Résumé
Background: Different viruses employ similar pathways for replication, revealing key intracellular hotspots to target with host-directed therapies and achieve a broad-spectrum antiviral activity. Plitidepsin is a clinically approved antitumoral agent that blocks the elongation factor eEF1A required for protein translation. This drug counteracts SARS-CoV-2 replication and shows a favorable safety profile in COVID-19 patients. Yet, the precise antiviral mechanism of action of plitidepsin remains unknown. Method(s): Here we used a deep quantitative proteomic analysis to measure the impact of plitidepsin on the proteome of SARS-CoV-2-infected Vero E6 cells. This was complemented with transmission electron microscopy assays, which unraveled the subcellular and morphological changes associated to plitidepsin treatment. In addition, we performed functional in vitro assays to dissect the antiviral activity of plitidepsin against SARS-CoV-2 and other viruses. Result(s): We found that this drug inhibited the synthesis of all SARS-CoV-2 proteins in a dose-dependent manner. These included the R1AB polyproteins, which facilitate the synthesis of non-structural proteins involved in the formation of double membrane vesicles (DMV) required for viral replication. Plitidepsin reduced DMV formation and the morphogenesis of new viruses, having a greater impact on viral than on host proteins. Less than 14% of the cellular proteome was significantly affected by plitidepsin, inducing the up-regulation of key molecules associated with protein biosynthesis, such as the translation initiation factors eIF4A2 and eIF2S3. Therefore, plitidepsin induced a compensatory state that rescued protein translation. This proteostatic response explains how cells preserve the cellular proteome after treatment with a translation inhibitor such as plitidepsin. In addition, it suggests that plitidepsin could inhibit other RNA-dependent and non-integrated DNA viruses, as we confirmed in vitro using Zika virus, Hepatitis C virus replicon and Herpes simplex virus. However, the compensatory proteostasis induced by plitidespin also explains why this drug failed to inhibit the replication of integrated DNA proviruses such as HIV-1. Conclusion(s): Unraveling the mechanism of action of host-directed therapies like plitidepsin is imperative to define the indications and antiviral profile of these compounds. This knowledge will be key to develop broad-spectrum treatments and have them ready to deploy when future pandemic viruses break through.