A Conserved Residue, Tyrosine (Y) 84, in H5N1 Influenza A Virus NS1 Regulates IFN Signaling Responses to Enhance Viral Infection

Abstract

The non-structural protein, NS1, is a virulence factor encoded by influenza A viruses (IAVs). In this report, we provide evidence that the conserved residue, tyrosine (Y) 84, in a conserved putative SH2-binding domain in A/Duck/Hubei/2004/L-1 [H5N1] NS1 is critical for limiting an interferon (IFN) response to infection. A phenylalanine (F) substitution of this Y84 residue abolishes NS1-mediated downregulation of IFN-inducible STAT phosphorylation, and surface IFNAR1 expression. Recombinant IAV (rIAV) [H1N1] expressing A/Grey Heron/Hong Kong/837/2004 [H5N1] NS1-Y84F (rWSN-GH-NS1-Y84F) replicates to lower titers in human lung epithelial cells and is more susceptible to the antiviral effects of IFN-β treatment compared with rIAV expressing the intact H5N1 NS1 (rWSN-GH-NS1-wt). Cells infected with rWSN-GH-NS1-Y84F express higher levels of IFN stimulated genes (ISGs) associated with an antiviral response compared with cells infected with rWSN-GH-NS1-wt. In mice, intranasal infection with rWSN-GH-NS1-Y84F resulted in a delay in onset of weight loss, reduced lung pathology, lower lung viral titers and higher ISG expression, compared with mice infected with rWSN-GH-NS1-wt. IFN-β treatment of mice infected with rWSN-GH-NS1-Y84F reduced lung viral titers and increased lung ISG expression, but did not alter viral titers and ISG expression in mice infected with rWSN-GH-NS1-wt. Viewed altogether, these data suggest that the virulence associated with this conserved Y84 residue in NS1 is, in part, due to its role in regulating the host IFN response.

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“…In primary human lung explants, IFN alfacon-1 inhibited avian H5N1 and pandemic H1N1 IAV replication, while upregulating the expression of several antiviral ISGs -PKR, OAS, and ISG15 [155]. Treatment of IAV-infected human lung A549 cells with IFN-β inhibits viral replication in a dose-dependent manner [237]. In the context of another acute virus infection, West Nile Virus, Type I IFNs have been shown to increase incorporation of microRNAs (miRNAs) into extracellular vesicles in A549 cells [238].…”

“…Type I IFNs have also been shown to inhibit acute influenza A virus (IAV) infections, in vitro [155,237]. In primary human lung explants, IFN alfacon-1 inhibited avian H5N1 and pandemic H1N1 IAV replication, while upregulating the expression of several antiviral ISGs -PKR, OAS, and ISG15 [155].…”

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“…Some of these viruses, such as those from Herpesviridae family, can establish latency and cause illness following reactivation [6]. These can suppress the cytokine and interferon responses by inhibiting the activation of transcription factors or receptors involved in signalling pathways [7] .…”

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“…The influenza A non-structural protein 1 (NS1) prevents the host innate immune response and cellular apoptosis of infected cells by suppressing IFN activation through multiple routes [118]. Also, NS1 regulates IFN-α/β receptor subunit 1 (IFNAR1) surface expression [119]. Due to suppression of the innate immune system, influenza A infection of neurones only leads to increases in tumor necrosis factor-α (TNF-α) release, not interleukin-6 (IL-6) or IFNs [120].…”