Role of interferon antagonist activity of rabies virus phosphoprotein in viral pathogenicity

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.

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 i.c. inoculation with 1,000 FFU of each virus (30). ++, lethal (all mice died within 7 days); +, lethal (all mice died within 14 days); −, not lethal (all mice survived). (B) Amino acid differences between Nishigahara and Ni-CE P proteins are highlighted. The previously identified nuclear export signal (NES), nuclear localization signal (NLS) (20), and STAT1-binding domain (36) are also indicated. AA, amino acids. (C) Propagation of Nishigahara, Ni-CE, and CE(NiP) strains in adult mouse brains. The virus titers in the mouse brains were determined as previously described (34). †, All Nishigahara-inoculated mice died after this time point.

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 cells were inoculated with each virus at an MOI of 0.01 and cultured in growth medium with or without human IFN-α (500 U/ml). The viral titer in the culture medium, collected 3 days p.i., was determined by a focus assay using mouse neuroblastoma NA cells. The data are the means ± standard deviations of three independent replicates. (B) IFN sensitivity index of each strain. The index is 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 (500 U/ml). Each bar represents the mean ± standard deviation of the results of three independent replicates. ns, not significant (P ≥ 0.05). (C) Viral protein synthesis of each strain growing in SK-N-SH cells treated without or with IFN-α. The cells were inoculated with each virus at an MOI of 0.01 and then were cultured with growth medium containing different concentrations of IFN-α (0, 20, 100, and 500 U/ml). The cell lysate was prepared at 2 days p.i. and analyzed by Western blotting using an anti-P protein rabbit serum or an anti-N protein mouse monoclonal antibody. Alpha-tubulin (α-Tubulin) was used as a loading control. The numbers in the boxes represent relative amounts of RV P or N protein standardized to the amount of the loading control. The amounts of each protein are shown as the ratio, considering the standardized amount in IFN-α-untreated cells (0 U/ml) as 1.00. MW, molecular weight (in thousands).

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 plasmid (pRL-TK) and then inoculated with each virus at an MOI of 3 at 24 h posttransfection. Six hours later, the cells were treated with IFN-α (2,000 U/ml) for 12 h and the cell lysates were subjected to the dual luciferase assay. The data represent firefly luciferase activity (GL) normalized to Renilla luciferase activity (RL), presented as the means ± standard deviations of the results of three independent replicates. (B, upper panel) SK-N-SH cells were transfected with the ISRE reporter and the control plasmids together with the expression plasmid encoding Nishigahara or Ni-CE P protein or Nishigahara N protein (pcDNA-Ni P, -CEP, and -Ni N, respectively). At 24 h posttransfection, the cells were treated with IFN-α (2,000 U/ml) for 6 h and the cell lysates were subjected to the dual luciferase assay. GL, firefly luciferase activity; RL, Renilla luciferase activity; ns, not significant (P ≥ 0.05). (A and B, lower panels) Western blot analysis of the cell lysates used for the luciferase assay was carried out using an anti-P protein rabbit serum or an anti-N protein mouse monoclonal antibody. Antitubulin (α-Tubulin) was used as the loading control. (C) Relative expression level of MxA mRNA in SK-N-SH cells infected with the Nishigahara, Ni-CE, or CE(NiP) strain. SK-N-SH cells were inoculated with each virus at an MOI of 3 and, 6 h later, were treated with IFN-α (2,000 U/ml) for 12 h. Total RNA was extracted from the cells and subjected to quantitative real-time RT-PCR. The expression levels of the MxA gene were normalized to the mRNA levels of GAPDH. Each bar represents the mean ± standard deviation of the results of three independent replicates. Ni, Nishigahara; WB, Western blot.

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 were fixed with 3.7% formaldehyde for 10 min and 90% methanol for 5 min before being immunostained for STAT1 (green) and RV N protein (red) and analyzed by CLSM. (B) Images such as those shown in panel A were used to calculate the ratios of nuclear to cytoplasmic fluorescence (Fn/c) of STAT1, which are shown as the means ± standard errors of the means of the results from >30 images. ns, not significant (P ≥ 0.05); Ni, Nishigahara.

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 or without IFN-α for 1 h. The cells were fixed with formaldehyde and methanol and immunostained for STAT1 (red) before being analyzed by CLSM. (B, C) Images such as those shown in panel A were analyzed to derive the ratio of nuclear to cytoplasmic fluorescence (Fn/c) values (mean ± standard error of the mean, n > 130, combined data from 3 separate assays) for STAT1 (B) or GFP-tagged P protein (C). Ni, Nishigahara.

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% methanol for 5 min. The fixed cells were subjected to immunostaining for RV P protein and analyzed by CLSM. Dot structures observed in the cytoplasm represent Negri bodies (12). (B) Images such as those shown in panel A were used to calculate the ratio of nuclear to cytoplasmic fluorescence (Fn/c) values, which are shown as the means ± standard errors of the means of the results from >30 images.

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 protein-STAT1 interaction was assessed by the appearance of blue colonies in the presence of X-Gal on a plate lacking Trp and Leu (upper panel) and by the expression of the His3 reporter gene on a plate lacking Trp, Leu, and His (lower panel). (B) SK-N-SH cells were inoculated with strain Ni-CE or CE(NiP) at an MOI of 0.1. At 18 h p.i., the cells were treated with IFN-α for 2 h before being lysed in RIPA buffer. The cell lysates were subjected to co-IP analysis with an anti-STAT1 antibody or control rabbit IgG. The precipitates and total lysate (input) were analyzed by Western blotting (WB). The asterisk represents an additional band probably resulting from binding of antibodies used for Western blotting to protein A/G. α-Tubulin, alpha-tubulin.

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 58. (B) To compare the subcellular localization of Ni-CE P(NES⁺)-GFP with that of Ni-CE P-GFP, SK-N-SH cells were transfected with plasmid pEGFP-N1 expressing the respective protein and images were collected 24 h posttransfection. (C) Vero cells were transfected to express Ni-CE P-GFP or Ni-CE P(NES⁺)-GFP (green) and treated with or without IFN-α before being fixed and immunostained for STAT1 (red) and analyzed by CLSM. (D, E) Images such as those shown in panel C were analyzed to derive the ratio of nuclear to cytoplasmic fluorescence (Fn/c) values (mean ± standard error of the mean, n > 30) for GFP-tagged P protein (D) or STAT1 (E). (F) SK-N-SH cells were transfected with the ISRE reporter and the control plasmids, together with the pEGFP-N1 plasmid expressing Ni-GFP, Ni-CE GFP, or Ni-CE P(NES⁺)-GFP. At 24 h posttransfection, the cells were treated with IFN-α (2,000 U/ml) for 6 h and the cell lysates were subjected to the dual luciferase assay. GL, firefly luciferase activity; RL, Renilla luciferase activity; ns, not significant (P ≥ 0.05).