*3.7. Dec-RVKR-cmk Inhibits ZIKV and JEV Infection in Mosquito Cells*

It is interesting to know whether dec-RVKR-cmk inhibits ZIKV and JEV infection only in mammalian cells or inhibits infection in a mosquito cell line. To address this question, firstly we examined the cytotoxicity of dec-RVKR-cmk to C6/36 cells by using luminescence-based cell viability assay. Likewise, Vero cells treated with dec-RVKR-cmk exhibited no cytotoxicity to C6/36 cell line at the concentrations of 1, 10, 50, and 100 μM (Figure 8a). Afterwards, C6/36 cells were infected with ZIKV (1 MOI) and then treated with 100 μM of dec-RVKR-cmk. Cell supernatant was harvested at 24 hpi for virus titer determination through plaque assay and cells were fixed for immunofluorescence imaging. Figure 8b indicates that a significant inhibition of 2.23 log10 ZIKV progeny titer was observed when compared to the DMSO-treated control. The fluorescence image (Figure 8c) revealed a ~39% reduction in immunonoreactive positive cells in the dec-RVKR-cmk treatment group as compared to the control (Figure 8d). These results suggest that dec-RVKR-cmk could inhibit flavivirus propagation in both mammalian cells and mosquito cells.

**Figure 8.** Dec-RVKR-cmk inhibits ZIKV and JEV infection in mosquito cell line C6/36. (**a**) Cytotoxicity of dec-RVKR-cmk in C6/36 cell line was determined by CellTiter-GLO One Solution Assay kit (Promega). (**b**) C6/36 cells were infected with ZIKV (1 MOI) followed by dec-RVKR-cmk treatment using the 100 μM concentration. Cell supernatant was used to determine ZIKV viral titer by plaque assay at 24 hpi. (**c**) Immunofluorescence images of ZIKV-infected c6/36 cells were acquired to quantify (**d**) ZIKV immunoreactive positive cells. Data are presented as the mean ± SEM from three independent experiments.

#### **4. Discussion**

Encephalitis, hemorrhagic disease, biphasic fever, jaundice, and flaccid paralysis are typical manifestations of flaviviruses in human beings [2]. Among mosquito-borne flaviviruses, ZIKA and JEV are medically important pathogens. JEV is known to be neurotropic and cause encephalitis, while ZIKV is considered to cause febrile illness. However, in a recent worldwide outbreak, ZIKV has been associated with neurological manifestations, including Guillain–Barré syndrome in adults and fetal microcephaly [27]. Continued rise in flavivirus infections across the world highlights an urgent need for antivirals to combat these challenges.

The objective of this study was to evaluate the antiviral activity of dec-RVKR-cmk against flavivirus. Processing of viral proteins by host cellular protease is a characteristic feature to achieve virus maturation in different viruses of various families. Dec-RVKR-cmk is reported to inhibit cleavage and fusion activity of various glycoproteins mediated by furin and to play a key role in activating several bacteria and viruses, including anthrax, botulinum, influenza A, measles, Ebola, HIV, HBV, and CHIKV [16,28–33]. Among flaviviruses, an important step in the production of infectious virions is the processing of prM protein to the anchored membrane M stump and the "pr" peptide that takes place in the TGN by the host proprotein convertase furin protease, prior to release from infected cell [18,34]. Therefore, we assessed furin inhibition activity of dec-RVKR-cmk against ZIKV and JEV. In this respect, our observations confirmed the critical role of furin in flavivirus maturation. We found that peptidyl CMK inhibited ZIKV and JEV more significantly in the later stage of their life cycle by preventing efficient cleavage of prM protein through the host proprotein convertase furin protease resulting in the effective arrest of subsequent viral infection.

The time-of-drug addition assay revealed that dec-RVKR-cmk worked more efficiently post infection but, interestingly, inhibition was also observed when dec-RVKR-cmk added at the time of infection. This antiviral activity of dec-RVKR-cmk was still observed even when the drug was added 12 hpi. Collectively, this suggests that dec-RVKR-cmk is more effective when added post infection. A dose-related inhibition of ZIKV and JEV in Vero cells was observed in terms of extracellular viral progeny titer, intracellular viral genome copies, and viral spreading from infected to bystander cells. These findings were consistent with the results of dec-RVKR-cmk against Chikungunya infection in muscle cells [18]. The effectiveness of dec-RVKR-cmk was observed against both ZIKV and JEV infections at different time points (24, 36, and 48 hpi) in terms of lowering viral titer and viral spreading from infected to neighboring cells. Maximum antiviral activity of dec-RVKR-cmk was perceived at 36 and 48 hpi. In the case of flavivirus, a single round of replication was reported to continue for 16 h [26]. Our study suggests that dec-RVKR-cmk could inhibit virus propagation in a one-step growth cycle, and a mechanism study suggests that it cannot inhibit virus replication, but exerts its inhibitory action on the virus release and next round infectivity. This might be due to inhibition of prM cleavage, which would affect viral packaging or result in accumulation of immature viral particles, while virus release and next round infectivity would be diminished.

Hepatitis C virus (HCV) is also an important member of Flaviviridae but has been placed in a genus separate from the other flaviviruses and it is not clear to what extent HCV envelope proteins behave like those of other members of the family Flaviviridae. As previously reported, proprotein convertases furin was responsible for proteolytic cleavage of pro-TGF-β1 into its bioactive form in HCV-infected cells that positively regulates HCV RNA replication [34]. Based on this study, dec-RVKR-cmk might be active against HCV but with a different mechanism of action.

The phenomenon of redundancy existing between furin and other proprotein convertases to overcome the side effects PCs inhibitor [23,24] suggesting dec-RVKR-cmk could not interfere with the normal cellular function of furin. These findings, together with the observed antiviral activity of dec-RVKR-cmk at a non-cytotoxic concentration in this study, support the possibility of the therapeutic application of the furin inhibitor against flavivirus infection, and our future study of this furin inhibitor will enable verification of therapeutic efficacy in an animal model.

In summary, this is the first study that shows the antiviral activity of dec-RVKR-cmk against flaviviruses (ZIKV and JEV). The observed IC50 and cytotoxicity profile together with time-of-addition and molecular mechanism data suggest dec-RVKR-cmk as a potential candidate for treatment.

**Author Contributions:** Conceptualization, M.I., J.Y., and S.C.; methodology, M.I.; software, M.K.S. and M.I.; validation, Z.C., X.W., Z.Z., Y.L., D.Z., B.Z. and Q.L.; writing—original draft preparation, M.I.; editing, J.Y. and S.C.; supervision, S.C. and J.Y.; project administration, S.C.; funding acquisition, S.C.

**Funding:** This work was supported by National Key Research and Development Program of China (2016YFD0500407), National Natural Science Foundation of China (31825025, 31572517, 31972721), Fundamental Research Funds for the Central Universities (2662018QD025), and Natural Science Foundation of Hubei Province (2019CFA010).

**Acknowledgments:** We acknowledge Huazhong Agricultural University for the provision of the biosafety laboratory. We are also grateful to Xiaowu Pang (College of Dentistry, Howard University, USA) for kindly providing the ZIKV-MR-766 strain.

**Conflicts of Interest:** The authors declare no conflict of interest.
