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J Virol. 2010 July; 84(13): 6699–6710.
Published online 2010 April 28. doi:  10.1128/JVI.00011-10
PMCID: PMC2903245

Role of Interferon Antagonist Activity of Rabies Virus Phosphoprotein in Viral Pathogenicity[down-pointing small open triangle]

Abstract

The fixed rabies virus (RV) strain Nishigahara kills adult mice after intracerebral inoculation, whereas the chicken embryo fibroblast cell-adapted strain Ni-CE causes nonlethal infection in adult mice. We previously reported that the chimeric CE(NiP) strain, which has the phosphoprotein (P protein) gene from the Nishigahara strain in the genetic background of the Ni-CE strain, causes lethal infection in adult mice, indicating that the P gene is responsible for the different pathogenicities of the Nishigahara and Ni-CE strains. Previous studies demonstrated that RV P protein binds to the interferon (IFN)-activated transcription factor STAT1 and blocks IFN signaling by preventing its translocation to the nucleus. In this study, we examine the molecular mechanism by which RV P protein determines viral pathogenicity by comparing the IFN antagonist activities of the Nishigahara and Ni-CE P proteins. The results, obtained from both RV-infected cells and cells transfected to express P protein only, show that Ni-CE P protein is significantly impaired for its capacity to block IFN-activated STAT1 nuclear translocation and, consequently, inhibits IFN signaling less efficiently than Nishigahara P protein. Further, it was demonstrated that a defect in the nuclear export of Ni-CE P protein correlates with a defect in its ability to cause the mislocalization of STAT1. These data provide the first evidence that the capacity of the RV P protein to inhibit STAT1 nuclear translocation and IFN signaling correlates with the viral pathogenicity.

The host immune response to viral infection is a key factor in defining viral pathogenicity and the outcome of the infection. This depends not only on the capacity of the host to mount an innate and/or adaptive immune response against the virus but also on the ability of the virus to evade/subvert this response (22).

The principal response of host cells to viral infection is the production of type I interferons (IFNs) (including alpha interferon [IFN-α] and IFN-β), which, on binding to IFN receptors on the cell surface, activate the JAK/STAT intracellular signaling pathway that culminates in the phosphorylation, heterodimerization, and nuclear translocation of the transcription factors signal transducer and activator of transcription 1 (STAT1) and STAT2. In the context of a complex called IFN-stimulated gene factor 3 (ISGF3), the activated STATs bind to promoters in the DNA that contain an IFN-stimulated response element (ISRE) sequence, resulting in the transcription of a plethora of IFN-stimulated genes (ISGs) encoding antiviral proteins which act to establish the antiviral state in cells (reviewed in reference 22).

To propagate efficiently in host cells, viruses have had to evolve multiple strategies to dampen the host IFN system, which appear to involve the expression of viral proteins with IFN antagonist functions. These IFN antagonists are reported to exert their effect by a variety of mechanisms, reflecting the diversity of host antiviral responses, but the STATs are known as common targets of viral IFN antagonists, presumably because of their pivotal role in IFN signaling. For example, nonsegmented negative-strand RNA viruses (order Mononegavirales, which is composed of four families, Paramyxoviridae, Rhabdoviridae, Filoviridae, and Bornaviridae) are known to express proteins that act as IFN antagonists by targeting STATs. These include the VP24 protein of Ebola virus (24), belonging to the family Filoviridae, and the V protein of Nipah virus (25), a member of the family Paramyxoviridae, which are reported to prevent STAT nuclear accumulation. Similarly, the phosphoprotein (P protein) of rabies virus (RV), a prototype of the family Rhabdoviridae, has been identified as its viral IFN antagonist and has the capacity to block multiple stages of STAT1-dependent IFN signaling. Namely, RV P protein binds to STAT1 and inhibits not only the nuclear import of IFN-activated STAT1 (4, 17, 36) but also the binding of ISGF3 to DNA containing the ISRE sequence (37). Furthermore, it was reported that RV P protein binds to STAT2 and inhibits its nuclear translocation induced by IFN-α (4).

During infection, RV efficiently replicates in neurons and causes lethal encephalomyelitis. As neurons are able to produce type I IFN in response to viral infection and are responsive to type I IFN (6), it appears that circumventing the host IFN system would be a key factor for pathogenicity of RV. However, there is little data to link the IFN antagonism by RV P protein with viral pathogenicity directly. We previously reported that the RV strain Nishigahara kills adult mice after intracerebral (i.c.) inoculation, whereas the Ni-CE strain, which we have established after 100 passages of Nishigahara strain in chicken embryo fibroblast cells, causes nonlethal infection in adult mice (30). Using a reverse genetics approach, we found that the chimeric virus strain CE(NiP), which has the P protein-encoding P gene from the Nishigahara strain in the genetic background of the Ni-CE strain, kills adult mice after i.c. inoculation (30). We recently demonstrated that a key difference between the virulent Nishigahara and CE(NiP) strains and the avirulent Ni-CE strain is their sensitivity to IFN-α: Nishigahara and CE(NiP) strains are more resistant to IFN-α than the Ni-CE strain (31). This finding led us to the hypothesis that the RV P protein is a key regulator of viral pathogenicity, dependent on its capacity to inhibit IFN signaling.

In the present study, we examine the molecular mechanisms underlying the role of P protein in RV pathogenicity by comparing the inhibitory effects of the Nishigahara and Ni-CE P proteins on type I IFN signaling. The results obtained from both RV-infected cells and transfected cells transiently expressing P protein demonstrate that the Ni-CE P protein is significantly impaired in its capacity to block IFN-activated STAT1 nuclear translocation and, consequently, inhibits IFN signaling less efficiently than does Nishigahara P protein. Further, we find that the nuclear export of Ni-CE P protein is defective and that this correlates with a defect in its ability to cause mislocalization of STAT1. This study provides the first evidence that RV P protein's ability to inhibit STAT1 nuclear translocation and IFN signaling correlates with the pathogenicity of RV.

