Phosphorylation of PB2 at serine 181 restricts viral replication and virulence of the highly pathogenic H5N1 avian influenza virus in mice

Jiao Hu; Zixiong Zeng; Xia Chen; Manyu Zhang; Zenglei Hu; Min Gu; Xiaoquan Wang; Ruyi Gao; Shunlin Hu; Yu Chen; Xiaowen Liu; Daxin Peng; Xiufan Liu

doi:10.1016/j.virs.2023.12.003

February 2024

Citation: Jiao Hu, Zixiong Zeng, Xia Chen, Manyu Zhang, Zenglei Hu, Min Gu, Xiaoquan Wang, Ruyi Gao, Shunlin Hu, Yu Chen, Xiaowen Liu, Daxin Peng, Xiufan Liu. Phosphorylation of PB2 at serine 181 restricts viral replication and virulence of the highly pathogenic H5N1 avian influenza virus in mice .VIROLOGICA SINICA, 2024, 39(1) : 97-112. http://dx.doi.org/10.1016/j.virs.2023.12.003

Phosphorylation of PB2 at serine 181 restricts viral replication and virulence of the highly pathogenic H5N1 avian influenza virus in mice

Jiao Hu ^a,c,d ,
Zixiong Zeng ^a,c,d ,
Xia Chen ^a,c,d ,
Manyu Zhang ^a,c,d ,
Zenglei Hu ^a,b ,
Min Gu ^a,c,d ,
Xiaoquan Wang ^a,c,d ,
Ruyi Gao ^a,c,d ,
Shunlin Hu ^a,c,d ,
Yu Chen ^a,c,d ,
Xiaowen Liu ^a,c,d ,
Daxin Peng ^a,c,d ,
Xiufan Liu ^{a,c,d
,,}

a. Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China;
b. Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, 225009, China;
c. Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, 225009, China;
d. Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agri-food Safety and Quality, Ministry of Agriculture of China (26116120), Yangzhou University, Yangzhou, 225009, China

Corresponding author: Xiufan Liu, xfliu@yzu.edu.cn
Received Date: 01 May 2023
Accepted Date: 12 December 2023
Available online: 14 December 2023

Abstract

Influenza A virus (IAV) continues to pose a pandemic threat to public health, resulting a high mortality rate annually and during pandemic years. Posttranslational modification of viral protein plays a substantial role in regulating IAV infection. Here, based on immunoprecipitation (IP)-based mass spectrometry (MS) and purified virus-coupled MS, a total of 89 phosphorylation sites distributed among 10 encoded viral proteins of IAV were identified, including 60 novel phosphorylation sites. Additionally, for the first time, we provide evidence that PB2 can also be acetylated at site K187. Notably, the PB2 S181 phosphorylation site was consistently identified in both IP-based MS and purified virus-based MS. Both S181 and K187 are exposed on the surface of the PB2 protein and are highly conserved in various IAV strains, suggesting their fundamental importance in the IAV life cycle. Bioinformatic analysis results demonstrated that S181E/A and K187Q/R mimic mutations do not significantly alter the PB2 protein structure. While continuous phosphorylation mimicked by the PB2 S181E mutation substantially decreases viral fitness in mice, PB2 K187Q mimetic acetylation slightly enhances viral virulence in mice. Mechanistically, PB2 S181E substantially impairs viral polymerase activity and viral replication, remarkably dampens protein stability and nuclear accumulation of PB2, and significantly weakens IAV-induced inflammatory responses. Therefore, our study further enriches the database of phosphorylation and acetylation sites of influenza viral proteins, laying a foundation for subsequent mechanistic studies. Meanwhile, the unraveled antiviral effect of PB2 S181E mimetic phosphorylation may provide a new target for the subsequent study of antiviral drugs.
- H5N1 influenza virus
- , PB2
- , Phosphorylation
- , Acetylation
- , Viral fitness
- , Mice

Electronic Supplementary Material

10.1016j.virs.2023.12.003-ESM.docx
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