Disruption of Rho GTPase Prenylation by Statins Inhibits Respiratory Syncytial Virus

Abstract

INTRODUCTIONRespiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections necessitating hospitalization in the pediatric population. Despite the significant healthcare burden associated with RSV, few options are available to prevent or treat infection. To address the lack of therapeutic strategies for RSV, we developed a high content screening platform to identify small molecule inhibitors of the virus. Following a screen of 2400 compounds, statins were found to significantly reduce RSV infection rates. Here we show that statin‐mediated disruption of isoprenoid biosynthesis and Rho GTPase prenylation blocks late events within the life cycle of RSV, including protein trafficking and viral assembly.METHODSTo screen for inhibitors of RSV, human laryngeal epidermoid carcinoma (HEp‐2) cells were infected with recombinant RSV engineered to express green fluorescent protein (GFP). Automated image analysis algorithms were used to quantify infection rate by measuring the proportion of GFP‐fluorescent cells. Statin‐induced changes to the life cycle of RSV were determined by immunofluorescence microscopy and temporal quantification of viral fusion and nucleoproteins. Effects on protein prenylation by statins and RSV were evaluated via click chemistry using azide‐modified isoprenoids with a biotin‐alkyne detection reagent. Rho GTPase activity was quantified by Western blot following affinity purification of active, GTP‐bound RhoA or Rac1.RESULTSInhibition of RSV by statins was dose‐dependent with an effective concentration (EC50) of 1 μM. Viral events occurring >12 hours post‐infection, including protein trafficking and assembly, were inhibited by statins, whereas early events, such as viral entry, were unaffected. Viral trafficking and assembly are thought to be mediated by Rho GTPases, a group of prenylated proteins which modulate dynamics of the actin cytoskeleton. We found that RhoA‐specific inhibition by Rhosin (50 μM) moderately reduced RSV infection rate (26 ± 6% reduction), but Rac1‐specific inhibition by EHT1864 (20 μM) elicited a profound effect (90 ± 2% reduction). Critically, RSV infection promoted Rac1 prenylation and activity, while statin treatment blocked Rac1 prenylation and reduced active, GTP‐bound protein by 2.5‐fold.CONCLUSIONSOur results demonstrate the importance of protein prenylation to RSV infection and perturbation of this process by statins. Inhibition of Rac1 prenylation and activity by statins serves to block viral protein trafficking and assembly. Based on our findings, we propose repurposing statins as a treatment for RSV as these drugs potently inhibit the virus, are US Food and Drug Administration approved and have favorable clinical safety profiles.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.