During influenza viral infection, the virus uses RNPs (nucleoprotein and RNAs complex) as a template and virion enzymes to generate mRNAs in nucleus of infected cells. The viral transcriptase “steals” 5’ cap structures from newly synthesized host mRNAs for each viral mRNA. Subsequently, the mRNA will be transported into the cytoplasm of infected host cell to engage in virus-specific protein synthesis such as non-structure protein NS1. The NS1 protein is a multifunctional protein and a virulence factor, which can be notionally divided into two distinct functional domains: N-terminal RNA-binding domain (RBD) (amino acids 1–73), and C-terminal effector domain (ED) (amino acids 85–230/237). The two domains of NS1 modulate host immune response factors, playing a central role in the “tug of war” between viral colonization and host defense. For instance, Chen et al. (2014) demonstrated that influenza virus protein NS1 binds to DDX2, an RNA helicase of host cell, which frees PB1 (viral polymerase) from binding to DDX21. Additionally, by using recombinant virus encoding mutant NS1 protein, the authors pinpointed 41/44 sites of the RBD of NS1 are responsible for the binding to DDX21, resulting in displacing PB1 from DDX21. Owning to influenza viral NS1, the PB1’s function for viral genome replication can be restored, particularly between 9 and 12 hr after infection (Chen et al., 2014).

In contrast to antagonize host immune factor, Chassey et al. (2013) identified a novel mechanism that NS1 enhances the activity of a host pro-viral factor adenosine deaminase acting on RNA 1 (ADAR1). ADRA1 belongs to double-stranded RNA binding domain protein family, which is the major target of NS1 (Chassey et al., 2013). The expression of ADAR1 is necessary for optimal viral protein synthesis and replication. Moreover, the host ADAR1 editing activity is enhanced by influenza A NS1 protein (Chassey et al., 2013).

In addition to binding to host DDX21 or ADAR1 protein for facilitating viral replicating, the influenza NS1 involves in antagonizing host immune response factors through a multitude of protein–protein and protein–RNA interactions. These interactions have been shown to limit the host innate interferon (IFN-β) production, block the function of cytoplasmic antiviral proteins, inhibit host DC maturation and migration, and mediate host apoptosis (Hale et al., 2008). The host IFN response is a potent antiviral mechanism against viral infection. The virus lacking the NS1 protein is only able to replicate in IFN-deficient cells or mice (Kochs et al., 2007), indicating an antagonistic effect of NS1 to IFN. Mechanistically, binding of NS1 to the E3 ubiquitin ligase TRIM25 prevents activation of RIG-I signaling and subsequent IFN induction. Cellular RNA processing is also targeted by NS1, through recognition of cleavage and polyadenylation specificity factor 30 (CPSF30), leading to inhibition of IFN - mRNA processing as well as that of other cellular mRNAs (Nemeroff et al., 1998). Secondly, NS1 can bind to cytoplasmic antiviral proteins including dsRNA-dependent serine/threonine protein kinase R (PKR), thus blocking an important arm of the IFN system. Min et al., (2007) shows that PKR would bind to NS1A proteins in the cytoplasm prior to their import into the nucleus, where the NS1A-mediated viral RNA synthesis occurs. However, PKR activation and decreased viral RNA synthesis are not coupled because enhanced early viral RNA synthesis after 123/124 virus infection also occurs in mouse PKR-/- cells (Min et al., 2007). Zhang et al. (2019) revealed a novel role of NS1 by demonstrating that NS1 protein targets on the nuclear RNA export factors, leading to the block of host mRNAs nuclear export (Zhang et al., 2019). Using a virus-like particle system, it is recently shown that NS1 also involves in improving viral genome packing in a dsRNA binding-dependent manner. A point mutation (R38A) at the N-terminal RBD impaired the incorporation of NS1 into the virion and the virus-like particle, as well as viral protein productions such as M1 and HA2 (Sha et al., 2020).

Prophylactic intervention, such as influenza vaccine, is regenerated every year to serve as a major strategy against influenza. However, therapeutic interventions with antiviral drugs such as neuraminidase inhibitors and viral polymerase inhibitors also play an essential role in treatment of influenza viral infections, especially for population with weaker immune system. Recently, antiviral drug resistant strains have become an increasing problem. Therefore, given the multifunctional roles of NS1 in suppressing host immune response and in promoting viral replication, NS1 protein is among the most promising novel druggable anti-influenza target.

Biochemical, cell-based, and nucleic acid-based approaches are being applied to identify compounds that inhibit the binding of NS1. For example, based on the identified mechanisms in dsRNA-NS1 protein interaction, therapeutic agents that can inhibit dsRNA-NS1 interactions could restore PKR and/or RIG-I activated IFN response. Moreover, because RNA processing is affected by the NS1’s recognition of CPSF30, the hydrophobic CPSF30-binding pocket in NS1 is an attractive target for drug discovery. Interestingly, a meta-analysis involving 2620 sequences of NS1 protein proposed several conserved pockets for small-molecule inhibition of NS1 function based on Q-SiteFinder binding site prediction algorithm (Darapaneni et al., 2009). There are a few high-throughput screen (HTS) assays have been carried out for NS1-targeted drug discovery. For instance, a screen was carried out with a collection of small-molecules composed of 27,520 mixtures of synthetic chemical compounds (Maroto et al., 2008). This study identified three compounds, which were shown to inhibit the NS1 binding to viral RNA. However, their structures were not reported. In addition to biochemistry screening, one study using MDCK cell-based platform identified four compounds from the National Cancer Institute Diversity Set library of ~2,000 compounds that restored levels of IFN-related mRNA to the level of cells that infected with a NS1 deletion mutation influenza (Basu et al., 2009).

Recently, on the basis of the conserved pockets of a number of influenza NS1 proteins, a study designed a short hairpin RNA (shRNA) that silence NS1 in vitro, leading to induced RIG-I activation and INF expression (Singh et al., 2020). This dual-functioning 5ʹ-PPP-NS1shRNA leads to antiviral state, which inhibit influenza virus replication in vitro (Singh et al., 2020). Moreover, the functions of 5ʹ-PPP-NS1shRNA as NS1 antagonist and RIG-I inducer were further examined in vivo in mice. The results suggest that administration of 5ʹ-PPP-NS1shRNA in prophylactic and therapeutic settings resulted in significant inhibition of viral replication following viral challenge in vivo in mice. Meanwhile, the mice lungs were harvested on day 4 post-infection for quantitative real-time PCR assay. As expected, the data show significantly increased gene expression of RIG-I, IFN-β, and IFN-λ, as well as a lowered NS1 expression (Singh et al., 2020). The abovementioned mechanistic findings and small-molecule screening assays signify that the NS1 protein is a valuable target for anti-influenza drug development.