EBOLA: niente si trasmette come la paura 4 dicembre 2014 Sala Congressi - Asse Didattico E Complesso Universitario di Monserrato La biologia del virus Ebola Alessandra Pani Dipartimento di Scienze Biomediche Università degli Studi di Cagliari La biologia del virus Ebola 1. Struttura del virione 2. Infezione e replicazione 3. Fattori di virulenza e di evasione dalla risposta immunitaria 4. Target molecolari per lo sviluppo di farmaci Filovirus • Filamentous or Bacillus form • Vary greatly in length (up to 14000 nm) Mean Unit Length Ebola - 1200 nm Marburg - 860 nm • Uniform diameter of 80 nm Genome and Proteins EBOV-Z MARV Non-segmented, negative-strand RNA Filovirus Genome – 19 kb 7 Genes Nucleotide and amino acid differences b/w • Marburg and Ebola – 55% • Ebola – 37 to 41% Overlaps • • 1 in Marburg genome – VP30 to VP24 2 in Ebola genome-VP35 to VP40 & GP to VP30 Mühlberger E., Future Virology 2007 EboV-Z GP cleaved by FURIN Hofmann-Winkler et al.,Viruses 2012 La biologia del virus Ebola 1. Struttura del virione 2. Infezione e replicazione 3. Fattori di virulenza e di evasione dalla risposta immunitaria 4. Target molecolari per lo sviluppo di farmaci Filovirus life cycle Dolnik et al., Cell. Mol. Life Sci. 2008 Dolnik et al., Cell. Mol. Life Sci. 2008 Miller & Chandran, Curr Opinion Virol 2012 Kondratowicz et al., Proc Natl Acad Sci USA 2011 Hofmann-Winkler, Viruses 2012 Lectins involved in Filovirus attachment GP-binding lectins Tissue distribution ASGPR-1 •hepatocytes hMGL •monocyte-derived immature dendritic cells •intermediate precursors of macrophages DC-SIGN •dendritic cells •fetal macrophages •decidual macrophages •alveolar macrophages •platelets •activated primary B-cells DC-SIGNR •endothelial cells in the placenta •endothelial cells in the liver and lymph node sinuses LSECtin •sinusoidal endothelial cells in liver, lymph nodes and bone marrow •thymic dendritic cells •monocyte-derived macrophages •peripheral blood cells Cellular endocytic pathways and factors implicated in Filovirus entry Bhattacharyya et al., Virology 2010 Saeed et al., PLoS Pathog 2010 Carette et al., Nature 2011 Cote et al., Nature 2011 Filovirus life cycle Dolnik et al., Cell. Mol. Life Sci. 2008 GP expression in Ebola virus Transcriptional RNA Editing At a series of 7 U’s on the GP RNA insert a non-template coded adenosine: • 20% of GP mRNA is edited GP • 80% of GP mRNA is not edited sGP EBOV GP gene encodes 2 products: 2 ORF (MARV GP gene encodes 1 product: 1 ORF) GP-RNA Editing • Over-expression of GP • Cytotoxicity and eventual cell death GP expression and cytotoxicity can be regulated by virus through transcriptional RNA editing and secretory-GP expression Volchkov et al., Science 2001 Filovirus life cycle Dolnik et al., Cell. Mol. Life Sci. 2008 La biologia del virus Ebola 1. Struttura del virione 2. Infezione e replicazione 3. Fattori di virulenza e di evasione dalla risposta immunitaria 4. Target molecolari per lo sviluppo di farmaci EboV virulence factors: GP and sGP GP 1. Specific region of GP induces cytotoxic effects in endothelial cells -Rapid release of vasoactive agents from infected cells -Induces cell rounding and detachment from extracellular matrices -Increases cell membrane permeability 2. Specific region of GP contribute to evasion of host immunity -Possible immunosuppressive sequence in GP2: blocks lymphocyte mitogen-stimulated proliferation in vitro sGP 1. Inhibits early activation of neutrophils -Binds to neutrophils via CD16b cell surface receptor 2. Sequesters neutralizing antibodies Yang et al., Nature Med 2000 Simmons et al., J Virol 2002 Marzi et al., Virology 2006 EboV virulence factors: VP35 and VP24 Dolnik et al., Cell. Mol. Life Sci. 2008 Ramanan et al., Viruses 2011 Wei Xu et al., Cell Host & Microbs 2014 VP35: No IFN-α/β VP24: No IFNantiviral effectors Multiple funtions of IFN-α and IFN-β 1 (antiviral-IFNs) 2 3 • Antiviral state of host cells • Activation of immune cells 5 • Induction of IFN-γ (immune-IFN) cell-mediated immune response 5 5 Basler et al., Proc Natl Acad Sci USA 2000 Basler & Amarasinghe, J Interferon Cytokine Res 2009 Ramanan et al., Viruses 2011 Wei Xu et al., Cell Host & Microbs 2014 4 5 La biologia del virus Ebola 1. Struttura del virione 2. Infezione e replicazione 3. Fattori di virulenza e di evasione dalla risposta immunitaria 4. Target molecolari per lo sviluppo di farmaci EboV molecular targets for the therapy and/or prevention of Ebola infections • • • • • • vGlycoprotein GP (adhesion/penetration) cNPC1 (necessary for uncoating) vRNApol L (transcription/replication) VP35 (transcription/replication and IFN inhibition) VP40 (transport/assembly) VP24 (assembly and IFN inhibition) Drugs and vaccines in clinical trials with support for further development according to the Centers for Disease Control and Prevention Animals Healthy Small group Large group of patients Trials volunteers of patients ZMapp GP: triple mAbs cocktail Brincidofovir RNA-pol (??): nucleoside analog; broad spectrum - DNA viruses TKM-Ebola RNApol: siRNA RNApol: nucleoside analog; chain terminator; anti-Flu approved Favipiravir AVI-7537 BCX4430 ChAd3 VSV-EBOV Approved for use VP24: anti-sense oligomer Drugs RNApol: nucleoside analogue; broad spectrum - RNA viruses Vaccines Kondratowicz & Maury, Future Microbiol 2012 Nature Reviews Drug Discovery 2014 Gao, Drug Discov Ther 2014 Cohen, Science 2014