MATERIALS AND METHODS

Cells and viruses.

Human neuroblastoma SK-N-SH cells (ATCC number HTB-11) and mouse neuroblastoma NA cells were maintained in Eagle's minimal essential medium supplemented with 10% fetal calf serum. Vero cells were grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum. For infection with the RV Nishigahara and Ni-CE strains, we used recombinant virus of the respective strain which had been recovered from cloned cDNA (30, 38). The chimeric CE(NiP) strain, which has the Nishigahara P protein gene (P gene) in the genetic background of strain Ni-CE, was previously generated by a reverse genetics system (Fig. (Fig.1A)1A) (30). Stocks of these RV strains were prepared in NA cells.

FIG. 1.
Characteristics of the RV strains and P proteins used in this study. (A) Genome organization and pathogenicities of the Nishigahara (Ni), Ni-CE, and chimeric CE(NiP) strains. The pathogenicity of each strain for adult mice was previously determined by ...

Propagation of RV in mouse brains.

Virus propagation in the mouse brain was examined as described previously (33, 34). Briefly, 6-week-old female ddY mice (Japan SLC, Inc., Japan) were inoculated intracerebrally with 100 focus forming units (FFU) of each strain. Two inoculated mice were euthanized daily, and then their brains were mixed and homogenized. The virus titer in the homogenates (calculated as FFU/g) was determined by a focus assay on confluent NA cells after 10-fold weight/volume dilution of the homogenates and collection of the supernatants. The viral foci were visualized by indirect fluorescent antibody staining with anti-RV N protein mouse monoclonal antibody 13-27 (14) after fixation of the cells with 2% paraformaldehyde for 30 min and 100% methanol for 2 min. This animal experiment was conducted in accordance with the Standards Relating to the Care and Management of Experimental Animals promulgated by Gifu University, Japan (allowance no. 08119).

IFN-α sensitivity of RV.

SK-N-SH cells were inoculated with each virus at a multiplicity of infection (MOI) of 0.01 and then were cultured in growth medium with or without 500 U/ml of human IFN-α2a (PBL InterferonSource, Piscataway, NJ). At 3 days postinoculation (p.i.), the supernatant of the culture medium was collected and stored at −80°C until virus titration. The virus titer in the culture medium (calculated as FFU/ml) was determined by a focus assay on confluent NA cells as described above. Preliminary experiments showed that the concentration of IFN-α used in this experiment (500 U/ml) did not affect the accuracy of this virus titration (data not shown). The IFN sensitivity index of each strain was determined by the logarithm of the virus titer in SK-N-SH cells (untreated) minus that of the titer in SK-N-SH cells treated with IFN-α.

To examine virus propagation in terms of viral protein synthesis levels in the presence of IFN-α, the SK-N-SH cells were inoculated with each virus at an MOI of 0.01 and then were cultured in growth medium containing 0 to 500 U/ml IFN-α. At 2 days p.i., the cells were lysed in lysis buffer (20 mM Tris [pH 8.0], 150 mM NaCl, 2 mM EDTA, 20 mM CHAPS {3-[(3-cholamidopropyl)-dimethylammonior]-1-propanesulfonate}, 4 mM p-APMSF [(p-4-amidinophenyl)methanesulfonyl fluoride hydrochloride monohydrate]) and the solubilized protein was analyzed by Western blotting as described below.

Western blotting.

Cell lysate samples were separated by sodium dodecyl sulfate (SDS)-10% polyacrylamide gel electrophoresis before transfer to a polyvinylidene difluoride (PVDF) membrane (Immobilon transfer membranes; Millipore, Billerica, MA). RV N and P proteins on the membrane were visualized by immunoblotting with anti-N protein monoclonal antibody 13-27 (14) and anti-P protein rabbit serum (kindly provided by Akihiko Kawai), respectively. Alpha-tubulin was also detected, as a loading control, by using anti-monoclonal antitubulin antibody (Sigma, Saint Louis, MO). The intensities of the protein bands were quantified with Image J software (http://rsbweb.nih.gov/ij/).

Plasmid constructions.

The Nishigahara P protein-expressing plasmid pcDNA-NiP, with cDNA corresponding to the Nishigahara P gene cloned into the pcDNA1.1/Amp vector (Invitrogen, Carlsbad, CA), has been described previously (38). The construct pcDNA-CEP, containing the cDNA for the Ni-CE P gene, was produced similarly. To express green fluorescent protein (GFP)-tagged P proteins of Nishigahara and Ni-CE strains (designated Ni P-GFP and Ni-CE P-GFP, respectively), we cloned the cDNA fragment of the P gene from the respective strain into the pEGFP-N1 vector (Clontech, Mountain View, CA). To express the Ni-CE P-GFP mutant with proline (Pro)-to-leucine (Leu) substitutions at positions 56 and 58 of the Ni-CE P protein [designated Ni-CE P(NES+)-GFP], we mutated the pEGFP-N1 plasmid expressing Ni-CE P-GFP by using a QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA). For the yeast two-hybrid analysis described below, we cloned the cDNA fragment of the Nishigahara or Ni-CE P gene into the plasmid vector pLex10 (pLexA) (kindly provided by Jacques Camonis), which contains the yeast-selectable TRP1 gene and the LexA DNA-binding domain (BD) coding sequence, to express BD-fused Nishigahara and Ni-CE P proteins. The resulting plasmids were designated pLex-Ni P and pLex-Ni-CE P, respectively.

ISRE reporter assay.

