Attività antivirale di amiodarone sul virus ebola: interferenza sull'ingresso del virus nelle cellule bersaglio

The Ebola virus disease (EVD) is caused by the Ebola virus (EBOV), and has an average mortality rate of 73 % with peaks up to 90 %. The 2014 EVD outbreak in West Africa is the largest one in the history of this disease with a mortality rate roughly of 60 %. At 26 January 2015 the CDC has estimated 21924 infected people of which 8737 died. This outbreak was declared by the World Health Organization and the United Nations as the most serious health problem of the XXI century. It was due to its rapid and uncontrolled viral spread, highlighting the problem of lack of preventive and/or therapeutic treatments. For these reasons the WHO accelerated the procedures required for the human experimentation and some clinical trials are currently ongoing on patients in the regions of the outbreak. The new findings on the molecular mechanism of EBOV entry into target cells have allowed the development of small molecules that can block viral infection at this level. Some of these molecules identified in vitro studies have shown good efficacy in vivo allowing its use in clinical trials. Molecules such as U18666A and desipramine, which act as inhibitors of EBOV infection in vitro, have been important for the identification of cellular factors required for EBOV entry into target cells. The first step of viral replication cycle is the binding of the viral glycoprotein GP to adhesion cellular factors and/or to cell surface receptors which are localized at the lipid rafts. These lipid rafts are rich in sphingomyelin (SM) that is required for viral particles adhesion and internalization by micropinocytosis. The lysosomal acid sphingomyelinase (L-aSMase) catalytic activity promotes the internalization process hydrolyzing SM to ceramide affecting membrane fluidity. In fact, the presence of ceramide allows the curvature of the membrane. Inside the cell viral particles proceed along the endocytic pathway through early endosomes to late endosomes (LE). At this level the EBOV GP is proteolitically processed by endosomal cathepsins L and B, leading to the exposure of the binding site, in the GP subunit 1, that allows the interaction with the intracellular receptor, the cholesterol transporter NPC-1. Subsequently the GP1-NPC-1 interaction a series of conformational changes in the GP subunit 2 are required for the fusion between the viral envelope and the endosomal membrane. In this way the nucleocapsid is released into the cytoplasm for the establishment of infection. Our group has recently studied the cellular effects of the amiodarone, an antiarrhythmic drug widely used in the clinical practice, that is able to induce cellular alterations at the level of endocytic pathway. In particular, it has been shown that it affects the LE morphology, the NPC-1 intracellular localization and it leads to an accumulation of SM by inhibition of the L-aSMase activity. Then amiodarone acts on cellular factors required for the EBOV internalization into the target cells. It has been also demonstrated that amiodarone has antiviral activity against the Severe Acute Respiratory Syndrome (SARS) virus and the hepatitis C virus. Considering the knowledge on the molecular mechanism of EBOV entry and the cellular alterations induced by amiodarone, the aim of this work was to evaluate the effect of amiodarone on the EBOV entry into target cells providing evidence to consider the possibility to employ amiodarone as a treatment for the EVD. In order to study the effects of amiodarone on EBOV entry, two recombinant systems were adopted to analyze this step under biosafety level 2 conditions. The first system is based on the Vesicular Stomatitis Virus (VSV) with the gene encoding the VSV-G glycoprotein replaced with the reporter gene encoding the green fluorescent protein GFP (VSVÎG-GFP). This virus was pseudotyped by the Zaire ebolavirus glycoprotein GP (GP pseudotyped virus). The second system is based on the fluorescent EBOV Virus-Like Particles (EBOVLP), that are made by the EBOV matrix protein VP40 fused to GFP, and the EBOV GP on their envelope. With these two systems it has been observed that the GP-mediated entry is inhibited by amiodarone in a dose-dependent manner. The inhibition is achieved at amiodarone concentrations found in plasma of patients orally treated, confirming the results recently published by Gehring and colleagues. Interestingly, this study demonstrate that amiodarone acts at four GP-mediate entry levels: i) decreases the GP-mediated binding by inhibit the L-aSMase activity; ii) slows down the VLP trafficking along the endocytic pathway; iii) affects the GP processing; iv) blocks GP-mediated fusion. However, other amiodarone effects on subsequent replication cycle steps cannot be excluded. Furthermore, we demonstrated the synergic effect of amiodarone and its major metabolite MDEA to inhibit GP-mediated infection. In collaboration with Prof. Mirazimi Public Health Agency of Sweden these results were confirmed on the Sudan ebolavirus infection. In this context, amiodarone had an effect on the infection of target cells efficiency. Interestingly, amiodarone strongly affect the infectious viral progeny release at the highest concentration found in the plasma of patients intravenous treated. These data confirm the antiviral effect of amiodarone on EBOV infection. With these information, the Italian non-governmental organization Emergency announced in November 2014 the start of amiodarone randomized controlled trial on Ebola patients in Sierra Leone, to evaluate the efficacy of amiodarone in the EVD treatment

