Identification of two residues within the NS1 of H7N9 influenza A virus that critically affect the protein stability and function

Ji-Long Chen,Zhou Yang,Xiaojuan Chi,Rong Zhang,Yuan Liao,Sicheng Shu,Yanhui Xie,Lanlan Zhang,Song Wang

doi:10.1186/s13567-018-0594-y

Abstract

The emerging avian-origin H7N9 influenza A virus, which causes mild to lethal human respiratory disease, continues to circulate in China, posing a great threat to public health. Influenza NS1 protein plays a key role in counteracting host innate immune responses, allowing the virus to efficiently replicate in the host. In this study, we compared NS1 amino acid sequences of H7N9 influenza A virus with those of other strains, and determined NS1 protein variability within the H7N9 virus and then evaluated the impact of amino acid substitutions on ability of the NS1 proteins to inhibit host innate immunity. Interestingly, the amino acid residue S212 was identified to have a profound effect on the primary function of NS1, since S212P substitution disabled H7N9 NS1 in suppressing the host RIG-I-dependent interferon response, as well as the ability to promote the virus replication. In addition, we identified another amino acid residue, I178, serving as a key site to keep NS1 protein high steady-state levels. When the isoleucine was replaced by valine at 178 site (I178V mutation), NS1 of H7N9 underwent rapid degradation through proteasome pathway. Furthermore, we observed that P212S and V178I mutation in NS1 of PR8 virus enhanced virulence and promoted the virus replication in vivo. Together, these results indicate that residues I178 and S212 within H7N9 NS1 protein are critical for stability and functioning of the NS1 protein respectively, and may contribute to the enhanced pathogenicity of H7N9 influenza virus.

Highlights

Influenza A virus (IAV), whose genome is organized into eight single stranded negative-sense RNA segments that encode for at least 18 viral proteins [1], belongs to Orthomyxoviridae family and is subdivided into various subtypes according to surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA)
Forced expression of H7N9 NS1 protein suppresses host innate immune response and promotes the viral replication Since H7N9 virus is highly pathogenic for human and IAV NS1 plays a key role in suppressing the host innate immunity, we analyzed the effect of forced expression of H7N9 NS1 on IAV-induced innate immune response and viral replication. 293T cells were transfected with plasmids expressing H7N9 NS1 or empty vector (EV) for 24 h, followed by WSN virus infection
Previous studies have shown that the H7N9 influenza virus is more virulent in human than the 2009 pandemic H1N1 influenza virus, and amino acid substitutions in H7N9 virus proteins may contribute to the enhanced virulence [6]

Summary

Introduction

Influenza A virus (IAV), whose genome is organized into eight single stranded negative-sense RNA segments that encode for at least 18 viral proteins [1], belongs to Orthomyxoviridae family and is subdivided into various subtypes according to surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). It has been suggested that several amino acid substitutions in PB2, PA, NP, HA, NA and NS1 may be responsible for promoting replication and pathogenicity of this virus in humans [8–14]. No sustained human-to-human transmission of H7N9 viruses has. Wang et al Vet Res (2018) 49:98 been observed, the genomic sequence changes in H7N9 virus, which may alter its virulence and augment the possibility of efficient transmission between humans, are still of considerable concern. It has been recently reported that some highly pathogenic H7N9 mutants were detected in birds, suggesting an increased threat to humans [14–16]

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Results

Conclusion