SK-N-SH cells grown in a 24-well tissue culture plate were transfected with 0.04 μg of pRL-TK (Promega, Madison, WI), which expresses the Renilla luciferase, and 0.25 μg of pISRE-Luc (Stratagene), which has an ISRE-containing promoter upstream of the firefly luciferase reporter gene, using Lipofectamine 2000 (Invitrogen). At 24 h after transfection, the cells were inoculated with the Nishigahara, Ni-CE, or CE(NiP) strain at an MOI of 3 and incubated for 6 h prior to treatment with or without 2,000 U/ml of IFN-α2a for 12 h. After lysis of the cells, the activities of firefly and Renilla luciferases were determined by using a dual-luciferase-reporter assay system (Promega, Madison, WI) according to the manufacturer's instructions. The ISRE activity was calculated as firefly luciferase activity normalized to Renilla luciferase activity.

In other experiments, SK-N-SH cells were transfected with 0.25 μg of pcDNA-NiP, pcDNA-CEP, or pcDNA-NiN expressing Nishigahara N protein (38) or an empty vector (pcDNA1.1/Amp), together with pRL-TK and pISRE-Luc. At 24 h after transfection, the cells were treated with or without 2,000 U/ml of IFN-α2a for 6 h and the activities of the firefly and Renilla luciferases were determined as described above. In some cases, pEGFP-N1 plasmids expressing Ni P-GFP, Ni-CE P-GFP, or Ni-CE P(NES+)-GFP were employed instead of these pcDNA1.1/Amp plasmids, using the same experimental conditions described above.

Real-time reverse transcription (RT)-PCR.

SK-N-SH cells grown in a 24-well tissue culture plate were inoculated with the Nishigahara, Ni-CE, or CE(NiP) strain at an MOI of 3 and incubated for 6 h prior to treatment with 2,000 U/ml of IFN-α2a for 12 h. The expression of myxovirus resistance A (MxA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes in the infected cells was analyzed by using a TaqMan gene expression Cells-to-CT kit (Ambion, Austin, TX) with specific TaqMan probes (MxA [Hs00182073] and GAPDH [4326317E]; Applied Biosystems, Carlsbad, CA) in an ABI 7300 real-time PCR system (Applied Biosystems). The expression levels of the MxA gene are indicated as the number of copies of specific mRNA per copy of human GAPDH mRNA. All assays were carried out in triplicate, and the results are expressed as means ± standard deviations.

Immunostaining.

SK-N-SH cells grown in an 8-chamber culture slide (BD Falcon; BD Biosciences, Franklin Lakes, NJ) were inoculated with each strain at an MOI of 0.01 and then were treated with or without IFN-α (4,000 U/ml) for 30 min at 24 h p.i. The cells were fixed with 3.7% formaldehyde for 10 min and 90% methanol for 5 min and immunostained with an anti-STAT1 rabbit antibody (sc-346; Santa Cruz Biotechnology, Santa Cruz, CA) and an anti-RV N protein mouse monoclonal antibody, followed by incubation with Alexa Fluor 488 anti-rabbit IgG (Invitrogen) (green) and Alexa Fluor 594 anti-mouse IgG (Invitrogen) (red). The stained samples were analyzed by confocal laser scanning microscopy (CLSM), using a Leica SP5 microscope with 63× glycerol immersion objective. In some experiments, RV P protein in infected SK-N-SH cells was immunostained by using anti-P protein rabbit serum and Alexa Fluor 488 anti-rabbit IgG.

Subcellular localization of GFP-tagged P protein and STAT1 protein.

Vero cells or SK-N-SH cells were grown on coverslips and transfected with the pEGFP-N1 plasmid expressing Ni P-GFP, Ni-CE P-GFP, or Ni-CE P(NES+)-GFP by using Lipofectamine 2000 as previously described (18, 26). For costaining of cells for STAT1, cells were treated with or without IFN-α for 1 h before fixation and permeabilization as described above. Cells were immunostained with anti-STAT1 (610185; BD Biosciences) followed by Alexa 568-coupled secondary antibody (Invitrogen) and imaged using a Leica SP5 microscope as described above.

Yeast two-hybrid analysis.

Yeast cells (L40 strain) containing His3- and LacZ-responsive reporter genes were transformed with pLex-Ni P or -Ni-CE P, together with the plasmid pGAD-STAT1 (36), which has the yeast-selectable LEU2 gene and expresses the GAL4 activation domain (AD)-fused STAT1. A combination of pLex-P CVS expressing BD-fused P protein from the RV CVS strain (36) and pGAD-STAT1 was also used as a positive control. The RV P protein-STAT1 interaction was assayed by the expression of the His3 reporter gene on a plate lacking the amino acids tryptophan (Trp), Leu, and histidine (His) and also by the appearance of blue colonies following X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactosidase) overlay as follows: an X-Gal mixture containing 0.5% agar, 0.1% SDS, 6% dimethylformamide, and 0.04% X-Gal was overlaid on fresh transformants grown on a plate lacking Trp and Leu. Blue colonies were detected after 60 min to 18 h at 30°C.

Coimmunoprecipitation (co-IP).

SK-N-SH cells in a 60-mm tissue culture dish were infected with the Ni-CE or CE(NiP) strain at an MOI of 0.1 and incubated for 18 h prior to treatment with 2,000 U/ml of IFN-α2a for 2 h. After the cells were washed once with ice-cold phosphate-buffered saline (PBS), the cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer (1% NP-40, 0.1% SDS, and 0.5% sodium deoxycholate in PBS) containing protease inhibitor cocktail (Complete mini; Roche, Germany) on ice for 20 min and centrifuged at 13,200 × g for 10 min at 4°C to remove cell debris. Supernatants were collected and precleared by incubation with 20 μl of Protein A/G Plus-Agarose immunoprecipitation reagent (Santa Cruz Biotechnology) and 2 μg of IgG from rabbit serum (Sigma) at 4°C for 45 min. After centrifugation at 800 × g for 1 min at 4°C, supernatants were incubated with 4 μg of anti-STAT1 rabbit antibody (sc-346; Santa Cruz Biotechnology) or rabbit IgG (control IgG) at 4°C for 2 h. The samples were incubated further with 20 μl of Protein A/G Plus-Agarose immunoprecipitation reagent at 4°C for 15 h. After centrifugation at 800 × g for 1 min at 4°C, the precipitated immune complexes were washed four times with RIPA buffer. Finally, the precipitated proteins were dissolved in sample buffer solution (2× 2-mercaptoethanol-added) (Wako Pure Chemical Industries, Japan) and analyzed by Western blotting using an anti-STAT1 rabbit antibody (sc-346; Santa Cruz Biotechnology) or anti-P protein rabbit serum as the primary antibody, with rabbit IgG TrueBlot (eBioscience, San Diego, CA) as the secondary antibody. These antibodies were diluted with Can Get Signal immunoreaction enhancer solution (Toyobo, Japan).

