1 / 26

Jason Mercer and Ari Helenius Science 320 , 531 (2008)

Jason Mercer and Ari Helenius Science 320 , 531 (2008). Infected child. Vaccinia virus and smallpox. L ive-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

spiro
Download Presentation

Jason Mercer and Ari Helenius Science 320 , 531 (2008)

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Jason Mercer and Ari Helenius Science 320, 531 (2008)

  2. Infected child Vaccinia virus and smallpox Live-virus vaccine against smallpox Actual causative agent of smallpox – Variola virus 2 million deathsannuallyuntil 1959 WHO total vaccination programm Last fatal case – 1978 Completely eradicated by 1979 Immune responses generated from a Vaccinia virus infection protects the person against a lethal smallpox infection.

  3. 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

  4. eGFP E/L promoter eYFP A5 core protein 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

  5. MVs bind to filopodia and move towards the cell body Average speed: 1.05 ±0.38 µm/min

  6. When MVs reach the cell body, large blebs are formed Infected cells Uninfected cells

  7. 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

  8. 65% Without inhibitor 100μM Blebbistatin Is blebbing necessary for infection? Blebbistatin – an inhibitor of myosin II – dependent blebbing Infection Blebs formation Blebs formation is involved in productive viral entry

  9. 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

  10. 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

  11. Inhibition of infection by PAK1 autoinhibitorydomain (AID) 86 ± 4 % 8 ± 3 % 91 ± 6 % Number of infected cells eGFP-EXPRESS-MV infected cells – green mCherry-AID or mCherry-AID(L107F) transfected cells- red Actin -blue

  12. PAK1 is required for blebbing

  13. PAK1 localized into membrane blebs during infection Cells, expressing eGFP-PAK1, actin is stained in red Uninfected 30 min post infection

  14. Rac 1 (PAK1 activating GTPase) is activated during entry

  15. Comparison of cellular factors involved in macropinocytosis, MV infectivity and MV inducedblebbing Perturbants and inhibitors pattern has shown that MV use macropinocytosis for entry

  16. Professional phagocytes use macropinocytosis to engulf apoptotic bodies Phosphatidylserine (PS) - phospholipid component of the membrane In normal cells PS is kept on the inner-leaflet In apoptotic cells PS is exposed on the surface Nature Reviews Microbiology 2, 802-808, 2004

  17. 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

  18. Inhibition of MV infectivity after treatment of virions with ANX5 Black bars – binding White bars - infectivity 90% PS is required for MV infectivity

  19. Viral membrane delipidation by NP40 Without NP40 Green – eYFP-CORE-MV Red - actin 0.5% NP40 Delipidated virions can bindcells, but do not induce blebbing or entry

  20. Virion lipid add-back Reconstitution of lipid bilayer on virions by incubation with PC based liposomes Liposome Phosphatidylcholine (PC)

  21. Phosphatydlserine is exposed on the MV surface, can be extracted by NP40, and restoredwith PS liposomes. EYFP-A5-MV A647-Annexin V Merge Untreated Lipid extraction by 0.5% NP40 Lipid extraction by 0.5% NP40 and restoration with liposomes

  22. Rac 1 activation by relipdated MVs

  23. Virion lipid add-back: Plaque formation

  24. The fate of vaccinia – infected cells: apoptosis or necrosis? Red – necrotic cells (PI) Green – apoptotic cells (PS-ANX5) MV spread is connected with apoptosis

  25. Why do Vaccinia MVs use macropinocytosis andapoptotic mimicry to enter host cells? -Internalization of particlestoo big for other viral endocytic mechanisms. - Entry intothe different cell types, because PS-mediated clearance of apoptotic material is common to mostcells -Avoidance of the innate immune response

  26. Thank you for your attention!

More Related