Amiodarone and metabolite MDEA inhibit Ebola virus infection by interfering with the viral entry process

Ebola virus disease (EVD) is one of the most lethal transmissible infections characterized by a high fatality rate, and a treatment has not been developed yet. Recently, it has been shown that cationic amphiphiles, among them the antiarrhythmic drug amiodarone, inhibit filovirus infection. In the present work, we investigated how amiodarone interferes with Ebola virus infection. Wild-type Sudan ebolavirus and recombinant vesicular stomatitis virus, pseudotyped with the Zaire ebolavirus glycoprotein, were used to gain further insight into the ability of amiodarone to affect Ebola virus infection. We show that amiodarone decreases Ebola virus infection at concentrations close to those found in the sera of patients treated for arrhythmias. The drug acts by interfering with the fusion of the viral envelope with the endosomal membrane. We also show that MDEA, the main amiodarone metabolite, contributes to the antiviral activity. Finally, studies with amiodarone analogues indicate that the antiviral activity is correlated with drug ability to accumulate into and interfere with the endocytic pathway. Considering that it is well tolerated, especially in the acute setting, amiodarone appears to deserve consideration for clinical use in EV

Attività antivirale di amiodarone sul virus ebola: interferenza sull'ingresso del virus nelle cellule bersaglio

The Ebola virus disease (EVD) is caused by the Ebola virus (EBOV), and has an average mortality rate of 73 % with peaks up to 90 %. The 2014 EVD outbreak in West Africa is the largest one in the history of this disease with a mortality rate roughly of 60 %. At 26 January 2015 the CDC has estimated 21924 infected people of which 8737 died. This outbreak was declared by the World Health Organization and the United Nations as the most serious health problem of the XXI century. It was due to its rapid and uncontrolled viral spread, highlighting the problem of lack of preventive and/or therapeutic treatments. For these reasons the WHO accelerated the procedures required for the human experimentation and some clinical trials are currently ongoing on patients in the regions of the outbreak. The new findings on the molecular mechanism of EBOV entry into target cells have allowed the development of small molecules that can block viral infection at this level. Some of these molecules identified in vitro studies have shown good efficacy in vivo allowing its use in clinical trials. Molecules such as U18666A and desipramine, which act as inhibitors of EBOV infection in vitro, have been important for the identification of cellular factors required for EBOV entry into target cells. The first step of viral replication cycle is the binding of the viral glycoprotein GP to adhesion cellular factors and/or to cell surface receptors which are localized at the lipid rafts. These lipid rafts are rich in sphingomyelin (SM) that is required for viral particles adhesion and internalization by micropinocytosis. The lysosomal acid sphingomyelinase (L-aSMase) catalytic activity promotes the internalization process hydrolyzing SM to ceramide affecting membrane fluidity. In fact, the presence of ceramide allows the curvature of the membrane. Inside the cell viral particles proceed along the endocytic pathway through early endosomes to late endosomes (LE). At this level the EBOV GP is proteolitically processed