Statistical analysis.

Student's t test was used to determine statistical significance. P values of <0.05 were considered statistically significant.

RESULTS

Propagation of Nishigahara, Ni-CE, and CE(NiP) strains in mouse brain.

The fixed RV strain Ni-CE causes nonlethal infection in adult mice after i.c. inoculation, whereas the Nishigahara strain and the CE(NiP) strain, which is a chimeric virus with the Nishigahara P gene in the genetic background of the Ni-CE strain, kill adult mice (Fig. (Fig.1A)1A) (30). To investigate the mechanisms underlying this difference, we initially examined the propagation of these RV strains in mouse brains. The viral titer of the Nishigahara strain quickly increased and reached 1.3 × 108 FFU/g at 3 days p.i. (Fig. (Fig.1C).1C). In contrast, the titer of the Ni-CE strain was less than 102 FFU/g at 3 days p.i. and reached a peak of only 1.3 × 104 FFU/g at 6 days p.i. before gradually decreasing during 8 to 10 days p.i. Importantly, strain CE(NiP) grew more efficiently than strain Ni-CE, reaching a titer of 7.9 × 104 FFU/g at 3 days p.i. The different propagation efficiencies of the Ni-CE and CE(NiP) strains at this early stage of infection (3 days p.i.) are consistent with the hypothesis that host innate immunity is involved in the mechanism by which the P gene determines viral pathogenicity.

IFN-α sensitivities of the Nishigahara, Ni-CE, and CE(NiP) strains.

Using mouse neuroblastoma NA cells, we previously demonstrated that the attenuated Ni-CE strain is more sensitive to IFN-α than the virulent Nishigahara strain or strain CE(NiP) (30). Next, we confirmed that this is a general phenomenon in that it can be observed in cell lines derived from different mammalian species. Specifically, we used the human neuroblastoma cell line SK-N-SH and compared the growth of the different RV strains in these cells in the absence or presence of IFN-α (500 U/ml) (Fig. (Fig.22 A). In the absence of IFN-α, the Nishigahara strain grew less efficiently than the Ni-CE strain, probably due to the fact that strain Nishigahara has been maintained by rabbit brain passages and is not well adapted to the cultured cells. In spite of this, when grown in the presence of IFN-α, the titer of the virulent Nishigahara strain (1.6 × 105 FFU/ml) was significantly higher than that of the avirulent Ni-CE strain (2.1 × 103 FFU/ml). Similarly, the titer of the virulent CE(NiP) strain in the presence of IFN-α (1.5 × 106 FFU/ml) was much higher than that of strain Ni-CE. To standardize the different capacities of these strains for adaptation to the cultured cells, we determined and compared their IFN sensitivity indexes, which represent the log difference between the titers with and without IFN-α treatment (Fig. (Fig.2B).2B). The IFN sensitivity index of strain Nishigahara (1.69 ± 0.11 [mean ± standard deviation]) was not different from that of the CE(NiP) strain (1.77 ± 0.10). In contrast, the index of strain Ni-CE (4.43 ± 0.09) was significantly higher than that of strain Nishigahara or strain CE(NiP) (P < 0.0001). This indicates that the Ni-CE strain is approximately 500-fold more sensitive to IFN-α than the Nishigahara and CE(NiP) strains.

FIG. 2.
The Nishigahara (Ni) and CE(NiP) strains are more resistant to IFN-α than the Ni-CE strain in infected human neuroblastoma cells. (A) Growth of each strain in human neuroblastoma SK-N-SH cells in the presence and absence of IFN-α. The ...

Next, we infected SK-N-SH cells with each RV strain and incubated the cells with culture medium containing different concentrations of IFN-α (0, 20, 100, and 500 U/ml). Western blotting revealed dose-dependent decreases in the expression levels of N and P proteins in SK-N-SH cells infected with strains Nishigahara, Ni-CE, and CE(NiP) (Fig. (Fig.2C).2C). However, the decrease in the Ni-CE-infected cells was clearly greater than those observed in cells infected with the Nishigahara or CE(NiP) strain. Quantification of the protein band intensities supported this observation (Fig. (Fig.2C).2C). These data indicate that in infected human cells, the virulent Nishigahara and CE(NiP) strains are less sensitive to IFN-α treatment than the Ni-CE strain and that the P gene determines the different IFN-α sensitivities of the RV strains.

Nishigahara P protein blocks IFN-α-induced ISRE activity more efficiently than Ni-CE P protein.

Vidy et al. (36) and Brzózka et al. (4) previously reported that RV P protein has the capacity to inhibit the IFN signaling pathway. As strains Ni-CE and CE(NiP) are genetically identical except for the P gene but differ in their sensitivity to IFN-α (Fig. (Fig.2),2), we hypothesized that the Nishigahara P protein but not the Ni-CE P protein would be able to efficiently block the IFN signaling pathway. To test this, we examined IFN-induced transcriptional activation in SK-N-SH cells infected with the Nishigahara, Ni-CE, and CE(NiP) strains, using a reporter gene assay with luciferase expression under the control of an ISRE-containing promoter (Fig. (Fig.3A).3A). As expected, ISRE-dependent luciferase expression was clearly activated by IFN-α treatment in mock-infected cells, but the luciferase expression was significantly lower in equivalently treated cells infected with the Nishigahara or CE(NiP) strain (P < 0.0001) (Fig. (Fig.3A,3A, top). Ni-CE infection also inhibited IFN-induced luciferase expression, but the inhibition was significantly weaker than that observed in cells infected with Nishigahara and CE(NiP) strains (P < 0.0001 and P < 0.001, respectively). Western blotting of cell lysates prepared for the reporter gene assay demonstrated that these strains expressed comparable amounts of P protein (Fig. (Fig.3A,3A, bottom). Similar results were obtained with equivalent reporter assays using NA cells infected with each strain (data not shown).

