*3.4. Brevilin A Inhibits Influenza Viral RNA Synthesis*

Transcription of vRNA produces mRNA and complementary RNA (cRNA). The former serve as the template for synthesis of viral proteins, and the latter are templates for synthesis of more vRNA for production of new virions [24]. We next performed RT-qPCR to evaluate the effect of brevilin A on viral RNA synthesis. MDCK cells were infected with PR8 at 1 MOI, then treated with brevilin A or DMSO. Total RNA was extracted at 6, 12, or 24 hpi and reverse transcription was performed with specific primers for viral NP gene. At 6 hpi, when the viral RNA synthesis was already completed [25], the levels of vRNA in brevilin A-treated samples were significantly reduced in comparison to those in vehicle treated cells by 50%. In contrast, the levels of mRNA were only decreased by ~20% and cRNA was not altered upon the treatment with brevilin A (Figure 4A). We next tested several later time points post-infection to determine the inhibitory effects of brevilin A during multiple replication cycles of IAV. The effect of brevilin A on cRNA levels was not significant at 12 or 24 hpi (Figure 4B). However, the inhibition of mRNA levels became notable at 24 hpi with a reduction of about 45% (Figure 4C). These results indicate that the inhibitory effect of brevilin A is intimately involved with the vRNA synthesis.

**Figure 4.** The inhibitory effects of brevilin A on the expression of virus RNA. MDCK cells were infected with PR8 at a MOI of 1, then treated with brevilin A or vehicle. Total RNA was extracted at 6 hpi (**A**), 12 hpi (**B**), or 24 hpi (**C**), and the vRNA, cRNA, and mRNA were quantified by RT-qPCR. \*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, and *p* < 0.001 are considered statistically significant, compared to vehicle.
