Date Published: November 29, 2018
Publisher: Public Library of Science
Author(s): Chutikarn Chaimayo, Megan Dunagan, Tsuyoshi Hayashi, Netty Santoso, Toru Takimoto, Daniel R. Perez.
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.
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 . Like many other viruses, the influenza virus relies on the host cellular machinery to facilitate its replication cycle. However, host cells recognize and respond to viral infection through inducing various anti-viral proteins and interferons (IFNs). To overcome the host defense system, many viruses express proteins to evade the innate immune response or target general host protein synthesis. General shutoff targeting host mRNAs limits competition from cellular transcripts and secures priority access of viral mRNAs to host translation machinery .
Influenza A viruses express two accessory proteins, NS1 and PA-X, to induce general host shutoff, which work through different mechanisms [7, 11]. Interaction of NS1 with CPSF30 prevents processing of pre-mRNA in the cell nucleus, while PA-X directly degrades RNA Pol II transcripts in both nucleus and cytoplasm of an infected cell [7, 8, 12, 19, 35, 36]. Both NS1 and PA-X contribute to antagonizing host antiviral and immune responses [4, 11, 14, 15, 37, 38]. However, their shutoff activities vary between virus isolates. Most seasonal human H1N1 and H3N2 viruses express shutoff-active NS1, but their PA-X shutoff activities are relatively low compared to avian-origin viruses due to various mutations in the N-terminal domain of PA gene [9, 18]. The 2009 pH1N1 strains express shutoff-inactive NS1 due to amino acid substitution in CPSF30 binding residues, while their PA-X shutoff activities are highly active due to their avian-origin PA gene [9, 12, 18, 39]. Influenza viruses rely on cellular machinery for genome replication and viral protein synthesis; therefore, the viruses likely need to adjust their shutoff activities to create an optimum cellular environment for efficient viral growth. In this study, we investigated the functional interplay between influenza PA-X and NS1 proteins on viral and host gene expression in infected human and avian cells and identified preferential cellular mRNA targets and the biological pathways associated with PA-X and NS1 shutoff activities. We found that i) PA-X strongly inhibits host protein synthesis in human and avian cultured cells, while the effect of active NS1 on general host protein synthesis was predominantly observed in avian cells (Fig 2); ii) PA-X preferentially targets cellular mRNAs involved in protein modification, metabolism and repair processes, while shutoff-active NS1 specifically targets genes involved in antiviral responses and strongly suppresses induction of IFN, interleukin, chemokine and class I MHC genes (Figs 4 and 5, S1 and S2 Tables); iii) shutoff-active NS1 together with PA-X predominantly repressed viral NS1 and NEP mRNA expression (Figs 8, 9 and S3); and iv) the virus expressing shutoff-active NS1 with a limited amount of PA-X (Cal [NS1_high-PAX_low]) whose shutoff activity mimics that of seasonal human viruses, is the most potent in antagonizing antiviral and innate immune responses in human cells (Figs 5 and 6).