Influenza A virus (IAV) must utilize the host cell machinery to replicate, but many of the mechanisms of IAV-host interaction remain poorly understood. Improved understanding of interactions between host factors and vRNP not only increases our basic knowledge of the molecular mechanisms of virus replication and pathogenicity but also provides insights into possible novel antiviral targets that are necessary due to the widespread emergence of drug-resistant IAV strains. Here, we have identified LYAR, a cell growth-regulating nucleolar protein, which interacts with viral RNP components and is important for efficient replication of IAVs and whose role in the IAV life cycle has never been reported. In addition, we further reveal the role of LYAR in viral RNA synthesis. Our results extend and improve current knowledge on the mechanisms of IAV transcription and replication.

The innate immune response is vital for host defense and must be tightly controlled, but the mechanisms responsible for its negative regulation are not fully understood. The cell growth-regulating nucleolar protein LYAR was found to promote replication of multiple viruses in our previous study. Here, we report that LYAR acts as a negative regulator of innate immune responses. We found that LYAR expression is induced by beta interferon (IFN-β) during virus infection. Further studies showed that LYAR interacts with phosphorylated IFN regulatory factor 3 (IRF3) to impede the DNA binding capacity of IRF3, thereby suppressing the transcription of IFN-β and downstream IFN-stimulated genes (ISGs). In addition, LYAR inhibits nuclear factor-κB (NF-κB)-mediated expression of proinflammatory cytokines. In summary, our study reveals the mechanism of LYAR in modulating IFN-β-mediated innate immune responses by targeting phosphorylated IRF3, which not only helps us to better understand the mechanisms of LYAR-regulated virus replication but also uncovers a novel role of LYAR in host innate immunity. IMPORTANCE Type I interferon (IFN-I) plays a critical role in the antiviral innate immune responses that protect the host against virus infection. The negative regulators of IFN-I are important not only for fine-tuning the antiviral responses to pathogens but also for preventing excessive inflammation. Identification of negative regulators and study of their modulation in innate immune responses will lead to new strategies for the control of both viral and inflammatory diseases. Here, we report for the first time that the cell growth-regulating nucleolar protein LYAR behaves as a repressor of host innate immune responses. We demonstrate that LYAR negatively regulates IFN-β-mediated immune responses by inhibiting the DNA binding ability of IFN regulatory factor 3 (IRF3). Our study reveals a common mechanism of LYAR in promoting different virus replication events and improves our knowledge of host negative regulation of innate immune responses.

The viral ribonucleoprotein (vRNP) of the influenza A virus (IAV) is responsible for the viral RNA transcription and replication in the nucleus and its functions rely on host factors. Previous study has indicated that eukaryotic translation elongation factor 1 delta (eEF1D) may associate with RNP subunits, but its roles in IAV replication are unclear. Herein, we showed that eEF1D was an inhibitor of IAV replication, because knockout of eEF1D resulted in a significant increase in virus yield. eEF1D interacted with RNP subunits PA, PB1, PB2, and also with NP in an RNA-dependent manner. Further studies revealed that eEF1D impeded the nuclear import of NP and PA-PB1 heterodimer of IAV, thereby suppressing the vRNP assembly, viral polymerase activity, and viral RNA synthesis. Together, our studies demonstrate eEF1D negatively regulating the IAV replication by inhibition of the nuclear import of RNP subunits, which not only uncovers a novel role of eEF1D in IAV replication but also provides new insights into the mechanisms of nuclear import of vRNP proteins. IMPORTANCE Influenza A virus is the major cause of influenza, a respiratory disease in humans and animals. Different from most other RNA viruses, the transcription and replication of IAV occur in the cell nucleus. Therefore, the vRNPs must be imported into the nucleus for viral transcription and replication, which requires participation of host proteins. However, the mechanisms of the IAV-host interactions involved in nuclear import remain poorly understood. Here, we identified eEF1D as a novel inhibitor for the influenza virus life cycle. Importantly, eEF1D impaired the interaction between NP and importin α5 and the interaction between PB1 and RanBP5, which impeded the nuclear import of vRNP. Our studies not only reveal the molecular mechanisms of the nuclear import of IAV vRNP, but also provide potential anti-influenza targets for antiviral development.

Influenza A viruses (IAVs) initiate infection by attaching Hemagglutinin (HA) on the viral envelope to sialic acid (SA) receptors on the cell surface. Importantly, HA of human IAVs has a higher affinity for α-2,6-linked SA receptors, and avian strains prefer α-2,3-linked SA receptors, whereas swine strains have a strong affinity for both SA receptors. Host gene CMAS and ST3GAL4 were found to be essential for IAV attachment and entry. Loss of CMAS and ST3GAL4 hindered the synthesis of sialic acid receptors, which in turn prevented the adsorption of IAV. Further, the knockout of CMAS had an effect on the adsorption of swine, avian and human IAVs. However, ST3GAL4 knockout prevented the adsorption of swine and avian IAV and the impact on avian IAV was more distinct, whereas it had no effect on the adsorption of human IAV. Collectively, our findings demonstrate that knocking out CMAS and ST3GAL4 negatively regulated IAV replication by inhibiting the synthesis of SA receptors, which also provides new insights into the production of gene-edited animals in the future.

Production of type I IFN is pivotal for the cellular antiviral immunity. Virus infection leads to the activation of transcription factor IRF3 and subsequent production of type I IFN to eliminate viral infection.