling, are separable. Our P mutants identify a short stretch of amino acids, from 114 to 140, critical for inhibition of STAT1 activation by IFN-β. Mutations within this region, whether 10-amino-acid deletions or any of several point mutations, abrogated P protein inhibition of IFN-β-induced gene expression. Loss of this function correlated with an inability of the mutants to coprecipitate with STAT1 and to inhibit IFN-β-induced STAT1 tyrosine phosphorylation. These data are consistent with previous studies that focused on defining a STAT1-binding domain (defined as residues 50 to 150 or 100 to 160) within the NiV V and W proteins (39, 40, 42), but here we narrow this region to only 27 amino acids that, when in the context of a full-length P protein, are required for STAT1 binding and inhibition of IFN signaling. Importantly, we find that the same mutations, when introduced into V or W, also fully abrogate STAT1 binding and inhibition of IFN-β-induced STAT1 phosphorylation. It will also be of interest to determine whether expression of the 114-to-140 region alone is sufficient for STAT1 binding and for inhibition of IFN signaling. Further exploration of the precise mechanism by which interaction of STAT1 with P, V, or W inhibits tyrosine phosphorylation is also warranted. Previous studies demonstrated that NiV V directed both STAT1 and STAT2 into high-molecular-weight complexes, whereas W sequestered STAT1 in the nucleus (40, 42). Whether the cytoplasmic P protein functions identically to V remains to be determined. Finally, it has been demonstrated that NiV P, V, and W interact with polo-like kinase 1 (PLK1), and this interaction results in V phosphorylation (30). Notably, the PLK1-binding site overlaps the STAT1-binding site on P, V, and W, and mutations that disrupt the V-PLK1 interaction also disrupt the V-STAT1 interaction. However, the same mutations do not affect P replication function (30). It will be of interest to determine which of the mutations described above also affect the P or V interaction with PLK1.

Many viruses target STAT1 to disrupt the upregulation of IFN-stimulated genes (16). The NiV P, V, and W proteins display a physical interaction with STAT1 that, in contrast to the case of the V proteins of SV5 and other rubulaviruses, does not result in the degradation of STAT1. Rather, the NiV proteins, when expressed individually, appear to sequester STAT1 away from the activating Janus kinases. However, this interaction is not unique among paramyxoviruses, as binding without STAT1 degradation has been described for the Sendai virus C proteins and the V proteins of measles virus (4, 32, 33) and rinderpest virus (31). The phosphoprotein of rabies virus, a member of the family Rhabdoviridae, can bind tyrosine-phosphorylated STAT1 (1, 46, 47). Our data here point to a stretch of 27 amino acids (114 to 140) as the STAT1-binding domain of NiV P, V, or W. The identification of this domain may provide the ability to predict such interactions among other viral proteins and may also provide more insight into the precise mechanism of NiV P's STAT1-inhibitory function.

The requirement of residues 81 to 113 for viral polymerase function is not readily explained by our experiments or by studies of other P proteins. During the course of viral RNA synthesis, the paramyxovirus phosphoprotein oligomerizes and interacts with both the N and L proteins (27). The loss of function seen after mutating residues 81 to 113 is not due to the lack of interaction with NiV N or with itself, as shown by coimmunoprecipitations (data not shown). In our systems, the interaction of P with L is not readily demonstrated because L is not expressed to levels detectable by Western blotting (data not shown). However, previous studies indicate that the L-interacting domain lies in a conserved region of the carboxy terminus of paramyxovirus phosphoproteins, and therefore it seems unlikely that mutations in the amino terminus (81-to-113 region) will abrogate L-P interaction (9, 43).

We chose to mutate glycines within the STAT1-binding domain to glutamic acids based on the observation of Hagmaier et al., who found that the presence of a glutamic acid at position 125 of the NiV V protein abrogated NiV V protein inhibition of IFN-induced gene expression and V interaction with STAT1 (18). However, the abilities of V to block IFN signaling and to interact with STAT1 could be restored by changing E125 to G125, the amino acid found in most available NiV sequences (18). Our results confirm this loss of function also in the context of the P and W proteins and show that other important glycine residues exist (glycine 121 and 127) in addition to G125 and that their replacement with glutamic acid results in this loss of function. However, this observation does not hold for all of the glycine residues in the region. The G120E mutant forms of NiV P, V, and W functioned as well as their WT counterparts in reporter assays and bound STAT1 equally well. Interestingly, the protein with the G135E substitution did not detectably bind STAT1 in our immunoprecipitation studies but inhibited ISG54-driven reporter induction, albeit less efficiently, suggesting that it may retain residual STAT1-binding activity that is not detectable by our coimmunoprecipitation assay. The mechanism for such loss of STAT1 binding remains unclear, but it is possible that the glycine-rich region affords flexibility required for STAT1 binding. Also possible is that the introduction of an acidic residue like glutamic acid creates an area that is too charged to bind STAT1. Future structural studies should further enhance our understanding of the mechanistic details of NiV inhibition of STAT1.

