Influenza virus ns1 protein induces apoptosis in cultured cells

Abstract

The importance of influenza viruses as worldwide pathogens in humans, domestic animals, and poultry is well recognized. Discerning how influenza viruses interact with the host at a cellular level is crucial for a better understanding of viral pathogenesis. Influenza viruses induce apoptosis through mechanisms involving the interplay of cellular and viral factors that may depend on the cell type. However, it is unclear which viral genes induce apoptosis. In these studies, we show that the expression of the nonstructural (NS) gene of influenza A virus is sufficient to induce apoptosis in MDCK and HeLa cells. Further studies showed that the multimerization domain of the NS1 protein but not the effector domain is required for apoptosis. However, this mutation is not sufficient to inhibit apoptosis using whole virus.

FIG. 1

Expression of NS induces apoptosis in HeLa cells. (A) Confluent cultures of HeLa cells expressing pUHD 10-3 containing the NS gene from A/Ty/Ont/66 were washed two times with PBS and then incubated for 5, 24, or 48 h in medium with (+) or without (−) anhydrotetracycline. Cell monolayers were lysed, and 5 mg of total protein was loaded per lane under reducing conditions and resolved by SDS-PAGE. After being transferred to nitrocellulose, proteins were probed for NS with a mouse monoclonal antibody against NS. Bands were detected by enhanced chemiluminescence as instructed by the manufacturer. The arrow indicates the location of NS1. Molecular size markers are indicated on the left. MDCK cells infected with A/Ty/Ont/66 at an MOI of 0.5 for 24 h served as a positive control (virus lane). (B) Confluent cultures of HeLa cells in 25-cm² flasks expressing pUHD 10-3 containing the NS gene from A/Ty/Ont/66 were washed two times with PBS and then incubated for 8 or 24 h in medium containing anhydrotetracycline (+) or anhydrotetracycline-free medium (−) containing 1% FBS at 37°C in 5% CO₂. DNA was collected and analyzed for DNA fragmentation by agarose gel analysis.

FIG. 2

Expression of NS induces apoptosis. (A) Confluent cultures of MDCK cells expressing pUHD 10-3 containing the NS gene from A/Ty/Ont/66 were washed two times with PBS and then incubated for 5, 24, or 48 h in medium without anhydrotetracycline. Cell monolayers were lysed, and 5 mg of total protein was loaded per lane under reducing conditions and resolved by SDS-PAGE. After being transferred to nitrocellulose, proteins were probed for NS with a mouse monoclonal antibody against NS. Bands were detected by enhanced chemiluminescence as instructed by the manufacturer. The arrow indicates the location of NS1. Molecular size markers are indicated on the left. MDCK cells infected with A/Ty/Ont/66 at an MOI of 0.5 for 24 h served as a positive control (virus lane), and cells expressing empty vector (pUHD) served as a negative control. Results for two different clones (D1 and D5) are shown. (B) Confluent cultures of MDCK cells in 25-cm² flasks expressing pUHD 10-3 containing the NS gene from A/Ty/Ont/66 or empty vector (pUHD) were washed two times with PBS and then incubated for 5, 24, or 48 h in anhydrotetracycline-free MEM containing 1% FBS at 37°C in 5% CO₂. DNA was collected and analyzed for DNA fragmentation by agarose gel analysis. Two different clones, D1 and D5, expressing NS protein were analyzed.

FIG. 3

The RNA-binding domain of NS1 is required for apoptosis. (A) Confluent cultures of MDCK cells expressing pUHD 10-3 containing the NS1 gene from A/Udom/72 or empty vector (pUHD) were washed two times with PBS and then incubated for 5, 24, or 48 h in anhydrotetracycline-free MEM containing 1% FBS at 37°C in 5% CO₂. DNA was collected and analyzed for DNA fragmentation by agarose gel analysis. Two different clones, C4 and C9, expressing NS1 protein were analyzed. (B) Confluent cultures of MDCK cells expressing pUHD 10-3 containing an NS mutant that fails to produce NS1 protein (NS13′SS DM clones E8 and G4), a mutation in the RNA-binding domain (M2), full-length NS (clone D1), or a deletion of the effector domain (NS1Δ117-161) were washed two times with PBS and then incubated for 5, 24, or 48 h in anhydrotetracycline-free MEM containing 1% FBS at 37°C in 5% CO₂. DNA was collected and analyzed for DNA fragmentation by agarose gel analysis. (C) Confluent cultures of MDCK cells expressing pUHD 10-3 containing the NS M2 (RNA-binding domain mutation), full-length NS gene from A/Ty/Ont/66, or NS1Δ117-161 were washed two times with PBS and then incubated for 5, 24, or 48 h in medium without anhydrotetracycline. Cell monolayers were lysed, and 5 mg of total protein was loaded per lane under reducing conditions and resolved by SDS-PAGE. After being transferred to nitrocellulose, proteins were probed for NS with a rabbit polyclonal antibody against NS. Bands were detected by enhanced chemiluminescence as instructed by the manufacturer. The arrow indicates the location of NS. Molecular size markers are indicated on the left. MDCK cells infected with A/Ty/Ont/66 at an MOI of 0.5 for 24 h served as a positive control (virus lane).

FIG. 4

Influenza viruses containing mutated NS1 RNA-binding sites induce apoptosis. Confluent cultures of MDCK cells were washed two times with PBS, incubated with MEM alone (lane 1) or MEM with A/WSN parental virus (lane 2), A/WSN NS1 RK 19/20 AA (lane 3), or NS1 RK 19/20 AD (lane 4) at an MOI of 2 and incubated for 1 h at 37°C in 5% CO₂. After the 1-h binding period, the cells were washed with PBS to remove residual virus and incubated for 24 or 48 h in MEM containing 5% BSA. DNA was collected and analyzed for DNA fragmentation by agarose gel analysis.