ABSTRACT
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.
ABSTRACT
Background: SARS-CoV-2 receptor angiontensin-conveting-enzyme 2 (ACE2) is also a protective factor that contributes to reduce inflammation and fibrosis in tissues. An active form of ACE2 can be released from the cell surface by host proteases ADAM17 and TMPRSS2, being the latter also necessary for viral entry. Due to its properties, the administration of soluble recombinant ACE2 has been proposed as a SARS-CoV-2 treatment. Here, we assess the role of ACE2 activity and antiviral immune response at the site of infection in nasopharyngeal swabs of SARS-CoV-2 patients, to unravel its effect on inflammation cascade and infection outcome. Methods: Soluble enzymatic activity of ACE2 was measured in nasopharyngeal swabs at the time of PCR positivity (mean time from symptom=4d) and 3 days after in a cohort of mild SARS-CoV-2 patients (n=40, mean age=42y) and in uninfected controls. Gene expression profiles of ACE2, its proteases, ADAM17 and TMPRRS2, and interferon-stimulated genes (ISGs), DDX58, CXCL10 and IL-6 were also evaluated by RT-qPCR. Results: Both ACE2 activity and mRNA expression decreased significantly during infection course in paired samples of SARS-CoV-2 infected subjects (p=0.048 and p<0.001, respectively), although differences between infected and uninfected subjects were only seen at mRNA level (p<0.001) Importantly, both ACE2 activity and mRNA expression showed a positive correlation with viral load (rho=0.352, p-value=0.0259), suggesting that viral infection is influencing ACE2 function. Similarly, infection downregulates TMPRSS2 expression (pvalue< 0.01), but not ADAM17, further indicating the viral-induced regulation of host receptors. In contrast to ACE2 data, a clear induction of IFN stimulated genes, CXCL10, IL-6 and DDX58 (RIG-I), is observed upon infection (p-value<0.05 in all cases), demonstrating that SARS-CoV-2 induces an antiviral response and suggesting that ACE2 is not an ISG. This increased expression of ISGs is directly linked to viral load (rho=0.6177, p-value<0.0001;rho=0.4026, p-value=0.0110;rho=0.3024, p-value=0.0613, respectively) but it is rapidly reversed during infection course. Conclusion: Overall, our results demonstrate the existence of mechanisms by which SARS-CoV-2 suppress ACE2 expression and function, intracellular viral detection and subsequent ISG induction, offering new insights into ACE2 dynamics and inflammatory response in the human upper respiratory tract that may contribute to understand the early antiviral host response to SARS-CoV-2 infection.