G45R mutation in the nonstructural protein 1 of A/Puerto Rico/8/1934 (H1N1) enhances viral replication independent of dsRNA-binding activity and type I interferon biology.

Challika Kaewborisuth,Richard J Webby,Mark Zanin,Hans Häcker,Porntippa Lekcharoensuk

doi:10.1186/s12985-016-0585-4

Challika Kaewborisuth, Richard J Webby + Show 3 more

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https://doi.org/10.1186/s12985-016-0585-4

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Abstract

BackgroundThe nonstructural protein 1 (NS1) of influenza A viruses can act as a viral replication enhancer by antagonizing type I interferon (IFN) induction and response in infected cells. We previously reported that A/Puerto Rico/8/1934 (H1N1) (PR8) containing the NS1 gene derived from A/swine/IA/15/1930 (H1N1) (IA30) replicated more efficiently than the wild type virus. Here, we identified amino acids in NS1 critical for enhancing viral replication.MethodsTo identify a key amino acid in NS1 which can increase the virus replication, growth kinetics of PR8 viruses encoding single mutation in NS1 were compared in A549 cells. NS1 mutant functions were studied using dsRNA-protein pull down, RIG-I mediated IFNβ-promoter activity assays and growth curve analysis in murine lung epithelial type I (Let1) cells.ResultsThe G45R mutation in the NS1 of PR8 (G45R/NS1) virus is critical for the enhanced viral replication in A549 cells. G45R/NS1 slightly decreased NS1 binding to dsRNA but did not interfere with its suppression of RIG-I-mediated type I IFN production. Likewise, replication of G45R/NS1 virus was increased in comparison to wild type virus in both wild type and type I interferon receptor null Let1 cells.ConclusionsThe non-conserved amino acid, R45, enhances viral replication which is apparently independent of dsRNA binding and suppression of type I IFN, suggesting a non-characterized function of NS1 for the enhanced viral replication. As G45R/NS1 virus induced the type I IFN induction and response in infected A549 cells, it is also interesting to investigate virus virulence for further studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0585-4) contains supplementary material, which is available to authorized users.

Highlights

The nonstructural protein 1 (NS1) of influenza A viruses can act as a viral replication enhancer by antagonizing type I interferon (IFN) induction and response in infected cells
The RNA-binding domain of IA/ 15/30 H1N1 virus (IA30) NS1 increased virus replication We reported previously that IA30 replicated to higher titers than PR8 [20] and it was mediated by the IA30 NS [21]
Chimera 1 contained the RNA-binding domain (RBD) derived from PR8 NS1 and the effector domain (ED) derived from IA30 NS1 whilst Chimera 2 contained the RBD derived from IA30 NS1 and the ED derived from PR8 (Fig. 1a)

Summary

Introduction

The nonstructural protein 1 (NS1) of influenza A viruses can act as a viral replication enhancer by antagonizing type I interferon (IFN) induction and response in infected cells. The non-structural protein 1 (NS1) of influenza A virus plays an important role in counteracting the IFNα/ β system [12], thereby preventing the generation of an antiviral state in the cell and facilitating efficient viral replication [13, 14]. This action of NS1 was shown at multiple levels in vitro and is different among influenza A virus strains [15]. The RBD has been proposed to sequester dsRNA from RIG-I [16] and dsRNA-activated antiviral enzymes such as OAS1 [17] and PKR [18, 19] to prevent their antiviral activities

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