FIG. 3.
The extent of inhibition of IFN-α-induced ISRE activity by infection with RV or by the expression of RV P protein differs between RV strains. (A, upper panel) SK-N-SH cells were transfected with the ISRE reporter plasmid (pISRE-Luc) and the control ...

We next examined whether single expression of the Nishigahara or Ni-CE P protein differentially affects IFN-induced luciferase expression by using SK-N-SH cells transfected to express the respective proteins (Fig. (Fig.3B).3B). Expression of the Nishigahara or Ni-CE P protein alone significantly decreased IFN-induced luciferase expression compared to the level of expression in the control (empty vector transfected) cells (P < 0.0001 and P < 0.01, respectively) (Fig. (Fig.3B,3B, top). However, the inhibitory effect of Nishigahara P protein was significantly greater than that of Ni-CE P protein (P < 0.001), even though the expression level of Ni-CE P protein was comparable to that of Nishigahara P protein (Fig. (Fig.3B,3B, bottom). Similar results were obtained in identical assays performed using NA cells (data not shown). In contrast, the expression of Nishigahara N protein alone did not affect ISRE activation in response to IFN-α (Fig. (Fig.3B,3B, top), showing that the observed inhibitory effect on IFN signaling is specific to the P protein.

To check whether infections of these RV strains have different effects on the transcription of an endogenous cellular ISG which is controlled by ISRE, we used real-time RT-PCR to compare the expression levels of the MxA gene in Nishigahara-, Ni-CE-, and CE(NiP)-infected SK-N-SH cells treated with IFN-α (Fig. (Fig.3C).3C). We found that the expression levels of the MxA gene in Nishigahara- and CE(NiP)-infected cells were significantly lower than the level in Ni-CE-infected cells (P < 0.0001 and P < 0.01, respectively) (Fig. 3A and B), consistent with the results from the ISRE reporter assays. These results demonstrate that Nishigahara P protein blocks IFN signaling more efficiently than Ni-CE P protein.

Infection with the Nishigahara or CE(NiP) but not the Ni-CE strain effectively inhibits IFN-induced nuclear translocation of STAT1.

The results of previous studies have indicated that the inhibitory effect of RV P protein on IFN signaling depends on a physical interaction of the P protein with the IFN-activated transcription factor STAT1 (4, 36). The P protein-STAT1 interaction does not affect the activation (i.e., phosphorylation) of STAT1 but, rather, appears to block its nuclear translocation (36). However, the significance of this in viral pathogenicity has not been assessed. Accordingly, to investigate the possibility that Nishigahara P protein but not Ni-CE P protein effectively inhibits the nuclear translocation of STAT1, we examined STAT1 subcellular localization in Nishigahara-, Ni-CE-, and CE(NiP)-infected SK-N-SH cells (treated with or without IFN-α) by immunofluorescence assay followed by CLSM analysis (Fig. (Fig.4A).4A). In mock-infected cells, STAT1 was localized mainly in the cytoplasm of IFN-α-untreated cells (Fig. (Fig.4A,4A, a) but became localized in the nucleus after IFN-α treatment (Fig. (Fig.4A,4A, e), indicating clear nuclear translocation of IFN-activated STAT1. In IFN-α-untreated, RV-infected cells, STAT1 was found to be mainly in the cytoplasm, regardless of the RV strain infected (Fig. (Fig.4A,4A, b to d). Importantly, in IFN-α-treated cells, Nishigahara and CE(NiP) infections resulted in inhibition of STAT1 nuclear translocation (Fig. (Fig.4A,4A, f and h, respectively), whereas Ni-CE infection had no apparent effect (Fig. (Fig.4A,4A, g). Comparable results for phosphorylated STAT1 in infected SK-N-SH cells were obtained by immunostaining (data not shown).

FIG. 4.
The subcellular localization of STAT1 in RV-infected SK-N-SH cells differs between the viral strains. (A) The cells were inoculated with each strain at an MOI of 0.01 and then were treated with IFN-α (4,000 U/ml) for 30 min at 24 h p.i. The cells ...

To quantify these effects, we analyzed digitized CLSM images as previously reported (16, 17), to calculate the ratio of nuclear to cytoplasmic fluorescence (Fn/c), corrected for background fluorescence, of STAT1 in the infected cells. In IFN-α-untreated cells, the Fn/c values of STAT1 were similar for mock-, Nishigahara-, Ni-CE-, and CE(NiP)-infected cells (Fig. (Fig.4B).4B). On the other hand, in IFN-α-treated cells, the Fn/c values for STAT1 in Ni-CE-infected cells were significantly greater than those in Nishigahara- or CE(NiP)-infected cells (P < 0.0001). These data strongly suggest that the P protein is responsible for the different inhibitory effects of Nishigahara and Ni-CE infections on IFN-induced STAT1 nuclear translocation.

Ni-CE P protein is defective in its capacity to inhibit STAT1 nuclear translocation.

