*3.2. OA-10 Interferes with Virus Entry*

To identify the OA-10 affected stage(s) during an influenza infection cycle, we first performed a virus binding (attachment) assay by coculture A549 cells with IAV at 4 ◦C to permit attachment yet avoid viral entry in the presence or absence of OA-10. The co-incubation of 80 μM OA-10 with H5N1 IAV at 4 ◦C for 1 h followed by removal of excess virus and 37 ◦C culture did not affect IAV infection (Figure S3), indicating that OA-10 does not affect IVA binding to cells and also does not directly inactivate IAV particles. We next performed time course studies for the inhibitory effects of OA-10. A549 cells were treated with OA-10 for 2 h prior to virus infection (pre-treatment), or for 1 h during the viral infection (co-treatment), or for 24 h after 1 h virus infection and removal (post-treatment), as shown in Figure 3A. Results in Figure 3B–D show that pre-treatment of 80 μM OA-10 did not reduce progeny virus yield and viral NP production. This result indicates that OA-10 does not impair the susceptibility of A549 cells to IAV infection. When cells were incubated with H5N1 IAV in the presence of 80 μM OA-10 for 1 h (co-treatment), a mild but significant reduction in progeny virus yield as well as NP protein expression was observed. This result indicates that OA-10 likely interacts with the virus early during the infection cycle. When cells were treated with 80 μM OA-10 for 24 h post H5N1 IAV infection (post-treatment), one log reduction in progeny virus yield was observed (Figure 3B),

which was also reflected by decreased NP production (Figure 3C,D). This result is consistent with the above results and further indicates that OA-10 likely exerts its antiviral effects during virus entry. In these assays, 15 μM peramivir exhibited more significant inhibition on H5N1 IAV's replication in both co-treatment and post-treatment modes, while 30 μM ribavirin exhibited inhibition only in the post-treatment mode, and its inhibition was significantly weaker than that of peramivir. In addition, the combination of 40 μM OA-10 and 15 μM ribavirin treatment showed a synergistic effect on reducing progeny virus titer and NP production, suggesting their complimentary inhibiting mechanisms against IAV replication (Figure 3B–D).

**Figure 3.** OA-10 exhibited inhibition on H5N1 IAV replication in co- and post-treatment modes. A549 cells grown in 24-well plates were treated with indicated compounds for 2 h prior to virus infection (pre-treatment), or for 1 h during viral infection (co-treatment), or for 24 h after 1 h virus infection and removal (post-treatment) (**A**). For three treatment models, 0.1 MOI of H5N1 IAV was used for infecting cells for 1 h. At 24 hpi, supernatants were collected for determining virus titer using the end point dilution assay (**B**), and the cells were subjected to viral NP protein analysis using IFA (**C**,**D**). Results shown in (**D**) are normalized NP protein levels based on the fluorescence optical densities (OD) of the images from three independent experiments. Software Image J was used to digitize image OD. Results from OA-10 or peramivir or ribavirin treated samples were compared to those from corresponding DMSO-treated control groups (set as 100%) (**D**). Representative IFA images of the three independent experiments are shown in C. Scale bar: 250 μm. \* *P* < 0.05, \*\* *P* < 0.01 and \*\*\* *P* < 0.001 compared to the respective virus (DMSO-treated) control.

It was estimated that from IAV entering into a cell to producing its progeny takes on average 6–8 h, depending on cell type [34]. To identify the exact stage(s) affected by OA-10 during such a IAV replication cycle, we treated the infected cells with OA-10 at four separate 2 h time intervals (0–2, 2–4, 4–6 or 6–8, and 0–8 h as a control) and monitored viral NP RNA and protein expression (Figure 4A). As shown in Figure 4B, a more significant inhibition of viral replication, as represented by decreased viral NP RNA level, was observed when OA-10 was added to the A549 cells during 0–2 and 2–4 hpi. A

similar profile was also observed in viral NP expression (Figure 4C,D). These results again indicate that OA-10 exerts its effect during the early stages of IAV infection; i.e., virus internalization, endosome fusion, RNA release and replication. Ribavirin inhibits viral RNA synthesis. As expected, it exerted better IAV inhibition when added to the infected A549 cells before 4 hpi and showed no inhibition when added after 4 hpi. On the other hand, peramivir blocks progeny virus release from infected cells, and did not exhibit any inhibition of either viral RNA synthesis or NP protein expression during a single infection cycle in this assay, although its antiviral activity was the most remarkable, as shown in Figure 3, when multiple infection cycles were studied.

**Figure 4.** OA-10 inhibits H5N1 IAV replication by targeting the earlier stage(s) of the viral infection cycle. A549 cells grown in 24-well plates were infected with 1.0 MOI of H5N1 IAV for 1 h. After two washes with PBS, OA-10 or ribavirin or peramivir was added at the indicated time points and removed after 2 h or 8 h. After each incubation period, the cells were incubated in fresh medium, harvested at 8 hpi and NP mRNA and protein analyzed by RT-PCR and IFA, respectively. (**A**) The experimental design of time-of-addition assay. (**B**) Relative virus NP mRNA level. (**C**,**D**) Viral NP protein expression, representative IFA images (**C**) and digitized NP expression (**D**) from three independent experiments. Scale bar: 250 μm. \* *P* < 0.05, \*\* *P* < 0.01 and \*\*\* *P* < 0.001 compared to the respective virus control.
