NEDDylation of PB2 Reduces Its Stability and Blocks the Replication of Influenza A Virus

Abstract

Post-translational modifications of viral proteins play important roles in regulating viral replication. Here we demonstrated that the PB2 of influenza A virus (IAV) can be modified by NEDD8. We revealed that E3 ligase HDM2 can promote PB2 NEDDylation. Overexpression of either NEDD8 or HDM2 can inhibit IAV replication, while knockdown of HDM2 has the opposite effect. Then we identified residue K699 in PB2 as the major NEDDylation site. We found that NEDDylation deficient PB2 mutant (PB2 K699R) has a longer half-life than wild-type PB2, indicating that NEDDylation of PB2 reduces its stability. We generated an IAV mutant in which PB2 was mutated to PB2 K699R (WSN-PB2 K699R) and examined the replication of WSN and WSN-PB2 K699R viruses in both MDCK and A549 cells and found that the replication of WSN-PB2 K699R was more efficient than wild-type WSN. In addition, we observed that overexpression of NEDD8 significantly inhibited the replication of WSN, but not WSN-PB2 K699R. The infection assay in mice showed that WSN-PB2 K699R exhibited enhanced virulence in mice compared to WSN, suggesting that NEDDylation of PB2 reduced IAV replication in vivo. In conclusion, we demonstrated that NEDDylation of PB2 by HDM2 negatively regulates IAV infection.

Figure 1. PB2 can be NEDDylated.

(a) HEK293T cells were transfected with plasmids encoding FLAG-NP, FLAG-PA, FLAG-PB2, or FLAG-PB1 with or without pEF-His-NEDD8. (b) HEK293T cells were transfected with FLAG-PB2 together with His-ubiquitin, His-SUMO1 or His-NEDD8, respectively. For (a) and (b), the cell lysates were harvested for His-pulldown assay followed by immunoblotting with anti-FLAG antibody. (c) HEK293T cells were transfected with pCMV FLAG-PB2 or empty vector as control. The cell lysates were subjected to immunoprecipitation with anti-FLAG agarose beads, and immunoblotted with anti-NEDD8 antibody. (d) A549 cells were infected with WSN virus at an MOI of 0.1 for 16 h. The cell lysates were subjected to immunoprecipitation with anti-PB2 antibody or rabbit IgG as negative control, and immunoblotted with anti-NEDD8 antibody. (e) A549 cells were infected with WSN virus at an MOI of 0.1 for 16 h. The cell lysates were subjected to immunoprecipitation with anti-NEDD8 antibody or rabbit IgG as negative control, and immunoblotted with anti-PB2 antibody. 1/20 of cell lysates was used as the input. The intensity of NEDDylated PB2 and input PB2 were measured by Quantity One. (f) pCMV myc-PB2 from 4 different IAV subtypes (A/WSN/1933 (H1N1); A/Guangdong/ST798/2008 (H3N2); A/Anhui/1/2013 (H7N9) and A/HK/2108/2003 (H9N2)) were separately co-transfected with or without pEF-His-NEDD8 into HEK293T cells. After 48 h, cell lysates were harvested for His-pulldown assay followed by immunoblotting with anti-Myc antibody. TCL: total cell lysate. (g) HEK293T cells were transfected with FLAG-PB2 together with or without His-NEDD8, and cell lysates were collected for His-pulldown assay. 1/10 of total cell lysates was used as input. The intensity of NEDDylated PB2 and input PB2 were measured by Quantity One. *Unmodified PB2 non-specifically pulled-down by anti-NEDD8 antibody.

Figure 2. E3 ligase HDM2 promotes PB2 NEDDylation, whereas NEDP1 is the deneddylase.

(a) HEK293T cells were transfected with His-NEDD8 and FLAG-PB2 along with Myc-XIAP, Myc-HDM2, Myc-TRIM40, Myc-Smurf1, Myc-RNF111 and HA-SCCRO, HA-RBX1 and HA-c-CBL expression plasmids, respectively. (b) HEK293T cells were transfected with His-NEDD8 and FLAG-PB2 along with Myc-HDM2 or Myc-HDM2 C464A expression plasmids. (c) HEK293T cells were transfected with FLAG-PB2, along with Myc-HDM2 or Myc-XIAP, and His-ubiquitin or His-NEDD8 expression plasmids. For (a), (b) and (c), cell lysates were harvested and subjected to His-pulldown assay, followed by immunoblotting with anti-FLAG antibody. Total cell lysates were immunoblotted with indicated antibodies. (d) HEK293T cells were co-transfected with Myc-HDM2 and FLAG-PB2 expression plasmids. The cell lysates were immunoprecipitated with anti-Myc agarose beads and immunoblotted with anti-FLAG antibody. (e) 293 T cells were transfected with control siRNA or HDM2 siRNA (#1 or #2) for 24 h and then transfected with FLAG-PB2 and His-NEDD8 expression plasmids for 36 h. Cell lysates were harvested and subjected to His-pulldown assay, followed by immunoblotting with anti-FLAG antibody. The total cell lysates were immunoblotted with indicated antibodies.