by endosomal cathepsins L and B, leading to the exposure of the binding site, in the GP subunit 1, that allows the interaction with the intracellular receptor, the cholesterol transporter NPC-1. Subsequently the GP1-NPC-1 interaction a series of conformational changes in the GP subunit 2 are required for the fusion between the viral envelope and the endosomal membrane. In this way the nucleocapsid is released into the cytoplasm for the establishment of infection. Our group has recently studied the cellular effects of the amiodarone, an antiarrhythmic drug widely used in the clinical practice, that is able to induce cellular alterations at the level of endocytic pathway. In particular, it has been shown that it affects the LE morphology, the NPC-1 intracellular localization and it leads to an accumulation of SM by inhibition of the L-aSMase activity. Then amiodarone acts on cellular factors required for the EBOV internalization into the target cells. It has been also demonstrated that amiodarone has antiviral activity against the Severe Acute Respiratory Syndrome (SARS) virus and the hepatitis C virus. Considering the knowledge on the molecular mechanism of EBOV entry and the cellular alterations induced by amiodarone, the aim of this work was to evaluate the effect of amiodarone on the EBOV entry into target cells providing evidence to consider the possibility to employ amiodarone as a treatment for the EVD. In order to study the effects of amiodarone on EBOV entry, two recombinant systems were adopted to analyze this step under biosafety level 2 conditions. The first system is based on the Vesicular Stomatitis Virus (VSV) with the gene encoding the VSV-G glycoprotein replaced with the reporter gene encoding the green fluorescent protein GFP (VSVÎG-GFP). This virus was pseudotyped by the Zaire ebolavirus glycoprotein GP (GP pseudotyped virus). The second system is based on the fluorescent EBOV Virus-Like Particles (EBOVLP), that are made by the EBOV matrix protein VP40 fused to GFP, and the EBOV GP on their envelope. With these two systems it has been observed that the GP-mediated entry is inhibited by amiodarone in a dose-dependent manner. The inhibition is achieved at amiodarone concentrations found in plasma of patients orally treated, confirming the results recently published by Gehring and colleagues. Interestingly, this study demonstrate that amiodarone acts at four GP-mediate entry levels: i) decreases the GP-mediated binding by inhibit the L-aSMase activity; ii) slows down the VLP trafficking along the endocytic pathway; iii) affects the GP processing; iv) blocks GP-mediated fusion. However, other amiodarone effects on subsequent replication cycle steps cannot be excluded. Furthermore, we demonstrated the synergic effect of amiodarone and its major metabolite MDEA to inhibit GP-mediated infection. In collaboration with Prof. Mirazimi Public Health Agency of Sweden these results were confirmed on the Sudan ebolavirus infection. In this context, amiodarone had an effect on the infection of target cells efficiency. Interestingly, amiodarone strongly affect the infectious viral progeny release at the highest concentration found in the plasma of patients intravenous treated. These data confirm the antiviral effect of amiodarone on EBOV infection. With these information, the Italian non-governmental organization Emergency announced in November 2014 the start of amiodarone randomized controlled trial on Ebola patients