A series of reports show that a hexapeptide present in measles virus phosphoprotein is required for its inhibition of STAT1 phosphorylation (4, 11, 13, 32). We found a similar sequence in the NiV P amino acid sequence, specifically, a tyrosine at position 116. We replaced Y116 with alanine or glutamic acid and observed a loss of function in IFN signaling assays (Fig. (Fig.5).5). The conservation of these residues among these viruses underlines the importance of the tyrosine at this position. Furthermore, Caignard et al. recently reported that the minimal region required for the interaction of measles virus V protein with STAT1 is residues 110 to 120, which includes Y110 and is very similar in position to the NiV P, V, or W STAT1-binding domain that we define here (3). However, alignment of the NiV and measles virus P genes shows very little identity outside the hexapeptide sequence. The functionality of our phenylalanine substitution and lack of rescue by replacement with phosphomimetic glutamic acid in NiV P suggest that tyrosine phosphorylation at this site is not critical for protein function and point more to a structural importance for this residue.

Using a newly established reverse genetics system, we successfully generated viruses in which C protein expression is expected to be eliminated, due to the mutation of two potential initiator AUG codons, as well as the introduction of a downstream, in-frame, stop codon. The G121E mutation, predicted to abrogate inhibition of STAT1 by P, V, and W, was built into this Cko background because the mutation would otherwise also result in a C protein amino acid change. The Cko virus is attenuated compared to the WT in both of the cell lines tested, and the G121E mutant displays similar growth kinetics, suggesting that the point mutation in P, V, and W does not further affect replication. Interestingly, the addition of the G121E mutation to a Cko background did not further impair replication, even in 293T cells, which are, unlike Vero cells, capable of producing IFN-α/β. This may be explained by the ability of NiV V and W to block the induction of IFN, which is not expected to be dependent on an interaction with STAT1 (41). Future experiments will determine the amounts of IFN produced by IFN-competent cells during infection.

Examination of the status of STAT1 during WT (data not shown) or Cko NiV infection revealed a striking phenotype in that all STAT1 appears to be nuclear but not tyrosine phosphorylated. Given that the W protein has been found to direct nonphosphorylated STAT1 to the nucleus, while the P and V proteins maintain STAT1 in the cytoplasm (39, 40, 42), it appears that W is a dominant factor that controls STAT1 activation in NiV-infected cells. In future studies, it will be of interest to determine whether viruses that lack W expression but retain WT P and V expression keep the ability to effectively prevent STAT1 activation. Introduction of the G121E mutation fully reversed the inhibition seen in the Cko virus-infected cells. As seen in uninfected cells, STAT1 was not phosphorylated and was cytoplasmic prior to IFN addition. Upon IFN-β treatment, STAT1 was tyrosine phosphorylated and nuclear. These data conclusively demonstrate that the NiV P gene encodes a function that regulates the trafficking and activation of STAT1. Further experiments are needed to more fully address (i) the impact of this function on cellular gene expression during the course of infection, (ii) the impact of this function on virus sensitivity to IFN, and (iii) the impact of this function on viral pathogenesis.

Acknowledgments

This work was supported in part by funds from the NIH, including U54 AI057158 (Northeast Biodefense Center-Lipkin) and AI059536 to C.F.B. and V.E.V., and from INSERM (France) and the Deutsche Forschungsgemeinschaft (SFB 593 to V.E.V.).

We thank David E. Levy (New York University) for providing the ISG54-CAT reporter plasmid. All experiments involving live NiVs were carried out in the INSERM Jean Merieux BSL4 laboratory in Lyon, France. We thank the biosafety team members for their support in conducting experiments.

Footnotes

[down-pointing small open triangle]Published ahead of print on 10 June 2009.