Next, we examined whether single expression of Nishigahara P protein but not Ni-CE P protein is sufficient to block the nuclear translocation of IFN-activated STAT1. We transfected Vero cells to express Nishigahara or Ni-CE P protein fused to GFP (Ni P-GFP and Ni-CE P-GFP, respectively) and treated the cells with or without IFN-α before fixation, immunostaining for STAT1, and CLSM analysis. In the IFN-α-untreated cells, STAT1 was more cytoplasmic than nuclear (Fig. (Fig.5A,5A, top, and B) and the localization did not differ significantly between Ni P-GFP- and Ni-CE P-GFP-expressing cells (Fig. (Fig.5B).5B). In contrast, in IFN-α-treated cells, STAT1 localization differed significantly between cells expressing Ni P-GFP and Ni-CE P-GFP (Fig. (Fig.5A,5A, bottom, and B). Specifically, STAT1 was retained in the cytoplasm in Ni P-GFP-expressing cells, consistent with previous observations for P proteins from other RV strains (4, 36). On the other hand, STAT1 in Ni-CE P-GFP-expressing cells was able to translocate to and accumulate within the nucleus such that the Fn/c of STAT1 in IFN-α-treated, Ni-CE P-GFP-expressing cells was approximately 7-fold higher than in equivalently treated cells expressing Ni P-GFP (P < 0.0001) (Fig. (Fig.5B).5B). Similar results were obtained from transfected Vero cells treated with IFN-γ and transfected SK-N-SH cells treated with IFN-α or IFN-γ (data not shown).

FIG. 5.
The Ni-CE P protein is defective for cytoplasmic localization and for its capacity to inhibit nuclear import of IFN-activated STAT1. (A) Vero cells were transfected to express the indicated GFP-tagged P protein (green) and, 18 h later, were treated with ...

Taken together with the data from virus-infected cells (Fig. (Fig.4),4), these data demonstrate that Nishigahara P protein is able to block the nuclear translocation of IFN-activated STAT1 more efficiently than Ni-CE P protein and indicate that the inhibitory effect of RV P protein on STAT1 translocation correlates with viral pathogenicity.

Nishigahara and Ni-CE P proteins show differing subcellular localization.

Sequence analysis indicated that the previously identified CRM1-dependent nuclear export signal (NES) (conforming to the motif LXXXLXXLXL, where L can be replaced with M, I, V, or F), which is principally responsible for nuclear exclusion of the P protein of the RV CVS-11 strain (20), is conserved in the Nishigahara P protein (49 LPEDMSRLHL 58; the motif is indicated in boldface) but not in the Ni-CE P protein, due to two amino acid substitutions (49 LPEDMSRPHP 58; substitutions are underlined) (Fig. (Fig.1B)1B) (30). To check whether the amino acid substitutions in the Ni-CE P protein affect its subcellular localization, we compared the distribution of Ni P-GFP and Ni-CE P-GFP in transfected Vero (Fig. (Fig.5A)5A) and SK-N-SH cells (not shown). We found that Ni P-GFP was localized in the cytoplasm (Fig. (Fig.5A,5A, top) and was almost completely excluded from the nucleus (Fn/c, 0.22 ± 0.13) (Fig. (Fig.5C).5C). In contrast, Ni-CE P-GFP was localized more diffusely between the nucleus and cytoplasm, resulting in 4-fold greater levels of accumulation in the nucleus (Fn/c: 0.87 ± 0.32) (P < 0.0001) (Fig. (Fig.5A,5A, top, and C) than for Ni P-GFP. Interestingly, Ni-CE P-GFP was found to become significantly more nuclear after IFN-α treatment (P < 0.0001) (Fig. (Fig.5A,5A, bottom, and C). In contrast, the nucleocytoplasmic localization of Ni P-GFP was unaffected by IFN-α treatment.

We also examined the nucleocytoplasmic distribution of Ni-CE P protein and Nishigahara P protein in RV-infected SK-N-SH cells by fixation and immunostaining for P protein. Nishigahara P protein was localized almost exclusively in the cytoplasm of Nishigahara- and CE(NiP)-infected cells (Fig. 6A and B), whereas Ni-CE P protein was more diffusely localized between the nucleus and cytoplasm (P < 0.0001). There was no obvious fluorescent signal in mock-infected cells (Fig. (Fig.6A),6A), confirming that the signals detected in infected cells were specific for the P protein. These results indicate that the Ni-CE P protein is defective in nuclear export due to the mutagenic inactivation of the NES and strongly suggest that this results in inhibition of the capacity of Ni-CE P protein to regulate STAT1 nuclear import.

FIG. 6.
The Nishigahara (Ni) and Ni-CE P proteins show differing subcellular localizations in infected SK-N-SH cells. (A) The cells were inoculated with each strain at an MOI of 0.01 and, after 24 h, were fixed with 3.7% formaldehyde for 10 min and 90% ...

Both Nishigahara and Ni-CE P protein can interact with STAT1.

Sequence analysis showed that the previously identified STAT1-binding domain (36) is conserved between the Nishigahara and Ni-CE P proteins (Fig. (Fig.1B).1B). This predicts that both P proteins should maintain the ability to bind to STAT1. To confirm the physical interaction between the Nishigahara and Ni-CE P proteins and STAT1, we used the yeast two-hybrid system. The yeast L40 strain that contains the two LexA-responsive reporter genes LacZ and His3 was cotransformed with the pLexA-Ni P or -Ni-CE P plasmid, which encode BD-fused Nishigahara and Ni-CE P proteins, respectively, and pGAD-STAT1 expressing AD-fused STAT1. In addition, we used a combination of pLexA-CVS P, which expresses BD-fused P protein of RV strain CVS, and pGAD-STAT1 as a positive control (36). We found that BD-fused P proteins derived from each of the CVS, Nishigahara, and Ni-CE strains, when coexpressed with AD-fused STAT1, were able to activate transcription of LacZ to comparable extents (Fig. (Fig.7A,7A, top) and could also activate His3 gene expression, resulting in comparable growth on a plate lacking Trp, Leu, and His (Fig. (Fig.7A,7A, bottom). These data demonstrate that both Nishigahara and Ni-CE P protein physically interact with STAT1.

FIG. 7.
Both the Nishigahara (Ni) and Ni-CE P proteins physically interact with STAT1. (A) Yeast cells (L40 strain) were cotransformed with plasmid pLex-CVS P, -Ni P, or -Ni-CE P and plasmid pGAD-STAT1 (+) or the empty pGAD plasmid (−). The P ...

