Double-stranded RNA binding of influenza B virus nonstructural NS1 protein inhibits protein kinase R but is not essential to antagonize production of alpha/beta interferon

Abstract

Expression of alpha/beta interferon (IFN-alpha/beta) in virus-infected vertebrate cells is a key event in the establishment of a sustained antiviral response, which is triggered by double-stranded RNA (dsRNA) produced during viral replication. These antiviral cytokines initiate the expression of cellular proteins with activities that limit the replication and spread of the invading viruses. Within this response, the dsRNA-dependent protein kinase R (PKR) that is expressed at constitutive levels and upregulated by IFN-alpha/beta acts as an important antiviral effector that can block the cellular translational machinery. We previously demonstrated that efficient replication of influenza B virus depends on the viral dsRNA-binding NS1 protein that inhibits the transcriptional activation of IFN-alpha/beta genes. Here we tested the postulate that the viral NS1 protein counteracts antiviral responses through sequestering intracellular dsRNA by analyzing a collection of recombinant influenza B viruses. As expected, viruses expressing dsRNA-binding-defective NS1 proteins were strongly attenuated for replication in IFN-competent hosts. Interestingly, these virus mutants failed to prevent activation of PKR but could effectively limit IFN induction. Conversely, a mutant virus expressing the N-terminal dsRNA-binding domain of NS1 prevented PKR activation, but not IFN induction, suggesting an important role for the NS1 C-terminal part in silencing the activation route of IFN-alpha/beta genes. Thus, our findings indicate an unexpected mechanistic dichotomy of the influenza B virus NS1 protein in the suppression of antiviral responses, which involves at least one activity that is largely separable from dsRNA binding.

FIG. 1.

Generation of recombinant influenza B viruses with mutations in the dsRNA-binding domain of the NS1 protein. (A) The diagram indicates the positions of basic amino acid residues within the dsRNA-binding domain of the NS1 protein. NS1 proteins with alanine exchange mutations at the indicated positions had been shown to have strong (+), weak (+/−), or no (−) dsRNA-binding activity (9). The respective mutations were introduced by directed mutagenesis into the portion of the pHW-NS-XhoI plasmid that encodes the NS gene. Each of these constructs was used in an eight-plasmid system for recovery of recombinant influenza B mutant virus expressing an NS1 protein with alanine residues at the indicated positions. (B) RT-PCR analysis of the viral NS segments. After the extraction of viral RNAs of the wild-type (WT) virus and the NS1 mutant viruses, the NS segments were reverse transcribed and amplified by PCR. The amplified NS cDNAs were distinguished by digestion at the introduced HindIII, PstI, NheI, PstI, or BseMI site. The positions of size markers (in kilobases) are indicated on the left. (C) Metabolic labeling of proteins in virus-infected MDCK cells. Cells were either mock infected or infected with the recombinant wild-type or NS1 mutant viruses at an MOI of 10 and incubated at 33°C. At 7 h p.i., cells were metabolically labeled with [³⁵S]methionine for 1 h. Cell extracts were prepared and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. The positions of the molecular mass markers (in kilodaltons) and the NP, NS1, and M1 proteins are indicated on the left and right, respectively.

FIG. 2.

Activation of IRF-3 in infected cells. (A) A549 cells were either mock infected or infected with wild-type (WT) virus or ΔNS1 virus at an MOI of 1. Cell extracts were prepared 6, 9, and 12 h p.i. and subjected to coimmunoprecipitation with a monoclonal IRF-3 antibody. The immunocomplexes were analyzed by immunoblotting using CBP-specific and IRF-3-specific antibodies. (B) MDCK cells were transfected with IRF-3 reporter plasmid and pRL-TK-luc for normalization. Twenty-four hours posttransfection, cells were mock infected or infected with wild-type (WT), ΔNS1, or NS1 mutant virus at an MOI of 1. Reporter activities were determined by the dual luciferase assay and are presented as the change in activation (n-fold) compared to the luciferase value of mock-infected cells. The graph shows average values of three experiments conducted in duplicate. Error bars indicate the standard deviations. (C) A549 cells were either mock infected or infected with wild-type, ΔNS1, or NS1 mutant viruses at an MOI of 1. Cell extracts were prepared 10 h p.i. and immunoprecipitated with an IRF-3-specific antibody followed by immunoblot analysis for CBP and IRF-3. Cell extracts were further analyzed for the presence of the viral NS1 protein. (D) MDCK cells expressing an EGFP-IRF-3 fusion protein were infected with wild-type, ΔNS1, or NS1 mutant viruses at an MOI of 1 and stained for viral NP protein at 8 h p.i. The percentage of cells with nuclear IRF-3 was calculated by counting cells with a clear NP signal and is indicated by the gray bars.

