# AG1478 Elicits a Novel Anti-Influenza Function via an EGFR-Independent, GBF1-Dependent Pathway
## 2 Results
### AG1478 Had a Broad-Spectrum Antiviral Activity across Different Cell Models Independent of IFN  
To further characterize this interesting finding, we tested antiviral effects of AG1478 in different cell models. AG1478 demonstrated potent antiviral activity against H1N1 (A/WSN/33) in 293T cells, an immortalized kidney epithelial cell line (Figure 2A), in human primary airway epithelial cells (Figure 2B), in mouse primary airway epithelial cells (Figure 2C), and in Raw264.7, a mouse macrophage cell line (Figure 2D). These results extended our previous finding in an immortalized airway epithelial cell model to other useful models of human and mouse. Importantly, antiviral effects of AG1478 were preserved in primary cells of mouse or human, implying that this in vitro finding can be translated to animal models or human subjects. Indeed, in a pilot study using an animal model of IAV infection, AG1478 treatment demonstrated a significant antiviral activity to reduce lung viral titer by \(\sim\)3.4 times as compared to the mice treated with a vehicle control (Figure 2E). In comparison, an FDA-approved anti-influenza drug oseltamivir reduced lung viral titer by \(\sim\)4.8 times (Figure 2E). Thus, AG1478 was also able to elicit an anti-influenza activity in vivo. Additionally, in the Beas2b cells, AG1478 effectively repressed a different IAV-H1N1 (A/California/07/09) (Figure 2F), IAV-H3N2 (A/Udorn/72) (Figure 2G), and  
Figure 1: Anti-influenza activity of AG1478 was EGFR independent. Anti-influenza activity of AG1478 was EGFR independent. (**A**) Beas2b cells were treated with 2 uM AG1478 and A/WSN/33 viruses at different MOI (MOI = 0.1, 0.5, 1). Media were collected at 24 h and used for the plaque assay. The assay was carried out with MDCK cells. The same experiments (MOI = 0.1) were also performed with (**B**) 10 uM Geftinbi or (**C**) 5 uM BIRX1382. (**D**) p-EGFR expression was quantified by Western blot analysis. ACTIN was used as a loading control. This is a representative image from three independent repeats. (**E**) siRNA knockdown of EGFR (siEGFR) vs. control siRNA with a random sequence (SiC) were analyzed by Western blot analysis. (**F**) Viral titers were analyzed by PFU assay from the cells transfected with siEGFR or SiC. For viral titer, data shown are PFU/mL, mean \(\pm\) SEM. \(n\) = 4, #: \(p\) < 0.05. NS: not significant. a type B Influenza virus or IBV (B/Brisbane/60/2008) (Figure 2H), suggesting that the antiviral activity of AG1478 was not strain-dependent. Because the IFN system is indispensable for cellular antiviral defense, we tested if the antiviral activity of AG1478 was mediated through IFN. We found that AG1478 alone did not induce any expression of type I IFN (IFN() (Figure 3A) or type III IFN (IFN\(\lambda\)1) (Figure 3B). Notably, type II IFN (IFN\(\gamma\)) was not expressed by epithelial cells (data not shown). Interestingly, AG1478 repressed IAV-induced IFN (Figure 3A,B), suggesting the antiviral activity of AG1478 may be independent of the IFN pathway. To confirm this finding, we specifically knocked out IRF3, the common upstream regulator of type I and III IFN pathways [14; 15] by CRISPR approach. The lack of IRF3 protein in IRF3-KO cells was confirmed by a Western blot analysis (Figure 3C). The lack of IRF3 significantly repressed IAV-induced IFN (IFN\(\beta\) or IFN\(\lambda\)1) (Figure 3D,E) and downstream IFN-regulated genes (MX1 or ISG15) (Figure 3F,G) as compared to wild-type cells. Consistently, the lack of the IFN pathway significantly elevated IAV production (Figure 3H). These data confirm that IRF3-KO cells were completely devoid of any IFN system. Interestingly, AG1478 still preserved its antiviral activity in these IRF3-KO cells (Figure 3I). Thus, the antiviral activity of AG1478 was not mediated through the IFN system. Figure 2: AG1478 had a broad-spectrum antiviral activity across different cell models.