Sec61 translocon inhibitor flavitransin blocks selectively dengue virus polyprotein insertion in the ER with pan-orthoflavivirus antiviral potency

Dengue virus (DENV) infections impose a significant healthcare threat worldwide. Here, we report the antiviral activity of cyclotriazadisulfonamide (CADA) derivative VGD020, renamed as flavitransin (FT), against all four DENV serotypes, Zika, and yellow fever virus in various cell types. FT exerts a post-entry antiviral effect, in line with its anticipated Sec61 inhibition. Cell-free in vitro translation assays demonstrate a direct inhibitory effect of FT on DENV polyprotein translocation across the endoplasmic reticulum (ER) membrane of both human and insect (vector) origin, with high selectivity for the N-terminal transmembrane domain of the capsid subunit of different orthoflaviviruses. Selection and genotyping of FT-resistant HCT116 cells reveal a unique A70V mutation in the Sec61α subunit. Finally, a high barrier to FT resistance development is observed for wild-type DENV. In conclusion, our data demonstrate that FT selectively interferes with the initiation of DENV polyprotein ER translocation and confirm the critical role of the ER in orthoflavivirus replication.