*3.5. Brevilin A Decreases the Levels of Viral mRNA and Proteins Expressed from the M and NS Segments*

Next, we investigated the expression pattern of viral proteins during IAV infection observed in the presence of brevilin A. For this purpose, MDCK cells were infected with PR8 (MOI = 1) and treated with brevilin A at a concentration of 8 μM. At the time point of 4 hpi, only the expressions of NA, M1, and NS1 were observed in the control cells. Treatment with brevilin A resulted in about 20% reduction of these proteins (Figure 5A). At 8 hpi, the expressions of viral NA, HA, and NP were not affected by brevilin A, and a modest reduction of M1 (~30%) was observed upon brevilin A treatment. However, the reduction of M2, NS1, and NS2 proteins was much more dramatic: about 70% in M2, 50% in NS1, and 70% in NS2, respectively (Figure 5A). The M2 protein is translated from M2 mRNA, which is produced from the alternative splicing of M1; the NS1 and NS2 proteins were respectively translated from NS1 and NS2 mRNA generated from the NS segment. We next performed RT-qPCR to determine the effect of brevilin A on the mRNA expressions of M1, M2, NS1, and NS2. We infected MDCK cells with PR8 virus and then treated cells with brevilin A. Whole-cell RNA was isolated from the infected cells at 6 hpi and quantified for mRNA using RT-qPCR assay. The M2 mRNA was markedly reduced following treatment with brevilin A, with a reduction of 55%–60%, whereas the reduction rates of M1, NS1, and NS2 mRNA levels were similar to that of NP (with a reduction of ~20%) compared to vehicle (Figure 5B–E). These results suggest that brevilin A reduces the mRNA and protein expressions of viral M and NS.

**Figure 5.** The inhibitory effects of brevilin A on the mRNA and protein expression of viral NS and M. (**A**) MDCK cells were infected with PR8 at a MOI of 1, and then treated with brevilin A or vehicle. At the indicated time points, cell lysates were analyzed by Western blot assay for the indicated antigens; (**B**–**E**) MDCK cells were infected with PR8 at a MOI of 1, and then treated with brevilin A (8 μM) at 1 hpi, total RNA was extracted from the infected cells at 6 hpi, and mRNA was quantified with RT-qPCR. \*\*, *p* < 0.01; \*\*\*, *p* < 0.001; \*\*\*\*, and *p* < 0.0001 are considered statistically significant, compared with vehicle.
