Sec61 translocon inhibitor Flavitransin blocks selectively dengue virus polyprotein insertion into the ER membrane with pan-flavivirus antiviral potency

Flavivirus infections by Dengue and Zika virus impose a significant healthcare threat worldwide. At present no FDA-approved specific antiviral treatment is available, and the safety of a vaccine against Dengue virus is still under debate. Here, we report the identification of the CADA derivative flavitransin (FT), with potent activity against DENV serotype 2 in various cell types. Moreover, FT showed consistent anti-flaviviral activity against all four DENV serotypes, and also against Zika and Yellow fever virus. Viral polyprotein biogenesis was completely abolished by FT treatment of DENV-infected cells. Drug profiling by a-time-of-drug-addition assay revealed a post-entry antiviral effect of FT, in line with its anticipated Sec61 inhibitory effect. Subsequent analysis of the individual viral proteins in transfected HEK293T cells indicated that FT suppresses the expression of the structural proteins (pre-membrane and envelope) only. Furthermore, cell free in vitro protein translation analysis demonstrated a direct inhibitory effect of FT on the co-translational translocation of the DENV polyprotein across the ER membrane. More specifically, FT inhibited the initiation of protein translocation into the ER that relies on the N-terminal transmembrane region of the capsid subunit of the DENV polyprotein, resulting in rerouting of the viral pre-protein to the cytosol for proteasomal degradation. Finally, selection and genotyping of FT-resistant HCT116 cells revealed a unique A70V mutation in the Sec61α subunit that conferred resistance to FT in infected cells. Long-term exposure of DENV to FT demonstrated a high barrier to resistance development. In conclusion, our data demonstrate that FT selectively interferes with the initiation of ER co-translational translocation of the DENV polyprotein and confirm the critical role of this translocation process in the flavivirus replication cycle.