in Sierra Leone, to evaluate the efficacy of amiodarone in the EVD treatment.Il virus Ebola (EBOV) è la causa di una febbre emorragica nell'uomo, nota come malattia da virus Ebola (EVD), che presenta un tasso di mortalità medio pari al 73 % con picchi fino al 90 %. L'epidemia di EVD tuttora in corso in Africa occidentale è la più vasta nella storia di questa malattia, e si osserva un tasso di mortalità attorno al 60 %. Per la sua diffusione rapida ed incontrollata con 21924 persone infette ed 8737 morti (al 26 gennaio 2015), è stata dichiarata dall'Organizzazione Mondiale della Sanità e dalle Nazioni Unite come il più grave problema sanitario del XXI secolo, evidenziando il problema della mancanza di trattamenti preventivi e/o terapeutici specifici. Alcuni trattamenti sono attualmente in sperimentazione sui pazienti delle regioni interessate dall' epidemia grazie ad una accelerazione delle procedure di autorizzazione necessarie per la sperimentazione umana previste dalla regolamentazione internazionale. Le scoperte recenti sul meccanismo molecolare alla base dell'ingresso di EBOV nella cellula bersaglio hanno consentito lo sviluppo di piccole molecole in grado di bloccare l'infezione agendo proprio a questo livello. Alcune delle molecole identificate con saggi in vitro hanno dimostrato una buona efficacia anche in vivo consentendone l'inclusione nei trial clinici. Molecole come U18666A e la desipramina, che agiscono da inibitori dell' infezione da EBOV in vitro, sono state importanti per l'identificazione di fattori cellulari richiesti per l'ingresso del virus nelle cellule bersaglio. Il ciclo replicativo virale ha inizio con il legame della glicoproteina virale GP a fattori cellulari di adesione e/o recettori localizzati in punti particolari della membrana plasmatica, i raft lipidici. Queste zone sono particolarmente ricche di sfingomielina (SM) la cui presenza e' importante sia per l'adesione del virus che per la sua internalizzazione per macropinocitosi. Quest'ultimo processo e' promosso dall'attivita' enzimatica della sfingomielinasi acida lisosomiale (L-aSMasi) che idrolizza la SM a ceramide. E' proprio la presenza di ceramide che permette la curvatura della membrana, modificandone la fluidià , cosi da macropinocitare la particella virale di EBOV adesa sulla superficie cellulare. Una volta all'interno della cellula questa procede lungo il pathway endocitico attraverso gli endosomi precoci per arrivare negli endosomi tardivi (LE) dove hanno luogo i processi chiave per l'instaurarsi dell'infezione. Il primo di questi e' il processamento sequenziale della GP da parte delle catepsine L e B che porta all'esposizione del sito di legame sulla subunità 1 con il recettore intracellulare, il trasportatore del colesterolo NPC-1. Successivamente, il legame che si instaura mette in atto una serie di cambiamenti conformazionali a carico della subunità 2 necessari all'inserimento del peptide fusogeno nella membrana endosomiale, all'avvicinamento del pericapside alla membrana endosomiale e alla formazione del poro di fusione fino alla completa fusione delle due membrane e liberazione del nucleocapside nel citoplasma. Il nostro gruppo ha recentemente studiato gli effetti cellulari di amiodarone, un farmaco antiaritmico largamente usato nella partica clinica, che è in grado di indurre alterazioni a livello del pathway endocitico cellulare, non correlate con l'attività antiaritmica. In particolare ha dimostrato che altera la morfologia