Specificity and functional interplay between influenza virus PA-X and NS1 shutoff activity.

Chutikarn Chaimayo,Tsuyoshi Hayashi,Megan Dunagan,Netty Santoso,Toru Takimoto,Daniel R Perez

doi:10.1371/journal.ppat.1007465

Chutikarn Chaimayo, Tsuyoshi Hayashi + Show 4 more

Open Access

https://doi.org/10.1371/journal.ppat.1007465

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Abstract

Influenza A viruses modulate host antiviral responses to promote viral growth and pathogenicity. Through viral PA-X and NS1 proteins, the virus is capable of suppressing host protein synthesis, termed “host shutoff.” Although both proteins are known to induce general shutoff, specificity of target genes and their functional interplay in mediating host shutoff are not fully elucidated. In this study, we generated four recombinant influenza A/California/04/2009 (pH1N1) viruses containing mutations affecting the expression of active PA-X and NS1. We analyzed viral growth, general shutoff activity, specificity of mRNA targets, and viral gene expressions. Our results showed that PA-X was the major contributor in reducing general host protein expression in the virus-infected cells. Intriguingly, our transcriptomic analysis from infected human airway A549 cells indicate that shutoff-active NS1 specifically targeted host mRNAs related to interferon (IFN) signaling pathways and cytokine release. Specificity of target mRNAs was less evident in PA-X, although it preferentially degraded genes associated with cellular protein metabolism and protein repair. Interestingly, in the presence of shutoff-active NS1, PA-X also degraded viral mRNAs, especially NS segments. The virus expressing shutoff-active NS1 with reduced amount of PA-X expression most efficiently suppressed antiviral and innate immune responses in human cells, indicating that influenza virus needs to optimize the contribution of these two shutoff proteins to circumvent host responses for its optimum growth.

Highlights

Influenza A virus is a major respiratory pathogen that frequently causes seasonal epidemics and periodic pandemics, resulting in half-a million deaths worldwide each year [1]
Influenza A virus expresses two proteins, NS1 and PA-X, to induce general shutoff. Their mechanism of action is different; NS1 interacts with CPSF30 and blocks the 3’end processing of host pre-mRNAs, while PA-X degrades target mRNAs through its endonuclease domain
Our data showed that PA-X was the major contributor of general shutoff of host protein synthesis, while NS1 targeted genes involved in host innate response and cytokine response

Summary

Introduction

Influenza A virus is a major respiratory pathogen that frequently causes seasonal epidemics and periodic pandemics, resulting in half-a million deaths worldwide each year [1]. A crystal structure of a complex composed of the NS1 effector domain (ED) and F2F3 fragment of CPSF30 indicates that two NS1 molecules and two CPSF30 are components of this complex, and NS1 residues F103, M106, K108, D125, and D189 play a critical role for CPSF30 interaction [8, 9]. These key NS1 residues are highly conserved among human isolates, they are less conserved among animal isolates. The NS1 of 2009 pandemic influenza viruses (pH1N1), including A/California/04/2009 (Cal) lack the ability to bind to CPSF30 due to mutations in the consensus CPSF30 binding residues (K108R, D125E and D189G), and are unable to induce general shutoff [9]

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