Genetic dissection of interferon-antagonistic functions of rabies virus phosphoprotein: inhibition of interferon regulatory factor 3 activation is important for pathogenicity

Abstract

The rabies virus (RV) phosphoprotein (P) is a type I interferon (IFN) antagonist preventing both transcriptional induction of IFN and IFN-mediated JAK/STAT signaling. In addition, P is an essential cofactor of the viral polymerase and is required for encapsidation of viral RNA into nucleoprotein during replication. By site-directed mutagenesis, we have identified a domain of P required for efficient inhibition of IFN induction. Phosphoproteins lacking amino acids (aa) 176 to 181, 182 to 186, or 176 to 186 were severely compromised in counteracting phosphorylation of IRF3 and IRF7 by TBK1 or IKKi while retaining the full capacity of preventing nuclear import of activated STATs and of supporting virus transcription and replication. Recombinant RV carrying the mutated phosphoproteins (the SAD ΔInd1, SAD ΔInd2, and SAD ΔInd1/2 viruses) activated IRF3 and beta IFN (IFN-β) transcription in infected cells but still blocked STAT-mediated expression of IFN-stimulated genes. Due to a somewhat higher transcription rate, the SAD ΔInd1 virus activated IRF3 more efficiently than the SAD ΔInd2 virus. After intracerebral injection into mouse brains at high doses, the SAD ΔInd1 virus was completely apathogenic for wild-type (wt) mice, while the SAD ΔInd2 virus was partially attenuated and caused a slower progression of lethal rabies than wt RV. Neurovirulence of IFN-resistant RV thus correlates with the capacity of the virus to prevent activation of IRF3 and IRF7.

FIG. 1.

RV P mutants with decreased ability to inhibit IFN-β transcription. HEK 293T cells were transfected with full-length RV P coding plasmids or the indicated P mutants using Lipofectamine 2000. (A) Dual-luciferase reporter gene assays were performed 24 h p.i. with p125-luc and pCMV-Renilla, and plasmids encoding ΔRIG-I, ΔMDA5, or TBK1. (B) ΔRIG-I and P constructs were expressed from transfected plasmids, and IFN-β mRNA levels were analyzed by qRT-PCR 24 h posttranscription. Averages of results from two experiments are depicted, with error bars representing standard deviations. Expression levels were controlled by SDS-PAGE by using antibodies against RV P and actin. (C) Dual-luciferase assay from cells transfected with IRF3 plus TBK1 (150 ng each), IRF7 plus TBK1 (150 ng each), IRF3-5D (300 ng), or IRF7-2D (300 ng) and expressing the indicated P constructs. (D) Phosphorylation and dimerization of IRF3 were analyzed for HEK 293T cells cotransfected with TBK1 (left) or IKKi (right) and with RV P expression vectors as indicated. Cell lysates were prepared 24 h posttransfection and submitted to native PAGE analysis. Only wt RV P was able to efficiently prevent IRF3 phosphorylation and dimerization.

FIG. 2.

(A) RV P mutants support gene expression from the RV pSDI-luc minigenome. BSR-T7/5 cells were transfected with empty vector, RV P, or the indicated P mutants along with plasmids encoding RV L, RV N, and Renilla luciferase. Luciferase activities and protein expression were determined at 48 h posttransfection. (B) Infectious titers of SAD PΔInd mutant viruses from supernatants of BSR-T7/5 cells infected at an MOI of 0.1. (C) Production of virus proteins in BSR-T7/5 cells infected at an MOI of 1. Cell extracts were harvested at days 1 and 2 after infection and submitted to Western blot analysis. Accumulation of viral proteins is comparable in cells infected with the SAD PΔInd and wt SAD L16 viruses, while protein accumulation is delayed in the SAD ΔPLP mutant. (D) Ability of SAD PΔInd viruses to inhibit IFN signaling. BSR-T7/5 cells were transfected with an ISRE luciferase reporter plasmid, infected at an MOI of 3, and stimulated at 24 h p.i. with 1,000 IU/ml of IFN-α A/D. Luciferase induction by IFN is observed only in mock- and SAD ΔPLP mutant-infected cells.

FIG. 3.

(A) Growth of SAD ΔInd mutant viruses on IFN-competent HEp-2 cells infected at an MOI of 0.05. While SAD ΔPLP virus is completely attenuated, the SAD ΔInd1 and SAD ΔInd2 mutants were able to amplify similar to SAD L16. (B) Activation of IRF3 in HEp-2 cells infected with the indicated viruses at an MOI of 1 for 24 h. Native PAGE and Western blot analysis revealed phosphorylated and dimerized IRF3 in the mutant viruses. (C) Nuclear localization of IRF3 in HEp-2 cells infected with SAD ΔInd viruses. Twenty-four hours after infection at an MOI of 1, HEp-2 cells were stained with antibodies specific for IRF3 and RV and with To-PRO-3 dye to delineate the nucleus. (D) Western blot analysis of HEp-2 cells infected with the indicated viruses at an MOI of 1 for 24 and 48 h. The indicated viral proteins, interferon-stimulated genes (MxA, IRF9), and the phosphorylation status of STATs were monitored using respective antibodies. For details, see Results. (E) Western blot analysis of HEp-2 cells infected with SAD PΔInd mutant viruses at an MOI of 1. The lysates were prepared 1 and 2 days after infection. Viral proteins were analyzed with respect to their expression levels using respective antibodies. The ratio of RV M relative to RV G (bottom; wt RV set to 1.0) was calculated using the Bio 1D software of the Fusion Molecular Imaging device.

FIG. 4.

(A and B) IFN induction in 293T cells after infection with mutant RV (MOI of 3). (A) Dual-luciferase assay from cells infected for 24 h. (B) qRT-PCR for IFN-β mRNA at 24 h p.i. Infection and P expression were controlled by Western blot analysis. (C) Transcription analysis of recombinant viruses. BSR-T7/5 cells were infected with the indicated viruses at an MOI of 3. RNA was harvested 24 h postinfection and analyzed by Northern blot hybridization with an N probe. The ratio between N gene mRNA and the genome RNA was calculated using ImageQuant and presented as the percentage of the wt SAD L16 ratio.

FIG. 5.

Attenuation in vivo of SAD ΔInd viruses. wt mice (A), IFNAR^−/− mice (B), and IFNGR^−/− mice (C) were intracranially infected with 10⁵ FFU of the indicated viruses, and survival was monitored. Whereas IFNAR^−/− mice rapidly succumbed to the infection with either RV, the SAD PΔInd2 mutant caused a delayed disease course, and the SAD ΔInd1 mutant was completely apathogenic in wt mice.