dei LE, la localizzazione di NPC-1 e porta ad un accumulo della SM inibendo la L-aSMasi, quindi agendo su fattori cellulari necessari per l'internalizzazione del virus EBOV nella cellula bersaglio. E' stato inoltre dimostrato che amiodarone ha attivita’ antivirale contro il virus della Severe Acute Respiratory Syndrome (SARS) e contro il virus dell'epatite C. Considerando le conoscenze sul meccanismo molecolare di ingresso di EBOV nella cellula bersaglio e le alterazioni cellulari indotte da amiodarone siamo andati ad analizzare l'effetto di questo farmaco sull'ingresso di EBOV. Quindi, lo scopo di questo lavoro è stato quello di valutare l'azione antivirale di amiodarone sul virus Ebola con la finalita’ di fornire le basi biologiche per un suo possibile utilizzo nel trattamento della EVD. Al fine di studiare gli effetti di amiodarone sull'ingresso di Ebola, sono stati adottati due sistemi ricombinanti che consentono di lavorare in condizioni di biosicurezza di livello 2 senza dover usare direttamente il virus, che richiederebbe una struttura di biosicurezza di livello 4. Il primo modello è basato sul virus della Stomatite Vescicolare (VSV) nel quale il gene codificante la glicoproteina VSV-G è stato sostituito con il gene reporter codificante la green fluorescent protein (GFP). Questo virus è stato pseudotipizzato con la glicoproteina GP di EBOV (VSVÎG-GP ). Il secondo modello utilizzato è quello delle particelle simil-virali di EBOV (EBOVLP) fluorescenti, costituite dalla proteina di matrice VP40 di EBOV fusa alla GFP e contenenti la GP di EBOV sul pericapside. Con questi sistemi è stato osservato che l'ingresso GP-mediato è inibito in maniera dose-dipendente da amiodarone a concentrazioni riscontrabili nel plasma dei pazienti trattati per via orale, confermando quanto recentemente pubblicato da Gehring e colleghi. Analizzando l'effetto di amiodarone addizionato a vari tempi rispetto l'infezione, è emerso che amiodarone agisce nelle fasi precoci dell'infezione come ipotizzato. In particolare, amiodarone agisce su diverse fasi del processo di ingresso di EBOV: i) diminuisce il legame GP-mediato per la sua inibizione sulla L-aSMasi; ii) rallenta il trafficking delle EBOVLP lungo il pathway endocitico; iii) blocca la fusione GP-mediata influenzando il processamento della GP da parte delle catepsine endosomiali. Inoltre, abbiamo dimostrato che amiodarone in combinazione con il suo principale metabolita MDEA inibisce più efficientemente l'infezione di EBOV che se usato singolarmente ai dosaggi riscontrati nel siero dei pazienti. Questi risultati sono stati confermati utilizzando il virus EBOV wild-type grazie alla collaborazione con il Prof. Mirazimi dell'Agenzia di Sanita Pubblica della Svezia. In tale contesto è stato dimostrato che amiodarone non solo ha un effetto sull'efficienza di infezione delle cellule bersaglio ma diminuisce il rilascio della progenie virale infettiva, fino quasi ad azzerarla alla massima concentrazione ritrovabile nel plasma dei pazienti trattati per via endovenosa. Questi dati confermano un'effettiva azione antivirale del farmaco supportando anche quanto osservato con i sistemi ricombinanti. Grazie a questi dati Emergency ha avviato alla fine dello scorso anno una sperimentazione con somministrazione compassionevole di amiodarone in Sierra Leone e a novembre 2014 è stata annunciata la programmazione di un trial clinico per la valutazione dell' efficacia di amiodarone nel trattamento della EVD

