D2I and F9Y Mutations in the NS1 Protein of Influenza A Virus Affect Viral Replication via Regulating Host Innate Immune Responses
Mengqi Yu,
Yanna Guo,
Lingcai Zhao,
Yuanlu Lu,
Qingzheng Liu,
Yinjing Li,
Lulu Deng,
Zhiyu Shi,
Haifeng Wang,
Samar Dankar,
Jihui Ping
Affiliations
Mengqi Yu
MOE Joint International Research Laboratory of Animal Health and Food Safety, Engineering Laboratory of Animal Immunity of Jiangsu Province, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
Yanna Guo
MOE Joint International Research Laboratory of Animal Health and Food Safety, Engineering Laboratory of Animal Immunity of Jiangsu Province, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
Lingcai Zhao
MOE Joint International Research Laboratory of Animal Health and Food Safety, Engineering Laboratory of Animal Immunity of Jiangsu Province, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
Yuanlu Lu
MOE Joint International Research Laboratory of Animal Health and Food Safety, Engineering Laboratory of Animal Immunity of Jiangsu Province, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
Qingzheng Liu
MOE Joint International Research Laboratory of Animal Health and Food Safety, Engineering Laboratory of Animal Immunity of Jiangsu Province, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
Yinjing Li
MOE Joint International Research Laboratory of Animal Health and Food Safety, Engineering Laboratory of Animal Immunity of Jiangsu Province, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
Lulu Deng
MOE Joint International Research Laboratory of Animal Health and Food Safety, Engineering Laboratory of Animal Immunity of Jiangsu Province, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
Zhiyu Shi
MOE Joint International Research Laboratory of Animal Health and Food Safety, Engineering Laboratory of Animal Immunity of Jiangsu Province, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
Haifeng Wang
Changjia (Shanghai) Agricultural Science and Technology Co., Ltd., Shanghai 201414, China
Samar Dankar
Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1V 8M5, Canada
Jihui Ping
MOE Joint International Research Laboratory of Animal Health and Food Safety, Engineering Laboratory of Animal Immunity of Jiangsu Province, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
Influenza A viruses (IAV) modulate host antiviral responses to promote viral growth and pathogenicity. The non-structural (NS1) protein of influenza A virus has played an indispensable role in the inhibition of host immune responses, especially in limiting interferon (IFN) production. In this study, random site mutations were introduced into the NS1 gene of A/WSN/1933 (WSN, H1N1) via an error prone PCR to construct a random mutant plasmid library. The NS1 random mutant virus library was generated by reverse genetics. To screen out the unidentified NS1 functional mutants, the library viruses were lung-to-lung passaged in mice and individual plaques were picked from the fourth passage in mice lungs. Sanger sequencing revealed that eight different kinds of mutations in the NS1 gene were obtained from the passaged library virus. We found that the NS1 F9Y mutation significantly enhanced viral growth in vitro (MDCK and A549 cells) and in vivo (BALB/c mice) as well as increased virulence in mice. The NS1 D2I mutation attenuated the viral replication and pathogenicity in both in vitro and in vivo models. Further studies demonstrated that the NS1 F9Y mutant virus exhibited systematic and selective inhibition of cytokine responses as well as inhibited the expression of IFN. In addition, the expression levels of innate immunity-related cytokines were significantly up-regulated after the rNS1 D2I virus infected A549 cells. Collectively, our results revealed that the two mutations in the N-terminal of the NS1 protein could alter the viral properties of IAV and provide additional evidence that the NS1 protein is a critical virulence factor. The two characterized NS1 mutations may serve as potential targets for antiviral drugs as well as attenuated vaccine development.