Figure 3. Overexpression of HDM2 and NEDD8 inhibits IAV replication.

(a,b) HEK293T cells were transfected with pEF-His-NEDD8 and empty vector or pEGFP-C2 as control for 24 h and then infected with WSN at an MOI of 0.5. The cells were harvested at 6 h post-infection, and the total RNA was extracted and subjected to RT-qPCR with primers specific for M1 vRNA and mRNA (a), the cell lysates were subjected to immnoblotting with indicated antibodies (b). (c,d) HEK293T cells were transfected with pCMV-Myc-HDM2 or pCMV-Myc-HDM2 C464A, respectively. At 24 h post-transfection, the cells were infected with WSN virus at an MOI of 0.5 for 10 h and 14 h respectively. The cell lysates were harvested and subjected to immunoblotting with the indicated antibodies (c). The supernatants of infected cells were collected for plaques assay to measure the virus titer (d). (e,f) HEK293T cells were transfected with control siRNA or si-HDM2 for 24 h and then transfected pCMV-Myc-HDM2, and then infected with WSN at an MOI of 0.5 for 10 h and 14 h respectively. The cell lysates were harvested for immunoblotting with the indicated antibodies (e). The supernatants of infected cells were collected for plaque assay to measure the virus titer (f). The data of d and f were expressed as the mean of triplicated samples from 2 independent experiments. *p < 0.05, **p < 0.01.

Figure 4. Residue K699 is the major NEDDylation site in PB2.

(a) HEK293T cells were transfected with expression plasmids encoding FLAG-PB2 or FLAG-PB2 with K-to-R mutations at the indicated positions together with pEF-His-NEDD8 for 48 h. (b) HEK293T cells were transfected with FLAG-PB2 or FLAG-PB2 K699R and His-ubiquitin expressing plasmids. For (a) and (b), cell lysates were harvested and subjected to His-pulldown assay, followed by immunoblotting with anti-FLAG antibody. Cell lysates were immunoblotted with indicated antibodies. (c) HEK293T cells were transfected with pCMV-Myc-PB2 (aa 600–759) for 48 h. Cell lysates were immunoprecipitated with anti-Myc beads. Purified Myc-PB2 (aa 600–759) was subjected to mass spectrometry analysis. b- and y-ion designations are shown on the figure.

Figure 5. NEDDylation of PB2 reduced its stability.

HEK293T cells were transfected with FLAG-PB2 or FLAG-PB2 K699R along with or without His-NEDD8 expression plasmids for 36 h. Cells were treated with 100 μg/ml of CHX at indicated times. The total cell lysates were harvested for immunoblotting with anti-FLAG antibody and anti-β-actin as control (a). Relative PB2 protein levels were quantified and the data represents the average of two independent experiments (b).

Figure 6. NEDDylation of PB2 hinders the replication of IAV.

(a) MDCK cells were infected with WSN or WSN-PB2 K699R at an MOI of 0.01. The supernatants were harvested for plaque assay. (b,c) A549 cells were transfected with or without His-NEDD8 for 24 h and then infected with WSN or WSN-PB2 K699R at an MOI of 0.1 for 16 h. The supernatants were collected for plaque assay (b). The experiment is repeated twice and results were found to be comparable, while all samples were assayed in triplicate. The presented data is the mean from one experiment. Cell lysates were harvested and subjected to immunoblotting with indicated antibodies (c). (d) 293T-IAV-Luc cells were infected with WSN or WSN-PB2 K699R at an MOI of 0.1 for 12 h. The cell lysates were harvested for luciferase assay. The experiment was repeated twice and results were found to be consistent. The presented data is the mean of three biological samples from one experiment. *p < 0.05.

Figure 7. WSN-PB2 K699R possesses higher virulence than WSN.

(a,b,c,d) BALB/c mice (7 weeks old, female) were infected intranasally with PBS (n = 10), WSN (n = 10) or WSN-PB2 K699R (n = 9), respectively (10000 PFU each). The body weight (a) and survival (b) of mice were monitored daily. At 3 and 5 dpi, the lungs of infected mice (n = 3) were collected to measure the virus titer (c) and as well as histopathological analysis (d). Representative histological images of lung tissues from mock-challenged mice (left panels), mice treated with WSN (middle panels) or WSN-PB2 K699R (right panels) were shown. All Experiments for the plague assay were repeated twice and results were found to be comparable. The presented data were expressed as the mean from one experiment. *p < 0.05; **p < 0.01; ***p < 0.001.