*3.3. LUMS1 Inhibits HCV Cellular Entry*

Next, to evaluate the effect of LUMS1 on HCV infection, we used cell culture-derived infectious HCV (HCVcc) expressing an NS5A-GFP fusion protein [21]. In this assay, the infection of liver-derived cell line Huh-7.5 by HCVcc was analyzed by determining the number of GFP-positive cells using automated confocal microscopy. The expression of the NS5A-GFP fusion protein, which served as a marker for productive HCV infection, was inhibited by LUMS1 in a dose-dependent manner with a calculated EC50 of 45.3 ± 18.6 nM and CC50 > 10 μM (Figure 4a). Representative images are shown in Figure 4b. To investigate whether LUMS1 can also interfere with some post entry event of viral replication cycle, we tested the effect of LUMS1 using subgenomic HCV replicon cells [21]. We observed that HCV replication was not affected by LUMS1 at a concentration as high as 10 μM, while it was significantly inhibited by sofosbuvir, a known inhibitor of HCV replication (Figure 4c). This suggested that LUMS1 interfered with HCV entry rather than HCV replication. Results of the HCVcc and replicon assays are summarized in Table 1. To further confirm that LUMS1 interferes with HCV E1/E2-mediated viral entry, the HCV pseudoparticle (HCVpp) system was employed [22]. LUMS1 inhibited the HCVpp with EC50 of 142.1 ± 23 nM demonstrating that LUMS1 specifically inhibited viral entry (Figure 4d).

**Table 1.** Overview of antiviral activity of LUMS1 and compounds used as positive control.