REFERENCES

1. Brzózka, K., S. Finke, and K.-K. Conzelmann. 2006. Inhibition of interferon signaling by rabies virus phosphoprotein P: activation-dependent binding of STAT1 and STAT2. J. Virol. 802675-2683. [PMC free article] [PubMed]
2. Buchholz, U. J., S. Finke, and K.-K. Conzelmann. 1999. Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter. J. Virol. 73251-259. [PMC free article] [PubMed]
3. Caignard, G., M. Bourai, Y. Jacob, F. Tangy, and P. O. Vidalain. 2009. Inhibition of IFN-α/β signaling by two discrete peptides within measles virus V protein that specifically bind STAT1 and STAT2. Virology 383112-120. [PubMed]
4. Caignard, G., M. Guerbois, J.-L. Labernardière, Y. Jacob, L. M. Jones, F. Wild, F. Tangy, and P.-O. Vidalain. 2007. Measles virus V protein blocks Jak1-mediated phosphorylation of STAT1 to escape IFN-α/β signaling. Virology 368351-362. [PubMed]
5. Chadha, M. S., J. Comer, L. Lowe, J. Robbins, P. Rota, P. Rollin, W. J. Bellini, T. Ksiazek, and A. C. Mishra. 2006. Nipah virus-associated encephalitis outbreak, Siliguri, India. Emerg. Infect. Dis. 12235-240. [PMC free article] [PubMed]
6. Childs, K., N. Stock, C. Ross, J. Andrejeva, L. Hilton, M. Skinner, R. Randall, and S. Goodbourn. 2007. mda-5, but not RIG-I, is a common target for paramyxovirus V proteins. Virology 359190-200. [PubMed]
7. Chua, K. B., W. J. Bellini, P. A. Rota, B. H. Harcourt, A. Tamin, S. K. Lam, T. G. Ksiazek, P. E. Rollin, S. R. Zaki, W.-J. Shieh, C. S. Goldsmith, D. J. Gubler, J. T. Roehrig, B. Eaton, A. R. Gould, J. Olson, H. Field, P. Daniels, A. E. Ling, C. J. Peters, L. J. Anderson, and B. W. J. Mahy. 2000. Nipah virus: a recently emergent deadly paramyxovirus. Science 2881432-1435. [PubMed]
8. Chua, K. B., C. Lek Koh, P. S. Hooi, K. F. Wee, J. H. Khong, B. H. Chua, Y. P. Chan, M. E. Lim, and S. K. Lam. 2002. Isolation of Nipah virus from Malaysian Island flying-foxes. Microbes Infect. 4145-151. [PubMed]
9. Curran, J., T. Pelet, and D. Kolakofsky. 1994. An acidic activation-like domain of the Sendai virus P protein is required for RNA synthesis and encapsidation. Virology 202875-884. [PubMed]
10. Darnell, J. J., I. M. Kerr, and G. R. Stark. 1994. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 2641415-1421. [PubMed]
11. Devaux, P., V. von Messling, W. Songsungthong, C. Springfeld, and R. Cattaneo. 2007. Tyrosine 110 in the measles virus phosphoprotein is required to block STAT1 phosphorylation. Virology 36072-83. [PubMed]
12. Epstein, J., S. Rahman, J. Zambriski, K. Halpin, G. Meehan, A. Jamaluddin, S. Hassan, H. Field, A. Hyatt, and P. Daszak. 2006. Feral cats and risk for Nipah virus transmission. Emerg. Infect. Dis. 121178-1179. [PMC free article] [PubMed]
13. Fontana, J. M., B. Bankamp, W. J. Bellini, and P. A. Rota. 2008. Regulation of interferon signaling by the C and V proteins from attenuated and wild-type strains of measles virus. Virology 37471-81. [PubMed]
14. Fontana, J. M., B. Bankamp, and P. A. Rota. 2008. Inhibition of interferon induction and signaling by paramyxoviruses. Immunol. Rev. 22546-67. [PubMed]
15. Freiberg, A., L. K. Dolores, S. Enterlein, and R. Flick. 2008. Establishment and characterization of plasmid-driven minigenome rescue systems for Nipah virus: RNA polymerase I- and T7-catalyzed generation of functional paramyxoviral RNA. Virology 37033-44. [PMC free article] [PubMed]
16. García-Sastre, A., and C. A. Biron. 2006. Type 1 interferons and the virus-host relationship: a lesson in detente. Science 312879-882. [PubMed]
17. Gurley, E., J. M. Montgomery, M. J. Hossain, M. Bell, and A. K. Azad. 2007. Person-to-person transmission of Nipah virus in a Bangladeshi community. Emerg. Infect. Dis. 131031-1037. [PMC free article] [PubMed]
