*3.2. Dec-RVKR-cmk Inhibits prM Cleavage during ZIKV and JEV Infection*

Flavivirus maturation is associated with the status of the M protein of viral particles, where the prM precursor protein is cleaved in TGN by the host proprotein convertase furin protease. Therefore, cleavage of the prM protein was analyzed by Western blotting (WB) at 36 hpi from Vero cells infected with JEV or ZIKV and treated or untreated with dec-RVKR-cmk. As expected, both dec-RVKR-cmk treated and untreated viral immune complexes showed almost identical bands of E and NS5 proteins, while a prominent thicker band of prM was detected in the dec-RVKR-cmk-treated cells when compared to untreated cells (Figure 2a,b), indicating a larger amount of prM protein accumulated in the treatment group of cells. After that, the relative quantification of detected signals of protein bands E and prM were analyzed by image J software. The proportion of prM was determined by dividing the prM adjusted signal over the E adjusted signal, and then we made a comparison of the prM/E ratio between the ZIKV or JEV treated sample and untreated control. Results revealed a significant increase in the prM/E index in the treated group compared to the untreated control (Figure 2c,d). Taken together, the data suggest that dec-RVKR-cmk exerts its inhibitory action on prM cleavage.

**Figure 2.** Dec-RVKR-cmk inhibits viral maturation process by preventing prM cleavage. Vero cells were infected with ZIKV-0.2 MOI and JEV-0.2 MOI followed by dec-RVKR-cmk treatment using 100 μM concentration. (**a**) ZIKV and (**b**) JEV viral proteins were analyzed using SDS-PAGE at 36 hpi from the peptidyl CMK-treated and untreated infected cells and then detected by Western blotting (WB) using relevant antibodies. Quantification of E and prM proteins were analyzed by image J software and the ratio of prM/E between the dec-RVKR-cmk-treated and untreated control group was compared for both (**c**) ZIKV and (**d**) JEV.
