Identification of the rabies virus alpha/beta interferon antagonist: phosphoprotein P interferes with phosphorylation of interferon regulatory factor 3

Abstract

Rabies virus (RV) of the Rhabdoviridae family grows in alpha/beta interferon (IFN)-competent cells, suggesting the existence of viral mechanisms preventing IFN gene expression. We here identify the viral phosphoprotein P as the responsible IFN antagonist. The critical involvement of P was first suggested by the observation that an RV expressing an enhanced green fluorescent protein (eGFP)-P fusion protein (SAD eGFP-P) (S. Finke, K. Brzozka, and K. K. Conzelmann, J. Virol. 78:12333-12343, 2004) was eliminated in IFN-competent HEp-2 cell cultures, in contrast to wild-type (wt) RV or an RV replicon lacking the genes for matrix protein and glycoprotein. SAD eGFP-P induced transcription of the IFN-beta gene and expression of the IFN-responsive MxA and STAT-1 genes. Similarly, an RV expressing low levels of P, which was generated by moving the P gene to a promoter-distal gene position (SAD DeltaPLP), lost the ability to prevent IFN induction. The analysis of RV mutants lacking expression of truncated P proteins P2, P3, or P4, which are expressed from internal AUG codons of the wt RV P open reading frame, further showed that full-length P is competent in suppressing IFN-beta gene expression. In contrast to wt RV, the IFN-inducing SAD DeltaPLP caused S386 phosphorylation, dimerization, and transcriptional activity of IFN regulatory factor 3 (IRF-3). Phosphorylation of IRF-3 by TANK-binding kinase-1 expressed from transfected plasmids was abolished in wt RV-infected cells or by cotransfection of P-encoding plasmids. Thus, RV P is necessary and sufficient to prevent a critical IFN response in virus-infected cells by targeting activation of IRF-3 by an upstream kinase.

FIG. 1.

SAD eGFP-P induces IFN-β gene expression. (A) Genome organization of wt SAD L16; SAD eGFP-P, expressing a P protein with an N-terminal eGFP-moiety; NPgrL virus, with M and G genes replaced with eGFP and DsRed genes; and SAD VB eGFP, containing an extra eGFP gene. (B) Whole-cell extracts of BSR T7/5 and HEp-2 cells were harvested at 48 h p.i. (MOI of 1) and analyzed by Western blotting for expression of the viral N, P, and M proteins. In contrast to infection of BSR cells (right panel), SAD eGFP-P produced low levels of N and eGFP-P protein in HEp-2 cells (left panel). (C) SAD eGFP-P infection of HEp-2 cells induces transcription of IFN-β mRNA as shown by RT-PCR (right panel). and up-regulation of MxA and STAT1 proteins as shown by Western blotting (left panel).

FIG. 2.

The level of P protein is crucial for growth of RV in IFN-competent cells. (A) Genome organization of recombinant RV. SAD PLP carries an extra copy of the P gene downstream of L (gene order, 3′-N-P-M-G-L-P-5′). In SAD ΔPLP, the P gene downstream of N in SAD PLP was deleted, resulting in a virus with a single P gene copy downstream of L (gene order, 3′-N-M-G-L-P-5′). (B) Northern blot hybridization with N and P gene-specific cDNA of RNA from BSR T7/5 cells infected for 24 h at an MOI of 1 with the indicated viruses. SAD ΔPLP virus produces lower levels of P mRNA than SAD L16. (C) Expression of RV N, P, M, and G proteins was analyzed in Western blots of cell extracts from BSR T7/5 at 24 h p.i. at an MOI of 1. (D and E) Single-step growth curves were performed on BSR T7/5 cells (D) and on HEp-2 cells (E) after infection with the indicated viruses at an MOI of 0.01. ffu, focus-forming units.

FIG. 3.