18. Hagmaier, K., N. Stock, S. Goodbourn, L.-F. Wang, and R. Randall. 2006. A single amino acid substitution in the V protein of Nipah virus alters its ability to block interferon signalling in cells from different species. J. Gen. Virol. 873649-3653. [PMC free article] [PubMed]
19. Halpin, K., B. Bankamp, B. H. Harcourt, W. J. Bellini, and P. A. Rota. 2004. Nipah virus conforms to the rule of six in a minigenome replication assay. J. Gen. Virol. 85701-707. [PubMed]
20. Harcourt, B. H., A. Tamin, K. Halpin, T. G. Ksiazek, P. E. Rollin, W. J. Bellini, and P. A. Rota. 2001. Molecular characterization of the polymerase gene and genomic termini of Nipah virus. Virology 287192-201. [PubMed]
21. Horvath, C. M. 2004. Weapons of STAT destruction. Interferon evasion by paramyxovirus V proteins. Eur. J. Biochem. 2714621-4628. [PubMed]
22. Hossain, M. J., E. S. Gurley, J. M. Montgomery, M. Bell, D. S. Carroll, V. P. Hsu, P. Formenty, A. Croisier, E. Bertherat, M. A. Faiz, A. K. Azad, R. Islam, M. A. R. Molla, T. G. Ksiazek, P. A. Rota, J. A. Comer, P. E. Rollin, S. P. Luby, and R. F. Breiman. 2008. Clinical presentation of Nipah virus infection in Bangladesh. Clin. Infect. Dis. 46977-984. [PubMed]
23. Hsu, V. P., M. Hossain, U. D. Parashar, M. M. Ali, T. Ksiazek, I. Kuzmin, M. Niezgoda, C. Rupprecht, J. Bresee, and R. Breiman. 2004. Nipah virus encephalitis reemergence, Bangladesh. Emerg. Infect. Dis. 102082-2087. [PMC free article] [PubMed]
24. Kash, J. C., E. Mühlberger, V. Carter, M. Grosch, O. Perwitasari, S. C. Proll, M. J. Thomas, F. Weber, H.-D. Klenk, and M. G. Katze. 2006. Global suppression of the host antiviral response by Ebola- and Marburgviruses: increased antagonism of the type I interferon response is associated with enhanced virulence. J. Virol. 803009-3020. [PMC free article] [PubMed]
25. Kato, A., Y. Sakai, T. Shioda, T. Kondo, M. Nakanishi, and Y. Nagai. 1996. Initiation of Sendai virus multiplication from transfected cDNA or RNA with negative or positive sense. Genes Cells 1569-579. [PubMed]
26. Kulkarni, S., V. Volchkova, C. F. Basler, P. Palese, V. E. Volchkov, and M. L. Shaw. 2009. Nipah virus edits its P gene at high frequency to express the V and W proteins. J. Virol. 833982-3987. [PMC free article] [PubMed]
27. Lamb, R. A., and G. D. Parks. 2007. Paramyxoviridae: the viruses and their replication, p. 1450-1496. In D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman, and S. E. Straus (ed.), Fields virology, 5th ed. Lippincott Williams & Wilkins, Philadelphia, PA.
28. Levy, D., D. Kessler, R. Pine, and J. J. Darnell. 1989. Cytoplasmic activation of ISGF3, the positive regulator of interferon-alpha-stimulated transcription, reconstituted in vitro. Genes Dev. 31362-1371. [PubMed]
29. Lo, M. S., R. M. Brazas, and M. J. Holtzman. 2005. Respiratory syncytial virus nonstructural proteins NS1 and NS2 mediate inhibition of Stat2 expression and alpha/beta interferon responsiveness. J. Virol. 799315-9319. [PMC free article] [PubMed]
30. Ludlow, L. E., M. K. Lo, J. J. Rodriguez, P. A. Rota, and C. M. Horvath. 2008. Henipavirus V protein association with polo-like kinase reveals functional overlap with STAT1 binding and interferon evasion. J. Virol. 826259-6271. [PMC free article] [PubMed]
31. Nanda, S. K., and M. D. Baron. 2006. Rinderpest virus blocks type I and type II interferon action: role of structural and nonstructural proteins. J. Virol. 807555-7568. [PMC free article] [PubMed]
32. Ohno, S., N. Ono, M. Takeda, K. Takeuchi, and Y. Yanagi. 2004. Dissection of measles virus V protein in relation to its ability to block alpha/beta interferon signal transduction. J. Gen. Virol. 852991-2999. [PubMed]
