CELL-REPORTS-D-25-03172

Hypothesis: Flavitrasin (FT) is a selective Sec61 inhibitor of dengue virus (DENV)/ Orthoflavivirus polyprotein translocation.
We report the antiviral activity of the CADA derivative VGD020, renamed Flavitransin, against DENV (DENV1-4) and other orthoflaviviruses (ZIKV and YFV).
Immunoblots and radioblots (cell-free translation-translocation assay) provide evidence for selective inhibition of Sec61-directed translocation of the viral polytopic preprotein by FT. For immunoblots cells were transfected or infected and treated with FT for 18h, cells were lyzed in NP40 and lysates were an analyzed by detection with different antibodies for DENV proteins or V5/FLAG/MYC tag. Clathrin or actin antibodies were used as cell loading control. Cell-free in vitro translation and translocation of DENV2 prM and E proteins in rabbit reticulocyte lysate (RRL) supplemented with ovine rough microsomes (RM) and [35S]-labeled methionine. Only fully translocated proteins in the ER lumen are protected from proteinase K (PK) treatment. Representative autoradiogram of DENV2 C14-prM166 and M21-E122 constructs. Samples were left untreated, or treated with FT or FTIN. Half of the translated material of each sample was additionally treated with PK
Flow cytometry data show that the C-terminus of flaviviral capsid protein determines sensitivity to Flavitransin. Data were aquired from transfection of HEK293T/HCT116 cells with plasmid construct encoding for DENV protein and flow cytometric analysis after 18h flavitransin treatment. Data were collected on CELESTA flow cytometer as mean fluorescence intensity (MFI) values for tGFP (FITC) and BFP (BV421) from at least 5,000 cell.
Sequencing data provided for the FT-resistant cells (nanopore sequencing) which display unique mutations in the Sec61α translocon subunit, CRISPR-Cas9 A70V knock-in (sanger sequencing data) and for FT-desensitized DENV.
For detailed prescriptions on experiments procedures were refer to the paper.