Journal: Nature Communications

Article Title: Targeting eEF1A reprograms translation and uncovers broad-spectrum antivirals against cap or m 6 A protein synthesis routes

doi: 10.1038/s41467-025-56151-y

Figure Lengend Snippet: A Expression of a bi-cistronic dual reporter plasmid that expresses firefly luciferase via a poliovirus IRES and renilla luciferase via cap-dependent translation 48 h after Vero E6 transfection ± 50 nM of plitidepsin. B Geometric mean expression (Geo Mean) of HIV-1 Gag-eGFP plasmid detected by FACS 48 h after transfection on Vero E6 cells ± 50 nM of plitidepsin ( P = 0.0002). C Infection of a luciferase single-cycle infectious HIV-1 construct pseudotyped with VSVg in the presence of the indicated antivirals ( P = 0.0315; P ≤ 0.0012). Vero E6 cells were pulsed with equal amount of pseudoviruses and cultured for two days to determine luciferase activity, in RLUs. D Infection of a GFP single-cycle infectious HIV-1 construct pseudotyped with VSVg in the presence of indicated antivirals for two days. Vero E6 cells were pulsed with an equal amount of pseudoviruses to determine Geo Mean GFP in HIV-1 GFP + cells by FACS ( P < 0.0001). E Representation of Biorender showing proteins upregulated by 50 nM plitidepsin involved in translation via m 6 A and readers identified. Graph shows infection of HIV-1 construct pseudotyped with VSVg as in C , but in the presence or absence of the indicated concentrations of CWI1-2 ( P = 0.0384; P = 0.0001), tegaserod and plitidepsin ( P = 0.0057). F Nanoluciferase produced by Vero E6 cells infected with a nanoluciferase SARS-CoV-2 reporter virus. Cells were treated with the indicated concentrations of CWI1-2, tegaserod and plitidepsin ( P < 0.0001) and assayed by luminometry 48 h post-infection. Values are expressed in RLUs. G Fold reduction of luciferase mRNA expression on Vero E6 cells 48 h in the presence of 50 nM plitidepsin after transfection of a capped 5’ mRNA, an uncapped with 50 % of m 6 A ( P < 0.0001) or an uncapped mRNA not modified ( P = 0.0002). A – G show mean and S.D. from at least n exp = 3 and n rep = 8, where statistical differences were measured with a two-sided Man–Whitney U test. HIV-1 human immunodeficiency virus type 1, VSVg vesicular stomatitis virus G protein, DTG Dolutegravir, AZT Zidovudine. Source Data file provides source data.

Article Snippet: 250 ng of the bicistronic reporter plasmid pcDNA3 RLUC POLIRES FLUC (Addgene plasmid #4524, kind gift from Nahum Sonenberg) was transfected with X-tremeGENE HP Transfection Reagent (Roche) into 6 × 10 4 Vero E6 cells per well seeded in 96 well plates 24 h before.

Techniques: Expressing, Plasmid Preparation, Luciferase, Transfection, Infection, Construct, Cell Culture, Activity Assay, Produced, Virus, Modification