D2I and F9Y Mutations in the NS1 Protein of Influenza A Virus Affect Viral Replication via Regulating Host Innate Immune Responses

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.

Antiviral responses versus virus-induced cellular shutoff: a game of thrones between influenza A virus NS1 and SARS-CoV-2 Nsp1

“…For SIV, the EPEV and GSEI motifs in the CTD of NS1 have been shown to increase viral replication and virulence in mice by interacting with the cellular PDZ domain that has a critical role is cell signaling, cellular polarity, and act as a scaffold to assemble protein complexes ( Figure 4A ) ( Harris and Lim, 2001 ; Wang et al., 2012 ). The mouse adaptive F9Y substitution increased the virulence of WSN virus in mice as well as replication efficiency in vivo and in vitro via enhancing the IFN antagonism properties ( Yu et al., 2022 ). In contrast, the mouse adapted D2I substitution in WSN attenuated the replication and virulence of WSN in vitro and in vivo via decreasing the IFN antagonistic ability, compared to the NS1-WT protein of WSN ( Yu et al., 2022 ).…”

“…The mouse adaptive F9Y substitution increased the virulence of WSN virus in mice as well as replication efficiency in vivo and in vitro via enhancing the IFN antagonism properties ( Yu et al., 2022 ). In contrast, the mouse adapted D2I substitution in WSN attenuated the replication and virulence of WSN in vitro and in vivo via decreasing the IFN antagonistic ability, compared to the NS1-WT protein of WSN ( Yu et al., 2022 ). These and other findings highlight the critical role of NS1 protein in mediating IAV replication, pathogenicity, and virulence.…”

Virus versus host: influenza A virus circumvents the immune responses

“…The NS1 protein can escape innate immunity through amino acid substitution ( Figure 2C ). The NS1 F9Y amino acid mutation of H1N1 introduced by error-prone PCR can significantly enhance virus growth in vitro and in vivo , increasing virus virulence in mice ( Yu et al, 2022 ). This characteristic mutation can target antiviral drugs and attenuate vaccine development.…”

Research progress on the nonstructural protein 1 (NS1) of influenza a virus

“…IRFs and NF-κB form an active transcriptional complex that activates the expression of IFN-I [ 80 ]. Furthermore, influenza virus lacking the NS1 protein limits interferon production [ 81 ] and mutating NS1 protein inhibits interferon production and subsequently virus replication [ 82 ]. The IAV NS1 prevents viral and dsRNA-mediated activation of IRF3 and NF-κB, thereby enhancing viral replication and transmission [ 17 , 39 ].…”