Next, to check the interaction of the Nishigahara and Ni-CE P proteins with STAT1 in infected cells, we carried out co-IP analysis (Fig. (Fig.7B).7B). Lysates of SK-N-SH cells infected with strain Ni-CE or CE(NiP) were subjected to IP with an anti-STAT1 antibody or control rabbit IgG. We found that both Ni-CE and Nishigahara P protein were detected in the precipitates after IP with an anti-STAT1 antibody (Fig. (Fig.7B,7B, middle) but not after IP with control IgG (Fig. (Fig.7B,7B, right). The amounts of Ni-CE and Nishigahara P proteins detected in the precipitates were comparable, indicating that the interactions of Ni-CE and Nishigahara P proteins with STAT1 occur with similar efficiencies.

The NES in RV P protein is important for IFN antagonism.

Despite the fact that the Ni-CE P protein maintains the ability to bind to STAT1, the P protein cannot block nuclear translocation of IFN-activated STAT1. Taken together with the data above showing that the Ni-CE P protein is diffusely localized between the nucleus and cytoplasm, probably due to the inactivation of NES, this strongly suggests that the NES in RV P protein is responsible for retention of the P protein-STAT1 complex in the cytoplasm to inhibit STAT1 nuclear translocation and, thereby, IFN signaling. To test this directly, we mutated the Ni-CE P-GFP to reinstate the functional NES by converting the Pro at positions 56 and 58 to Leu, producing Ni-CE P(NES+)-GFP (Fig. (Fig.8A).8A). We examined the subcellular localization of Ni-CE P(NES+)-GFP, as well as its ability to inhibit STAT1 nuclear translocation. In contrast to Ni-CE P-GFP, Ni-CE P(NES+)-GFP was distributed mainly in the cytoplasm in live SK-N-SH cells (Fig. (Fig.8B).8B). Also, we found that Ni-CE P(NES+)-GFP was significantly more cytoplasmic than Ni-CE P-GFP in Vero cells both treated and untreated with IFN-α (P < 0.0001) (Fig. 8C and D). Importantly, nuclear translocation of IFN-activated STAT1 was inhibited in Vero cells expressing Ni-CE P(NES+)-GFP but not in Vero cells expressing Ni-CE P-GFP (Fig. 8C and E).

FIG. 8.
The NES in RV P protein plays an important role in its IFN antagonism. (A) In order to restore the NES activity to the Ni-CE P protein [producing Ni-CE P(NES+)-GFP], Pro-to-Leu substitutions were introduced into Ni-CE P-GFP at positions 56 and ...

Next, we compared IFN-α-induced ISRE activities in SK-N-SH cells transfected to express Ni P-GFP, Ni-CE P-GFP, or Ni-CE P(NES+)-GFP (Fig. (Fig.8F).8F). We found that the expression of Ni-CE P(NES+)-GFP suppressed ISRE activity more efficiently than Ni-CE P-GFP (P < 0.01). Notably, there was no statistically significant difference between the activities in Ni P-GFP- and Ni-CE P(NES+)-GFP-expressing cells. These results indicate that the NES on the RV P protein plays an important role in the inhibition of STAT1 nuclear translocation and, thereby, of IFN signaling.

DISCUSSION

RV is a neurotropic virus that causes encephalomyelitis with a high mortality rate (almost 100%) in humans and other mammals, for which no effective cure has been established, resulting in approximately 55,000 human fatalities in Asia and Africa per annum (11). To develop an effective cure, it is important to fully understand the molecular mechanism by which RV circumvents host immune response and, consequently, causes the lethal neurological disease. To date, RV glycoprotein (G protein), which participates in binding to host cells, has been shown to play important roles in the viral pathogenicity (7, 10, 27, 32-35), but little is known about the contribution of other RV proteins to pathogenicity.

The RV P protein can inhibit IFN signaling by physically interacting with STAT1, which has been hypothesized to relate to inhibition of STAT1 nuclear localization (4, 36). Thus, it has been assumed that these “IFN antagonist” proteins enable viruses to evade the innate immune system and thereby contribute to the viral pathogenicity. However, the importance of these mechanisms in viral pathogenicity remains unclear. Here, using both RV-infected cells and cells transfected to express only the RV P protein, we show for the first time that the capacity of the RV P protein to inhibit type I IFN signaling correlates with viral pathogenicity.

We found that the CE(NiP) strain grows more efficiently in the mouse brain than does the Ni-CE strain at the early stage of infection (3 days p.i.) (Fig. (Fig.1C).1C). One possible explanation for this difference is that the P protein from the virulent Nishigahara strain functions to efficiently evade innate immunity, whereas the P protein from the attenuated Ni-CE strain is impaired in this function. In the present study, we tested this hypothesis, demonstrating that Nishigahara P protein inhibits type I IFN-activated STAT1 nuclear translocation and IFN signaling more efficiently than does Ni-CE P protein. Therefore, the difference in this P protein function appears to be directly related to the different pathogenicities of the two strains.

Brzózka et al. (3) demonstrated that the RV P protein also inhibits type I IFN induction by interfering with the phosphorylation of the transcription factor interferon regulatory factor 3 (IRF-3) by TANK-binding kinase 1. We found that infection of human neuroblastoma SYM-I cells (9) with either the Ni-CE or CE(NiP) strain induces equivalent levels of IFN-β gene transcription (data not shown). Further, single expression of the Nishigahara and Ni-CE P proteins in SYM-I cells was equally inhibitory for the activation of the IRF-3-dependent IFN-β promoter induced by infection with Newcastle disease virus (13). These findings strongly suggest that the different pathogenicities of strains Nishigahara and Ni-CE do not relate to inhibition of IFN induction.

