title: Vaccinia Virus Infection Requires Maturation of Macropinosomes.
abstract: The prototypic poxvirus, vaccinia virus (VACV), occurs in two infectious forms, mature virions (MVs) and extracellular virions (EVs). Both enter HeLa cells by inducing macropinocytic uptake. Using confocal microscopy, live-cell imaging, targeted RNAi screening and perturbants of endosome maturation, we analyzed the properties and maturation pathway of the macropinocytic vacuoles containing VACV MVs in HeLa cells. The vacuoles first acquired markers of early endosomes [Rab5, early endosome antigen 1 and phosphatidylinositol(3)P]. Prior to release of virus cores into the cytoplasm, they contained markers of late endosomes and lysosomes (Rab7a, lysosome-associated membrane protein 1 and sorting nexin 3). RNAi screening of endocytic cell factors emphasized the importance of late compartments for VACV infection. Follow-up perturbation analysis showed that infection required Rab7a and PIKfyve, confirming that VACV is a late-penetrating virus dependent on macropinosome maturation. VACV EV infection was inhibited by depletion of many of the same factors, indicating that both infectious particle forms share the need for late vacuolar conditions for penetration.
authors: ETH Zürich Institute of Biochemistry, Otto-Stern-Weg 3, 8093 Zürich, Switzerland.
authors: Zaira
authors: Z
authors: Rizopoulos
authors: ETH Zürich Institute of Biochemistry, Otto-Stern-Weg 3, 8093 Zürich, Switzerland.
authors: Giuseppe
authors: G
authors: Balistreri
authors: MRC-Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK.
authors: Samuel
authors: S
authors: Kilcher
authors: MRC-Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK.
authors: Caroline K
authors: CK
authors: Martin
authors: ETH Zürich Institute of Biochemistry, Otto-Stern-Weg 3, 8093 Zürich, Switzerland.
authors: Mohammedyaseen
authors: M
authors: Syedbasha
authors: ETH Zürich Institute of Biochemistry, Otto-Stern-Weg 3, 8093 Zürich, Switzerland.
authors: Ari
authors: A
authors: Helenius
authors: MRC-Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK.
authors: Jason
authors: J
authors: Mercer
mesh_terms: D011992:Endosomes / Q000378:metabolism / Q000821:virology; D006367:HeLa Cells; D006801:Humans; D052118:Lysosomal-Associated Membrane Protein 1 / Q000235:genetics / Q000378:metabolism; D010587:Phagocytosis; D010588:Phagosomes / Q000378:metabolism* / Q000821:virology; D058305:Sorting Nexins / Q000235:genetics / Q000378:metabolism; D014616:Vaccinia virus / Q000378:metabolism / Q000472:pathogenicity*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
chemical_list: D052118:Lysosomal-Associated Membrane Protein 1; D058305:Sorting Nexins; D000090802:rab7 GTP-Binding Proteins; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins
keywords: PIKfyve; Poxvirus; Rab conversion; endocytosis; macropinocytosis; phosphoinositide exchange; virus entry