The level of P protein is crucial for IFN-β gene expression. (A) SAD ΔPLP effectively induces transcription of IFN-β mRNA in HEp-2 cells as shown by RT-PCR. Mock-infected cells were stimulated by poly(I · C) transfection. RNA was isolated 20 h after infection at an MOI of 1. For RT-PCR, primers specific for IFN-β or β-actin (as a loading control) were used. (B) SAD ΔPLP infection induces an antiviral response and up-regulates expression of MxA and STAT1 as shown by Western blotting at 48 h p.i. Only in wt SAD L16-infected cells were abundant amounts of RV N and P proteins detected.(C) Infection (MOI of 1) with SAD ΔPLP of U3A cells lacking STAT-1 leads to accumulation of viral proteins, in contrast to the case for the parental STAT-1-containing 2fTGH cells.

FIG. 4.

Mutation of internal AUG start codons does not abrogate IFN-antagonistic activity of P. (A) Schematic representation of mutations introduced into the P protein. Solid vertical bars represent authentic methionine codons. Lack of the bar indicates substitution with isoleucine codons. Dotted vertical bars represent the AUG codon of the hypothetical C of P ORF + 1. In SAD PΔC, the AUG codon was mutated to ACG. (B) Protein synthesis of RV encoding mutated P proteins. BSR T7/5 cells were infected with the indicated viruses at an MOI of 1. Cell extracts were analyzed by Western blotting at 24 h p.i. P and N proteins were detected by mouse polyclonal anti-P serum and rabbit polyclonal serum S50, respectively. (C) Single-step growth curves of RVs lacking short forms of P protein on BSR T7/5 and HEp-2 cells infected at an MOI of 0.01. RV SAD P1xxx, lacking P2, P3, and P4, grew productively in HEp-2 cells.

FIG. 5.

Rabies virus targets the IRF-3 activation pathway. (A) Vero cells were transfected with reporter plasmid p125luc, p55C1Bluc, p55A2luc, or pAP1luc and infected at an MOI of 3 with SAD L16 or SAD ΔPLP. Cell lysates were analyzed using the dual luciferase reporter system (Promega) for luciferase activity at 48 h p.i. In contrast to SAD ΔPLP, SAD L16 is able to prevent expression of firefly luciferase from plasmids containing the IFN-β promoter (p125luc) and the IRF-3 binding site (p55C1Bluc). In contrast to IRF-3, the activities of NF-κB and AP-1 are not further stimulated in SAD ΔPLP-infected cells compared to SAD L16-infected cells. Incubation of cells with tumor necrosis factor alpha was used as a positive control. Error bars indicate standard deviations. (B) Dimerization and Ser386 phosphorylation of IRF-3 in SAD ΔPLP-infected cells. HEp-2 cells were infected at an MOI of 1 and cell extracts were analyzed at 24 h p.i. by native PAGE and Western blotting. In contrast to SAD ΔPLP, wt SAD L16 prevents IRF-3 dimerization and phosphorylation on Ser386. The same cell lysates were analyzed by SDS-PAGE for expression of viral N and P proteins.

FIG. 6.

Expression of P is sufficient to prevent TBK-1-mediated IFN-β induction. (A) HEK 293 cells were transfected with expression plasmids encoding the indicated genes and reporter plasmids harboring the firefly luciferase gene under control of the IFN-β promoter and renilla luciferase controlled by the CMV promoter. Luciferase activities were determined at 48 h posttransfection. Cotransfection of P or of P1xxx with TBK-1 inhibited activation of the IFN-β promoter almost completely. The P protein of BRSV (RSV P) was used as a control that is not able to inhibit TBK-1-mediated activation of the p125luc reporter plasmid (left panel). The inhibition of TBK-1-mediated activation of IRF-3 by RV P is dose dependent as revealed by luciferase expression from p55C1Bluc (right panel). Error bars indicate standard deviations. (B) Expression of RV P inhibits TBK-1-mediated dimerization of endogenous IRF-3 and phosphorylation of IRF-3 at Ser386. Cell extracts were harvested at 24 h posttransfection of TBK-1 or TBK-1 and P-encoding plasmids and were analyzed by native PAGE and Western blotting. Expression of Flag-tagged TBK-1 (fl-TBK1), RV P, and β-actin was confirmed by SDS-PAGE and Western blot analysis of the same lysates.