D2I and F9Y Mutations in the NS1 Protein of Influenza A Virus Affect Viral Replication via Regulating Host Innate Immune Responses
Open Access
- 1 June 2022
- Vol. 14 (6), 1206
- https://doi.org/10.3390/v14061206
Abstract
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.Keywords
Funding Information
- National Key Research and Development Program of China (2021YFD1800205)
- National Natural Science Foundation of China (31772775)
- the State Key Laboratory of Veterinary Biotechnology (SKLVBF202103)
This publication has 33 references indexed in Scilit:
- Multifunctional Adaptive NS1 Mutations Are Selected upon Human Influenza Virus Evolution in the MousePLOS ONE, 2012
- Influenza Virus Non-Structural Protein 1 (NS1) Disrupts Interferon SignalingPLOS ONE, 2010
- NS2/NEP protein regulates transcription and replication of the influenza virus RNA genomeJournal of General Virology, 2009
- Influenza A Virus Inhibits Type I IFN Signaling via NF-κB-Dependent Induction of SOCS-3 ExpressionPLoS Pathogens, 2008
- X-ray structure of NS1 from a highly pathogenic H5N1 influenza virusNature, 2008
- Structural basis for suppression of a host antiviral response by influenza A virusProceedings of the National Academy of Sciences of the United States of America, 2008
- Influenza A virus abrogates IFN‐γ response in respiratory epithelial cells by disruption of the Jak/Stat pathwayEuropean Journal of Immunology, 2008
- Inhibition of Retinoic Acid-Inducible Gene I-Mediated Induction of Beta Interferon by the NS1 Protein of Influenza A VirusJournal of Virology, 2007
- RIG-I-Mediated Antiviral Responses to Single-Stranded RNA Bearing 5'-PhosphatesScience, 2006
- The primary function of RNA binding by the influenza A virus NS1 protein in infected cells: Inhibiting the 2′-5′ oligo (A) synthetase/RNase L pathwayProceedings of the National Academy of Sciences of the United States of America, 2006