# AG1478 Elicits a Novel Anti-Influenza Function via an EGFR-Independent, GBF1-Dependent Pathway
## 2 Results
### GBF1 Interacted with Selected Viral Proteins  
To further understand the mechanistic basis of the antiviral effect of AG1478, we tested its effect on viral transcription by real-time PCR measurements of individual viral RNAs (Supplemental Figure S2A,B), on viral protein translation by Western blot analysis (Supplemental Figure S2C), and on viral genome replication by a minigenome assay (Supplemental Figure S2D). Surprisingly, AG1478 did not affect any of these processes. By immunoprecipitation, GBF1 was found to interact with selected viral proteins: M1, NP, and PA, but not HA and M2 (Figure 6A). All of these interacting proteins were responsible for nucleoplasmic trafficking of vRNP [5]. Interestingly, a greater amount of GBF1 was precipitated when the cells were treated with AG1478 as compared with the control (Figure 6A), even though equal amounts of proteins from each sample were loaded. Although the absolute amount of each precipitated vRNP protein was not significantly changed by the treatment of AG1478 (Figure 6A), their ratio to precipitated GBF1 was decreased by greater than 2-fold when treated with AG1478 (Figure 6B), suggesting that the interaction between GBF1 and vRNP protein was indeed impaired by the treatment of AG1478. As a control, we also analyzed input proteins before IP analysis. As shown in Supplemental Figure S3, total GBF1 proteins were increased by AG1478 treatment irrespective of viral infection, which was consistent with IP data (Figure 6A) and suggested that AG1478 may affect GBF1 protein production. In addition, both HA and M2 were present in the input proteins (Supplemental Figure S3) but absent from immunoprecipitation (Figure 6A). Because AG1478 altered the cellular distribution of GBF1 from Golgi-cytoplasmic to Golgi-centric (Figures 4F and 5E), we reasoned that the immunoprecipitation on a whole cell lysate might not be sufficient to capture the overall effect of AG1478. Thus, we performed an immune-fluorescence co-staining using the antibodies against viral M1 protein and GBF1. Significant co-localization of M1 and GBF1, as demonstrated by yellowish co-staining pixels, was indeed observed in the absence of AG1478. However, when the cells were treated with AG1478, M1 and GBF1 were completely dissociated (Figure 6C). Quantittively, in the cells without AG1478, greater than 60% of cells showed co-localization of M1 and GBF1. In contrast, less than 4% of AG1478-treated cells showed co-localization (Figure 6D). This dramatic effect by AG1478 was further confirmed by using super-resolution microscopy (Figure 6E). In the enlarged images, a large number of yellow-colored spots were readily visible in IAV-infected cells but were almost completely absent in AG1478-treated cells.  
Figure 6: GBF1 interacted with viral proteins. (**A**) Beas2b cells were treated with 2 \(\upmu\)M AG1478 and infected with A/WSN/1933 for 24 h. GBF1 antibody was used for immunoprecipitation. The precipitants were analyzed for GBF1, NP, M1, PA, HA, and M2. (**B**) Quantification of the ratio of IAV protein over GBF1 in (**A**). These were the representation from three independent repeats. (**C**) GBF1 (red) and M1 (green) were co-localized (yellow) in the absence of AG1478, but completely dissociated in the presence of AG1478. DAPI (blue) was used for the nuclear staining. Images were taken under 63\(\times\) magnification. Scale bar: 5 \(\upmu\)m. (**D**) Quantification of percentage of cells having co-localization signals. Data shown are mean \(\pm\) SEM. \(n\) = 10, #: \(p\) < 0.05. (**E**) Super-resolution microscopy under 100\(\times\) magnification. Scale bar: 5 \(\upmu\)m. The co-localization of GBF1 (red) and M1 (green) was shown by yellowish colored dots in the enlarged images. All were representative images from three independent repeats.