Fig. 7: Generation and verification of the SARS-CoV-2 replicon.

A Schematic of the SARS-CoV-2 replicon. Viral genome expressed under tetracycline-inducible/CMV chimeric promoter with parts of E and S proteins deleted to attenuate virus infectivity. The replicon is stably integrated into HeLa cells through the Flp-In recombinase system. The main modifications introduced into the SARS-CoV-2 genome are highlighted. B HeLa-replicon or WT HeLa cells were exposed to doxycycline at 3 µg/mL for 24 hours, and luminescence was measured after lysis and supplementation with LgBiT and substrate furimazine. Mean ± SD are shown from 8 replicates, *p < 0.05. C Representative confocal images of the HaloTag stained with JFX650 dye and nucleus stained with NucSpot 470 in doxycycline-induced HeLa-replicon. Representative confocal images of doxycycline-induced HeLa-replicon showing expression of various markers. Left: confocal single plane images, right: 3-D rendering of z-stack. D mCerulean3 (∆Nsp2), E Nsp16-sfCherry2₁₁, F mNeonGreen2₁₁-M were co-stained with indicated nuclear dye, which is either NucSpot 470 or propidium iodide (PI). G Remdesivir inhibit doxycycline-induced HeLa-Replicon activation in a concentration-dependent manner. Replicon-containing cells were incubated with 3 µg/mL doxycycline as well as remdesivir at indicated concentrations for 24 hours before addition of LgBiT and furimazine (substrate) to quantify the generated luminescence (RLU refers to relative luminescence units). H Effects of compounds on doxycycline-induced HeLa-Replicon activation in a concentration-dependent manner. Replicon-containing cells were incubated with 3 µg/mL doxycycline as well as cordycepin, BTZ-1, and ADEP-42 at indicated concentrations for 24 hours before addition of LgBiT and furimazine (substrate) to quantify the generated luminescence (RLU refers to relative luminescence units). I Western blot analysis of AMPK, mTOR and Akt in HeLa-replicon cells treated with DMSO or cordycepin. Replicon cells were induced with 3 µg/mL doxycycline for 24 hours and treated with either DMSO or 30 µM cordycepin. Lysates were stained with AMPKα, phospho-AMPKα (Thr172), mTOR, phospho-mTOR (Ser2448), Akt, phospho-Akt (Ser473), and GAPDH antibodies. The numbers below the blots are quantified protein levels normalized against GAPDH and expressed relative to no doxycycline samples. MW markers are given on the right in kDa.