Journal: PLoS Pathogens

Article Title: Influenza B Virus Ribonucleoprotein Is a Potent Activator of the Antiviral Kinase PKR

doi: 10.1371/journal.ppat.1000473

Figure Lengend Snippet: (A) The schematic diagram shows basic amino acid residues in the dsRNA-binding domain of the NS1 protein. NS1 proteins with alanine exchange mutations at the indicated positions have been shown to have strong (+), weak (+/−) or no (−) dsRNA binding activity . Recombinant influenza B viruses expressing NS1 proteins with abolished dsRNA-binding and mutant virus #1 did not inhibit PKR activation and eIF2α phosphorylation (−), whereas viruses expressing dsRNA binding NS1 proteins inhibited PKR as WT virus (+) . (B) PKR +/+ or PKR −/− MEFs were infected with WT virus, delNS1 virus or NS1 mutant viruses #1, #2, #3, #4, #6 or #7 at an MOI of 0.1. Virus titers were determined at the indicated time points and represent the average of two independent experiments performed as duplicates. Error bars indicate the standard deviation. (C) For infection studies in mice the indicated representative recombinant influenza B/Lee viruses were chosen according to their ability to block PKR activation. Groups of eight-week-old female PKR −/− and wild type C57BL6 mice were anesthetized and infected intranasally with 1×10 5 ffu of the indicated recombinant influenza B/Lee virus. For viral lung titrations, three mice were sacrificed at day 3 and at day 6 post-infection and virus titers were determined in lung homogenates. Error bars indicate the standard deviation. Statistical analysis indicated significant differences between WT and mutant virus titers. *, p<0.05; **, p<0.01; n.d., not detectable. Other recombinant viruses were not tested in this setting.

Article Snippet: The precipitated proteins were analyzed as indicated by SDS gel electrophoresis and immunoblotting using the primary B/NS1-specific rabbit antiserum, mouse anti-PKR antibody 71/10 (Ribogene) or rabbit anti-PKR antibody (Epitomics), mouse anti-NP antibody (AbD Serotec), rabbit anti-phospho-PKR (Thr446) antibody (Cell Signaling), mouse anti-tubulin antibody (Sigma) and suitable secondary horseradish peroxidase (HRP)-conjugated IgG together with an enhanced chemiluminiscence protocol (Pierce).

Techniques: Binding Assay, Activity Assay, Recombinant, Expressing, Mutagenesis, Activation Assay, Infection, Standard Deviation, Blocking Assay

Journal: PLoS Pathogens

Article Title: Influenza B Virus Ribonucleoprotein Is a Potent Activator of the Antiviral Kinase PKR

doi: 10.1371/journal.ppat.1000473

Figure Lengend Snippet: (A) Human A549 lung epithelial cells were mock treated or infected with WT virus or the NS1 mutant viruses #1, #2, #3, #4, #6 or #7 at an MOI of 1. Lysates were subjected to immunoprecipitation (IP) with NS1 antiserum (α) or pre-serum (ctrl). The precipitated complexes were analyzed by immunoblotting with antibodies specific for PKR (upper panel) and NS1 (lower panel). Whole cell lysates were also analyzed with antibodies specific for PKR, tubulin and NS1 as indicated. (B) A549 cells were mock treated or infected with WT virus at an MOI of 1. Lysates were prepared that were either mock treated (−) or treated with the indicated amounts of RNase III for 10 minutes at 33°C prior to immunoprecipitation with NS1 antiserum (α) or pre-serum (ctrl). The precipitates were analyzed as described in panel 2A (panel to the left). Aliquots of the lysates were also analyzed directly by immunoblotting for PKR, tubulin and NS1 as indicated (panels to the right).

Article Snippet: The precipitated proteins were analyzed as indicated by SDS gel electrophoresis and immunoblotting using the primary B/NS1-specific rabbit antiserum, mouse anti-PKR antibody 71/10 (Ribogene) or rabbit anti-PKR antibody (Epitomics), mouse anti-NP antibody (AbD Serotec), rabbit anti-phospho-PKR (Thr446) antibody (Cell Signaling), mouse anti-tubulin antibody (Sigma) and suitable secondary horseradish peroxidase (HRP)-conjugated IgG together with an enhanced chemiluminiscence protocol (Pierce).

