# AG1478 Elicits a Novel Anti-Influenza Function via an EGFR-Independent, GBF1-Dependent Pathway
## 3 Discussion  
Because of its ability to modulate multiple ARFs and to control vesicle and lipid trafficking [24, 25], GBF1 is an ideal host factor to be hijacked by positive-strand RNA viruses (PSRVs) to establish specialized membranous compartments for efficient RNA replication and for successful evasion of cellular pattern recognition receptors and innate defense [26]. GBF1 has been reported to play a key function for the replication of a number of PSRVs including _Coronaviridae_[27, 28], _Flaviviridae_[24, 29, 30, 31, 32], _Picornaviridae_[33, 34, 35, 36, 37], and _Togaviridae_[38]. However, the role of GBF1 has rarely been studied on non-PSRVs. The only two available references in this area are about the high-throughput screening of cellular responses to vesicular stomatitis virus [39] or of protein interactome by IAV proteins [19]. GBF1 was identified as one of the 91 host factors that significantly affect IAV genome production, but no follow-up analysis was performed [19]. To our best knowledge, the present study is perhaps the only mechanistic study of GBF1 function on a non-PSRV. Different from its role in PSRVs, GBF1 did not affect IAV replication. This is consistent with the spatial separation of IAV replication (nucleus) and GBF1 protein (cytosol/Golgi). Instead, GBF1 was found to interact with several viral proteins including M1, NP, and PA, all of which are important for vRNP assembly and trafficking. Because vRNP is believed to be transported to the budding location via membrane-bound vesicles, IAV may hijack GBF1 for this task. Indeed, GBF1 was found in our study to be co-localized with M1 protein, a master regulator of intracellular vRNP trafficking. The only other well-characterized GBF1-interacting viral protein is the non-structural protein 3A in poliovirus or cossackievirus. 3A is a small hydrophobic membrane protein and was shown to inhibit GBF1 [40, 41], thereby interfering with ARF1-GBF1-mediated endoplasmic reticulum-to-Golgi complex transport. For these PSRVs, GBF1 was proposed to redirected to recruit other supportive host factor (e.g., PI4K\(\alpha\)) for viral replication [42]. In our study, although the nature of the interaction between GBF1 and M1 is not clear, ectopic GBF1 overexpression was found to enhance and GBF1 knockdown was found to inhibit viral production. The inhibition of viral production by AG1478-elicited alteration of GBF1 localization further supports the favorable role of GBF1 in facilitating viral production. AG1478 itself has also demonstrated antiviral activities against Encephalomyocarditis Virus (EMCV) [18], Hepatitis C Virus (HCV) [18], Marburg virus [43], Lassa virus [43], and Ebola virus [43]. For EMCV and HCV, Phosphatidylinositol 4-Kinase III\(\alpha\) (PI4K\(\alpha\)) was found to be the target of AG1478 based on its structural similarity to a known PI4K\(\alpha\) inhibitor--AL-9. For Marburg, Lassa, and Ebola viruses, >12.5 \(\upmu\)M of AG1478 was required to elicit antiviral activity [43]. However, no precise mode of action was elucidated. Since AG1478 completely inhibited EGFR activity at low \(\upmu\)M range, its EGFR inhibitory activity was not likely responsible for the antiviral activity against these hemorrhagic fever viruses. In this study, we are the first to link the anti-influenza activity of AG1478 with GBF1, another non-EGFR target. Thus, although AG1478 is a well-recognized EGFR inhibitor, its antiviral activity appears to be EGFR-independent. The effect of AG1478 on GBF1 was first reported by [PERSON] et al. in an image-based phenotypic screen to identify the small molecule regulator of intracellular traffic [16]. In their study using a 1 h short-term treatment protocol [16], AG1478 elicited its effect on Golgi dispersion through the conserved catalytic Sec7 domain of GBF1 [16]. The Sec7 domain has also been demonstrated to be responsible for the interaction between GBF1 and two other inhibitors--GCA and BFA.