33. Palosaari, H., J.-P. Parisien, J. J. Rodriguez, C. M. Ulane, and C. M. Horvath. 2003. STAT protein interference and suppression of cytokine signal transduction by measles virus V protein. J. Virol. 777635-7644. [PMC free article] [PubMed]
34. Perez, M., A. Sanchez, B. Cubitt, D. Rosario, and J. C. de la Torre. 2003. A reverse genetics system for Borna disease virus. J. Gen. Virol. 843099-3104. [PubMed]
35. Qureshi, S. A., M. Salditt-Georgieff, and J. E. Darnell, Jr. 1995. Tyrosine-phosphorylated Stat1 and Stat2 plus a 48-kDa protein all contact DNA in forming interferon-stimulated-gene factor 3. Proc. Natl. Acad. Sci. USA 923829-3833. [PMC free article] [PubMed]
36. Reid, S. P., L. W. Leung, A. L. Hartman, O. Martinez, M. L. Shaw, C. Carbonnelle, V. E. Volchkov, S. T. Nichol, and C. F. Basler. 2006. Ebola virus VP24 binds karyopherin α1 and blocks STAT1 nuclear accumulation. J. Virol. 805156-5167. [PMC free article] [PubMed]
37. Reid, S. P., C. Valmas, O. Martinez, F. M. Sanchez, and C. F. Basler. 2007. Ebola virus VP24 proteins inhibit the interaction of NPI-1 subfamily karyopherin α proteins with activated STAT1. J. Virol. 8113469-13477. [PMC free article] [PubMed]
38. Reynes, J.-M., D. Counor, S. Ong, C. Faure, V. Seng, S. Molia, J. Walston, M. C. Georges-Courbot, V. Deubel, and J.-L. Sarthon. 2005. Nipah virus in Lyle's flying foxes, Cambodia. Emerg. Infect. Dis. 111042-1047. [PMC free article] [PubMed]
39. Rodriguez, J. J., C. D. Cruz, and C. M. Horvath. 2004. Identification of the nuclear export signal and STAT-binding domains of the Nipah virus V protein reveals mechanisms underlying interferon evasion. J. Virol. 785358-5367. [PMC free article] [PubMed]
40. Rodriguez, J. J., J.-P. Parisien, and C. M. Horvath. 2002. Nipah virus V protein evades alpha and gamma interferons by preventing STAT1 and STAT2 activation and nuclear accumulation. J. Virol. 7611476-11483. [PMC free article] [PubMed]
41. Shaw, M. L., W. B. Cardenas, D. Zamarin, P. Palese, and C. F. Basler. 2005. Nuclear localization of the Nipah virus W protein allows for inhibition of both virus- and Toll-like receptor 3-triggered signaling pathways. J. Virol. 796078-6088. [PMC free article] [PubMed]
42. Shaw, M. L., A. García-Sastre, P. Palese, and C. F. Basler. 2004. Nipah virus V and W proteins have a common STAT1-binding domain yet inhibit STAT1 activation from the cytoplasmic and nuclear compartments, respectively. J. Virol. 785633-5641. [PMC free article] [PubMed]
43. Smallwood, S., K. W. Ryan, and S. A. Moyer. 1994. Deletion analysis defines a carboxyl-proximal region of Sendai virus P protein that binds to the polymerase L protein. Virology 202154-163. [PubMed]
44. Stark, G. R., I. M. Kerr, B. R. G. Williams, R. H. Silverman, and R. D. Schreiber. 1998. How cells respond to interferons. Annu. Rev. Biochem. 67227-264. [PubMed]
45. Thomas, S. M., R. A. Lamb, and R. G. Paterson. 1988. Two mRNAs that differ by two nontemplated nucleotides encode the amino coterminal proteins P and V of the paramyxovirus SV5. Cell 54891-902. [PubMed]
46. Vidy, A., M. Chelbi-Alix, and D. Blondel. 2005. Rabies virus P protein interacts with STAT1 and inhibits interferon signal transduction pathways. J. Virol. 7914411-14420. [PMC free article] [PubMed]
47. Vidy, A., J. El Bougrini, M. K. Chelbi-Alix, and D. Blondel. 2007. The nucleocytoplasmic rabies virus P protein counteracts interferon signaling by inhibiting both nuclear accumulation and DNA binding of STAT1. J. Virol. 814255-4263. [PMC free article] [PubMed]
48. Wang, L.-F., B. H. Harcourt, M. Yu, A. Tamin, P. A. Rota, W. J. Bellini, and B. T. Eaton. 2001. Molecular biology of Hendra and Nipah viruses. Microbes Infect. 3279-287. [PubMed]

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