Influenza virus NS1 protein counteracts PKR-mediated inhibition of replication

Abstract

The availability of an influenza virus NS1 gene knockout virus (delNS1 virus) allowed us to establish the significance of the biological relationship between the influenza virus NS1 protein and double-stranded-RNA-activated protein kinase (PKR) in the life cycle and pathogenicity of influenza virus. Our results show that the lack of functional PKR permits the delNS1 virus to replicate in otherwise nonpermissive hosts, suggesting that the major function of the influenza virus NS1 protein is to counteract or prevent the PKR-mediated antiviral response.

FIG. 1

(A) Western blot of PKR in infected W138 cells. Cells were mock treated, transfected with dsRNA, or infected with delNS1 or PR8 virus at an MOI of 2. For dsRNA transfection, 50 μg of poly(I)-poly(C) RNA was transfected using 30 μl of DOTAP transfection reagent according to the manufacturer's protocol (Boehringer, Mannheim, Germany). Twenty-four hours postinfection or posttransfection, respectively, cells were lysed, and equivalent amounts of cell extracts were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis. PKR-specific bands were detected by the PKR-specific antibody K-17 (Santa Cruz Biotechnology catalogue no. sc 707; Santa Cruz, Calif.). The upper band corresponds to phosphorylated (active) PKR. The lower band corresponds to unphosphorylated (inactive) PKR (18). The two PKR bands are indicated at the right. Lane 1, mock; lane 2, dsRNA; lane 3, delNS1 virus; lane 4, PR8 virus. (B) Immunoprecipitation of phosphorylated PKR of infected HeLa cells. A total of 10⁶ HeLa cells were mock treated or infected with influenza delNS1 or PR8 virus at an MOI of 0.5. At 5 h postinfection, the cells were washed with a phosphate-free buffer and incubated for 2 h in Dulbecco modified Eagle medium lacking both phosphate and pyruvate (Sigma), containing 500 μCi of [³²P]orthophosphate (Amersham). After being labeled, the cells were washed twice with cold phosphate-buffered saline and 10 mM EDTA (without Ca²⁺ and Mg²⁺) and lysed for 10 min on ice in lysis buffer. One quarter of the extract was used for immunoprecipitation carried out with 2 μg of PKR antibody B-10 (Santa Cruz Biotechnology catalog no. sc 1215), per ml followed by the addition of 30 μl of protein G-agarose (in a 50/50 ratio) at 4°C. The beads were washed according to the manufacturer's protocol with wash buffer containing PBSTDS (Oncogene, Cambridge, Mass.), heated for 2 min at 95°C and analyzed on a sodium dodecyl sulfate–10% polyacrylamide gel electrophoresis gel. The size of the bands was determined by a size marker (Benchmark, GIBCO-BRL). The bands of phosphorylated PKR were visualized by autoradiography for 7 days and quantified by laser densitometry. Lane 1, mock; lane 2, delNS1 virus; lane 3, PR8 virus. The size marker is indicated at the left. The PKR band is indicated at the right.

FIG. 2

Titers of influenza PR8 and delNS1 virus in PKR^+/+ and PKR^−/− mice. Female mice at 7 to 9 weeks of age were used for infection with 10⁵ PFU of wt PR8 or delNS1 virus. Virus was applied intranasally in a volume of 50 μl under ether anesthesia. To determine viral replication in the respiratory tracts, three mice of each group were sacrificed at day 2, 4, or 6 after inoculation. Lungs were removed and homogenized in 3 ml of phosphate-buffered saline. The quantity of virus in homogenates from each mouse was determined by titration on Vero cells. Virus titers are expressed as the number of PFU per milliliter of tissue extract. (A) Titers in PKR^+/+ mice. (B) Titers in PKR^−/− mice. The standard error of the mean is indicated.

FIG. 3

Body weights of PKR^+/+ and PKR^−/− mice which were infected with influenza delNS1 virus. Infection was carried out as described in the legend to Fig. 3. Each group comprised four mice. The standard error of the mean is indicated.