Techniques: Infection, Mutagenesis, Immunoprecipitation, Western Blot

Journal: PLoS Pathogens

Article Title: Influenza B Virus Ribonucleoprotein Is a Potent Activator of the Antiviral Kinase PKR

doi: 10.1371/journal.ppat.1000473

Figure Lengend Snippet: (A) A549 cells were infected with WT virus or mutant virus #4 at an MOI of 1. Cells were lyzed 12 hours p.i. and subjected to centrifugation through a continuous 5 to 50% sucrose gradient. 16 fractions were taken from top to bottom. Fractions 1 to 9 were analyzed by immunoblotting with antibodies specific for PKR and the viral NP and NS1 proteins. Also, RNA was extracted from gradient fractions 1 to 9 and was subjected to dot blot hybridization with probes specific for HA vRNA and NS vRNA, respectively (panels “HA and NS vRNA”). Whole cell lysates were analyzed by immunoblotting with antibodies specific for phospho-PKR, total PKR, viral NP, viral NS1 and tubulin as indicated (right panel,“lysate”). (B) A549 cells were mock treated or infected with WT virus or virus mutant #4 as described in panel A. Lysates were prepared and subjected to immunoprecipitation with anti-PKR (α) or control antibody (ctrl). The precipitated complexes were analyzed by immunoblotting for PKR and NS1 proteins. RNA was isolated from an identical set of PKR immunoprecipitates of cells infected with the mutant virus and subjected to dot blot analysis with an RNA-probe specific for HA vRNA.

Article Snippet: The precipitated proteins were analyzed as indicated by SDS gel electrophoresis and immunoblotting using the primary B/NS1-specific rabbit antiserum, mouse anti-PKR antibody 71/10 (Ribogene) or rabbit anti-PKR antibody (Epitomics), mouse anti-NP antibody (AbD Serotec), rabbit anti-phospho-PKR (Thr446) antibody (Cell Signaling), mouse anti-tubulin antibody (Sigma) and suitable secondary horseradish peroxidase (HRP)-conjugated IgG together with an enhanced chemiluminiscence protocol (Pierce).

Techniques: Infection, Mutagenesis, Centrifugation, Western Blot, Dot Blot, Hybridization, Immunoprecipitation, Isolation

Journal: PLoS Pathogens

Article Title: Influenza B Virus Ribonucleoprotein Is a Potent Activator of the Antiviral Kinase PKR

doi: 10.1371/journal.ppat.1000473

Figure Lengend Snippet: (A) A549 cells grown on glass cover slips were infected with WT or mutant virus #4 at an MOI of 1. Cells were mock treated or complemented with LMB starting at 3 hours p.i.. At 8, 12 and 16 hours p.i., cells were fixed and stained for NP (shown in red color), and also for the NS1 protein at the 16 h time-point (shown in green color). Microscopic sample analysis was conducted by confocal laser scanning microscopy. Scale bar, 10 µm. (B) A549 cells grown in culture dishes were infected with WT virus or mutant virus #4 at an MOI of 1. Cells were mock treated or complemented with LMB starting 3 hours p.i. and lyzed 8, 12 and 16 hours p.i.. Whole cell lysates were analyzed by immunoblotting with antibodies specific for phospho-PKR, total PKR, NP, NS1 and tubulin as indicated.

Article Snippet: The precipitated proteins were analyzed as indicated by SDS gel electrophoresis and immunoblotting using the primary B/NS1-specific rabbit antiserum, mouse anti-PKR antibody 71/10 (Ribogene) or rabbit anti-PKR antibody (Epitomics), mouse anti-NP antibody (AbD Serotec), rabbit anti-phospho-PKR (Thr446) antibody (Cell Signaling), mouse anti-tubulin antibody (Sigma) and suitable secondary horseradish peroxidase (HRP)-conjugated IgG together with an enhanced chemiluminiscence protocol (Pierce).

Techniques: Infection, Mutagenesis, Staining, Confocal Laser Scanning Microscopy, Western Blot

Journal: PLoS Pathogens

Article Title: Influenza B Virus Ribonucleoprotein Is a Potent Activator of the Antiviral Kinase PKR

doi: 10.1371/journal.ppat.1000473

Figure Lengend Snippet: The three panels show the localization of the genomic vRNA/RNP complexes with structured 5′- and 3′-ends together with the phosphorylation status of PKR in early (panel A) and late phases (panels B and C) of virus infection. Viral RNP is located in the nucleus early in infection and is not accessible for cytosolic PKR (panel A). Upon nuclear export at later stages, vRNP complexes stimulate PKR activation in the absence of functional NS1 protein, leading to an inhibition of viral proteins synthesis and replication (panel B). However, the presence of functional viral NS1 protein suppresses this antiviral reaction thereby supporting high-level viral propagation (panel C).

Article Snippet: The precipitated proteins were analyzed as indicated by SDS gel electrophoresis and immunoblotting using the primary B/NS1-specific rabbit antiserum, mouse anti-PKR antibody 71/10 (Ribogene) or rabbit anti-PKR antibody (Epitomics), mouse anti-NP antibody (AbD Serotec), rabbit anti-phospho-PKR (Thr446) antibody (Cell Signaling), mouse anti-tubulin antibody (Sigma) and suitable secondary horseradish peroxidase (HRP)-conjugated IgG together with an enhanced chemiluminiscence protocol (Pierce).

Techniques: Infection, Activation Assay, Functional Assay, Inhibition