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Jason Mercer and Ari Helenius Science 320, 531 (2008) Vaccinia virus and smallpox Live-virus vaccine against smallpox Actual causative agent of smallpox – Variola virus 2 million deaths annually until 1959 WHO total vaccination programm Last fatal case – 1978 Completely eradicated by 1979 Infected child Immune responses generated from a Vaccinia virus infection protects the person against a lethal smallpox infection. Vaccinia virus Poxvirus family dsDNA genome 190 kB over 200 proteins Vaccinia virus infection is very mild and is typically asymptomatic in healthy individuals Renewed interest in 21st century: -as a vector for immunization against other viruses. -due to concerns about smallpox being used as an agent for bioterrorism Two infectious forms of vaccinia virus: Intracellular mature virus (MV) majority of infectious progeny, remains in the cell until cell lysis Extracellular enveloped virus (EV) surrounded by an additional, TGN-derived membrane Constructs used in this study EGFP-EXPRESS-MV EYFP-CORE-MV E/L promoter eYFP eGFP A5 core protein MVs bind to filopodia and move towards the cell body Average speed: 1.05 ± 0.38 µm/min When MVs reach the cell body, large blebs are formed Uninfected cells Infected cells Is there a correlation between formation of blebs and MOI? The quantity of blebbing cells but not the number of blebs per cell is MOI dependent Is blebbing necessary for infection? Blebbistatin – an inhibitor of myosin II – dependent blebbing Blebs formation Infection 65% Without inhibitor 100μM Blebbistatin Blebs formation is involved in productive viral entry Which cellular factors are required for MV blebbing and entry? PAK1 (p21-activated kinase 1) Key regulator of the actin cytoskeleton, adhesion and cell motility Autoinhibited dimer Activated monomer PAK1 knockdown: siRNAs, dominant negative mutants, (AID) autoinhibitory domain expression siRNA mediated knockdown of PAK1 68% 74% Do PAK1 siRNAs specifically block viral entry or downstream processes as well? Acid-mediated endocytosis by-pass assay, viral endocytosis is subverted, viral cores are delivered directly in cytosol under pH 5.0 PAK1 is necessary for prefusion events Inhibition of infection by PAK1 autoinhibitory domain (AID) 86 ± 4 % 8±3% Number of infected cells 91 ± 6 % eGFP-EXPRESS-MV infected cells – green mCherry-AID or mCherry-AID(L107F) transfected cells- red Actin - blue PAK1 is required for blebbing PAK1 localized into membrane blebs during infection Cells, expressing eGFP-PAK1, actin is stained in red Uninfected 30 min post infection Rac 1 (PAK1 activating GTPase) is activated during entry Comparison of cellular factors involved in macropinocytosis, MV infectivity and MV induced blebbing Perturbants and inhibitors pattern has shown that MV use macropinocytosis for entry Professional phagocytes use macropinocytosis to engulf apoptotic bodies Phosphatidylserine (PS) - phospholipid component of the membrane In normal cells PS is kept on the innerleaflet In apoptotic cells PS is exposed on the surface Nature Reviews Microbiology 2, 802-808, 2004 The MV membrane is enriched in phosphatidylserine MVs are similar with apoptotic bodies in size Blocking of cell surface or virion PS with annexin V Inhibition of MV infectivity after treatment of virions with ANX5 Black bars – binding White bars - infectivity 90% PS is required for MV infectivity Viral membrane delipidation by NP40 Without NP40 Green – eYFP-CORE-MV Red - actin 0.5% NP40 Delipidated virions can bind cells, but do not induce blebbing or entry Virion lipid add-back Reconstitution of lipid bilayer on virions by incubation with PC based liposomes Liposome Phosphatidylcholine (PC) Phosphatydlserine is exposed on the MV surface, can be extracted by NP40, and restored with PS liposomes. EYFP-A5-MV Untreated Lipid extraction by 0.5% NP40 Lipid extraction by 0.5% NP40 and restoration with liposomes A647-Annexin V Merge Rac 1 activation by relipdated MVs Virion lipid add-back: Plaque formation The fate of vaccinia – infected cells: apoptosis or necrosis? Red – necrotic cells (PI) Green – apoptotic cells (PS-ANX5) MV spread is connected with apoptosis Why do Vaccinia MVs use macropinocytosis and apoptotic mimicry to enter host cells? - Internalization of particles too big for other viral endocytic mechanisms. - Entry into the different cell types, because PS-mediated clearance of apoptotic material is common to most cells - Avoidance of the innate immune response Thank you for your attention!