The Ubiquitin-Conjugating System: Multiple Roles in Viral Replication and Infection

Through the combined action of ubiquitinating and deubiquitinating enzymes, conjugation of ubiquitin to a target protein acts as a reversible post-translational modification functionally similar to phosphorylation. Indeed, ubiquitination is more and more recognized as a central process for the fine regulation of many cellular pathways. Due to their nature as obligate intracellular parasites, viruses rely on the most conserved host cell machineries for their own replication. Thus, it is not surprising that members from almost every viral family are challenged by ubiquitin mediated mechanisms in different steps of their life cycle and have evolved in order to by-pass or exploit the cellular ubiquitin conjugating system to maximize their chance to establish a successful infection. In this review we will present several examples of the complex interplay that links viruses and the ubiquitin conjugation machinery, with a special focus on the mechanisms evolved by the human immunodeficiency virus to escape from cellular restriction factors and to exit from infected cells

Amiodarone inhibits Ebola virus replication in vitro

Ebola virus (EboV) is the etiologic agent of an haemorrhagic fever characterised by a high mortality rate. No therapeutics are available for prevention or treatment of this disease. Recently, it has been demonstrated that viral entry into target cells requires the activity of the acid sphingomyelinase (aSMase), cathepsins and the presence of the intracellular receptor Niemann-Pick C1 (NPC1) protein. The antiarrhythmic drug Amiodarone (Amio) and its main metabolite MDEA are able to perturb the cellular endocytic pathway, with the generation of large vacuoles and mislocalization of cellular proteins, such as NPC1, and lipids. The aim of this work is to evaluate if Amio/MDEA are able to interfere with EboV infection. We evaluated the effect of the compounds on viral entry by confocal microscopy and citofluorimetric analysis of Vero cells incubated with: i) Ebola virus-like particles (EboVLPs) characterised by the VP40 matrix protein fused in frame to the GFP, and ii) Ebola-glycoprotein (GP)-pseudotyped VSV∆G (EboVSV) expressing the GFP in place of its native envelope glycoprotein. In addition, we analysed the Amio effect on key enzymatic activities required for EboV entry. Finally, the effect of the compounds were evaluated on wild-type EboV entry into target cells and viral progeny release under BSL4 condition. Our results demonstrate that Amio is able to inhibit EboVSV replication in a dose-dependent manner. By using the EboVLPs we observed that Amio reduces the binding of VLPs to the cell surface in pre-treated cells, due to the inhibition of the aSMase activity, and their trafficking through the endocytic pathway. Moreover our observations with EboVSV suggest an inhibition of the viral envelope fusion with the endosomal membranes. No effect on cellular cathepsins activity was observed in treated cells. Significantly, the combination of 2 µM Amio/MDEA (average serum concentration found in patients treated for arrhythmia), or 10µM Amio, significantly inhibit wild-type EboV replication in vitro by affecting both the number of infected cells and viral progeny release. Our data suggest that Amio and MDEA inhibit Ebola virus infection by interfering with the entry step. Noteworthy, Amio already exerts its effect at concentrations reached in treated patients

Amiodarone affects Ebola virus binding and entry into target cells

Ebola Virus Disease is one of the most lethal transmissible infections characterized by a high fatality rate. Several research studies have aimed to identify effective antiviral agents. Amiodarone, a drug used for the treatment of arrhythmias, has been shown to inhibit filovirus infection in vitro by acting at the early step of the viral replication cycle. Here we demonstrate that amiodarone reduces virus binding to target cells and slows down the progression of the viral particles along the endocytic pathway. Overall our data support the notion that amiodarone interferes with Ebola virus infection by affecting cellular pathways/targets involved in the viral entry process

Amiodarone inhibits Ebola virus infection by Blocking Viral Entry Into Target Cells Through Late Endosomes

Objectives: Ebola Virus Disease is one of the most lethal transmissible infections characterized by a high fatality rate. Recently, we demonstrated that the antiarrhythmic drug amiodarone inhibits Ebola virus infection interfering with the fusion of the viral envelope with the endosomal membrane. In the present work we further investigated the antiviral activity of amiodarone on Ebola virus infection. Methods: In addition to the wild type Sudan ebolavirus (SUDV), two recombinant systems were used, the Vesicular Stomatitis virus (VSV) and Ebola virus-like particles (EBOVLPs), to clarify the effect of amiodarone on Ebola virus infection. Both recombinant VSV and EBOVLPs were pseudotyped with the Zaire ebolavirus glycoprotein (GP). Results: Amiodarone time addition experiments on Vero cells infected with the SUDV show that amiodarone strongly inhibits Ebola virus infection if the drug is present during the adhesion and/or post-adhesion times. The inhibition of SUDV infection decreases if the drug is added at later time points after viral infection. To better characterize the fate of viral particles during the entry step, we optimized a protocol for the production and purification of fluorescent EBOVLPs. Preliminary experiments with EBOVLPs confirmed our previous results supporting a block of the viral particles inside the endosomes with a GP-mediated mechanism. In addition, results with the VSV-GP suggest that amiodarone decreases the ability of SUDV to bind to target cells after drug pre-treatment. Conclusions: These findings suggest that amiodarone is able to inhibit Ebola virus infection targeting several steps of viral entry phase providing further insights on the ability of amiodarone of inhibiting Ebola virus infection in vitro