FIG. 3.

dsRNA binding by the NS1 protein is not decisive for the inhibition of IFN-α/β induction. (A) MDCK-C3 cells that contain a stable firefly luciferase reporter gene under the control of the IFN-β promoter were infected with recombinant wild-type (WT), ΔNS1, or the NS1 mutant viruses at an MOI of 1. Luciferase activity was determined 8 h p.i. and is presented as the change in activation (n-fold) compared to the value for mock-infected cells. The graph shows average values of a typical experiment conducted in duplicate, which has been repeated five times. Error bars indicate the standard deviations. (B and C) A549 cells were mock infected or infected with wild-type, ΔNS1, or NS1 mutant viruses at an MOI of 1. Eighteen hours p.i., the supernatant was removed, and IFN-β (B) and IFN-α (C) levels were quantitated by ELISAs. The graphs show average values of three experiments conducted in duplicate. Error bars indicate the standard deviations. hu, human.

FIG. 4.

Replication of recombinant influenza B viruses in embryonated chicken eggs. Six- and eleven-day-old embryonated chicken eggs were inoculated with 1,000 infectious units of wild-type (WT), ΔNS1, or NS1 mutant virus and incubated for 72 h at 33°C. Virus titers were determined as described in Materials and Methods. The indicated values represent the averages of at least three independent experiments. Error bars indicate the standard deviations.

FIG. 5.

The dsRNA-binding activity of the NS1 protein is necessary to inhibit activation of PKR. A549 cells were either mock infected or infected with wild-type (WT), ΔNS1, or NS1 mutant viruses at an MOI of 1. Cell extracts were prepared 10 h p.i. and were analyzed by immunoblotting with antibodies specific for phospho-PKR (P-PKR) (A), total PKR (B), phospho-eIF2α (P-eIF2α) (C), total eIF2α (D), viral NP protein (E), and viral NS1 protein (F).

FIG. 6.

Generation of a recombinant influenza B virus with a C-terminally truncated NS1 protein (NS1-104). (A) Within the plasmid pHW-Lee-NS-XhoI, the amino acid codons 105 and 106 of the NS1 open reading frame were mutated to TAG and TGA, generating two translational stop codons and introducing a genetic tag site. This construct was used along with the other seven plasmids encoding the residual viral gene segments for recovery of the recombinant influenza B NS1-104 virus. After the extraction of viral RNAs from the wild-type (WT) virus or the NS1-104 virus, the NS segments were reverse transcribed (RT) (+) and amplified by PCR. The obtained NS cDNAs were distinguished by restriction analysis of the EcoRV tag site. The positions of size markers (in kilobases) are indicated on the left. (B) Metabolic labeling of proteins in virus-infected MDCK cells. Cells were either mock infected or infected with the recombinant wild-type (WT) or NS1-104 virus at an MOI of 10 and incubated at 33°C. Cells were metabolically labeled at 7 h p.i. with [³⁵S]methionine for 1 h. Cell extracts were prepared and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. The positions of the molecular mass markers (in kilodaltons) and of the NP, NS1, NS1-104, and M1 proteins are indicated on the left and right, respectively.

FIG. 7.

The NS1-104 protein supports viral replication and inhibition of PKR but not inhibition of IFN-α/β. (A) MDCK-C3 cells that contain a stable IFN-β promoter luciferase reporter gene were infected with recombinant wild-type (WT), ΔNS1, or NS1-104 virus at an MOI of 1. Luciferase activity was determined 8 h p.i. and is presented as the change in activation or induction (n-fold) compared to the value for luciferase activity in mock-infected cells. The graph shows average values of a typical experiment conducted in duplicate, which has been repeated five times. Error bars indicate the standard deviations. (B) MDCK cells were transfected with IRF-3 reporter plasmid and pRL-TK-luc for normalization. Twenty-four hours posttransfection, cells were mock infected or infected with wild-type, ΔNS1, or NS1-104 virus at an MOI of 1. Luciferase activities were determined 14 h p.i., and promoter induction is presented as the change in activation (n-fold) compared to the luciferase activity in mock-infected cells. The graph shows average values of two experiments conducted in duplicate. Error bars indicate the standard deviations. (C and D) A549 cells were mock infected or infected with wild-type, ΔNS1, or NS1-104 virus at an MOI of 1. Eighteen hours p.i., the supernatants were removed, and IFN-β (C) and IFN-α levels (D) were quantitated by ELISAs. The graphs show the average values of three experiments conducted in duplicate. Error bars indicate the standard deviations. hu, human. (E) Six- and eleven-day-old embryonated chicken eggs were inoculated with 10³ infectious units of wild-type or NS1-104 virus and incubated for 72 h at 33°C, and virus titers were determined. The indicated values represent the averages of at least three independent experiments. Error bars indicate the standard deviations. (F) A549 cells were either mock infected or infected with wild-type, ΔNS1, or NS1-104 virus at an MOI of 1. Cell extracts were prepared 10 h p.i. and were analyzed by immunoblotting with antibodies specific for phospho-PKR (P-PKR), total PKR, and the viral NP protein.