As the inhibition of IFN-activated STAT1 nuclear accumulation by P protein depends on the association of these molecules (4, 36), one possible explanation for the differences between the Nishigahara and Ni-CE P proteins was that the Ni-CE P protein might be unable to interact with STAT1. However, sequence analysis demonstrated that the C-terminal region within amino acid positions 267 to 297, previously identified as the STAT1-binding domain (36), is perfectly conserved between the Nishigahara and Ni-CE P proteins (Fig. (Fig.1B)1B) (30). Importantly, the results of our yeast two-hybrid and co-IP analyses confirmed that both the Nishigahara and Ni-CE P proteins bind effectively to STAT1 (Fig. (Fig.7).7). Therefore, it appears that the different effects of the Nishigahara and Ni-CE P proteins on IFN signaling do not involve any significant differences in the capacity to interact with STAT1.

The P protein from the RV CVS strain is a nucleocytoplasmic shuttling protein with three distinct signals for nuclear transport: two NESs, corresponding to amino acids 49 to 58 (20) and 227 to 232 (16) and proximal to the N and C terminus, respectively, and a nuclear localization signal (NLS), including residues 211 to 214 and 260 (20). The NESs conform to a motif recognized by the nuclear export transport molecule CRM1, characterized by the presence of several hydrophobic residues (LXXXLXXLXL, where L can be replaced with M, I, V, or F). At steady state, the full-length P protein is almost entirely cytoplasmic, because the N-terminal NES constitutes the predominant signal (16, 20), but following treatment with leptomycin B (LMB), a specific inhibitor of CRM1-mediated nuclear export, RV P protein is able to accumulate in the nucleus because of the presence of the NLS (20). Importantly, the N-terminal NES (49 LPEDMSRLHL 58; the motif is indicated in bold) and C-terminal NLS (211 KKYK 214 and 260 R) are absolutely conserved in Nishigahara P protein. In contrast, the NES motif is destroyed in the Ni-CE P protein, because of amino acid substitutions at positions 56 and 58 (49 LPEDMSRPHP 58; the substitutions are underlined), while the C-terminal NLS is perfectly conserved (Fig. (Fig.1B).1B). That these mutations efficiently inactivate the NES is indicated by the fact that Ni-CE P protein is able to localize in the nucleus and cytoplasm, whereas Nishigahara P protein is almost entirely excluded from the nucleus. Importantly, this difference was observed in both transfected (Fig. 5A and C) and infected cells (Fig. (Fig.6),6), providing the first evidence that the P protein can undergo nucleocytoplasmic trafficking in RV-infected cells, with significance for viral pathogenicity.

Interestingly, we also observed that Ni-CE P-GFP became significantly more nuclear after IFN-α treatment (Fig. 5A and C), suggesting that Ni-CE P protein is “piggybacked” into the nucleus due to the active nuclear import of the associated IFN-activated STAT1. In contrast, in Nishigahara P protein-expressing cells, nether STAT1 nor Ni P-GFP showed any enhancement of nuclear accumulation following IFN-α treatment (Fig. 5A and C). This strongly suggests that the P protein-STAT1 complex is excluded from the nucleus by the active nuclear export of the Nishigahara P protein component via its N-terminal NES. Consistent with this, we demonstrated that reconstitution of NES in Ni-CE P protein conferred the ability to inhibit IFN-activated STAT1 nuclear translocation (Fig. 8C and E) and, consequently, to suppress IFN-α-induced ISRE activity (Fig. (Fig.8F).8F). These findings highlight the importance of the N-terminal NES in the IFN antagonist activity of RV P protein. A previous report using transfected cells showed that the capacity of P protein to block STAT1 nuclear accumulation was disabled by global inhibition of nuclear export by LMB treatment and that a truncated form of P protein (P3) that is actively localized to the nucleus (20) does not block the nuclear accumulation of STAT1 (37). The present report is the first to show that mutations within the N-terminal NES can disable nuclear export of P protein specifically, both in transfected and infected cells, as well as disabling its capacity to block STAT1 nuclear accumulation in these systems.

Despite the observations described above, Ni-CE P protein still modestly suppressed IFN-α-induced ISRE activity (Fig. 3A and B). Vidy et al. (37) previously reported that intranuclear RV P protein can inhibit the interaction of the STAT1-containining transcription factor complex ISGF3 with ISRE-containing DNA, to inhibit transcriptional activation. This function of the nuclear fraction of Ni-CE P protein (Fig. (Fig.5A5A and and6)6) might account for the residual activity observed.

Although RNA viruses have no clear requirement to interact with the cell nucleus, as their basic life cycle occurs entirely in the cytoplasm, it is interesting to note that many proteins encoded by RNA viruses are known to traffic into and/or out of the nucleus (1, 2, 5, 8, 15, 19, 21, 23, 28, 29), indicating that trafficking of these proteins is important to viral infection by permitting the virus to modulate nuclear processes, such as gene transcription. The present study provides fundamental information regarding the mechanism by which RV P protein determines viral pathogenicity, including evidence that the nucleocytoplasmic shuttling of RV P protein that causes retention of IFN-activated STAT1 in the cytoplasm correlates with viral pathogenicity. Also, our data identify a clear example showing the importance of IFN antagonism in viral pathogenicity, with significance for numerous medically important viruses and for the development of novel therapeutic approaches.

Acknowledgments

We thank Akihiko Kawai (Research Institute for Production and Development, Japan) for providing an anti-RV P protein rabbit serum. We are grateful to Jacques Camonis (Institut Curie, France) for the plasmids pLexA and pGAD. We acknowledge Cassandra David for assistance with tissue culture, and we express our appreciation for the facilities and technical assistance of Monash Micro Imaging, Monash University, Victoria, Australia.

This study was partially supported by a grant (project code no. I-AD14-2009-11-01) from the National Veterinary Research and Quarantine Service, Ministry for Food, Agriculture, Forestry, and Fisheries, South Korea, in 2008, and by NHMRC project grant no. 535838.

Footnotes

[down-pointing small open triangle]Published ahead of print on 28 April 2010.

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