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NEDDylation of PB2 Reduces Its Stability and Blocks the Replication of Influenza A Virus
Tinghong Zhang1,2, Zhen Ye1,2,3, Xiaohai Yang1,2,4
1CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing, China.
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.
Insights
Post-translational modification of influenza A virus (IAV) PB2 protein by NEDD8, promoted by HDM2, inhibits viral replication. This finding reveals a new regulatory mechanism for IAV infection.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Post-translational modifications regulate viral protein function and replication.
- The role of NEDD8 modification in influenza A virus (IAV) replication is not well understood.
Purpose of the Study:
- To investigate the role of NEDD8 modification of IAV PB2 protein in viral replication.
- To identify the E3 ligase responsible for PB2 NEDDylation and the specific modification site.
Main Methods:
- Co-immunoprecipitation and Western blotting to detect PB2 NEDDylation.
- Site-directed mutagenesis to identify the NEDDylation site (K699).
- Viral replication assays in cell culture (MDCK, A549) and in vivo mouse models.
- Overexpression and knockdown experiments for NEDD8 and HDM2.
Main Results:
- HDM2 promotes the NEDDylation of IAV PB2 protein at residue K699.
- NEDD8 or HDM2 overexpression inhibited IAV replication, while HDM2 knockdown enhanced it.
- PB2 K699R mutant showed increased stability and enhanced viral replication compared to wild-type IAV.
- The PB2 K699R mutant exhibited increased virulence in mice, indicating reduced viral replication in vivo.
Conclusions:
- NEDDylation of IAV PB2 by HDM2 is a novel mechanism that negatively regulates viral replication.
- Targeting the PB2 NEDDylation pathway could be a potential strategy for antiviral therapy against influenza A virus.
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