title: A clathrin independent macropinocytosis-like entry mechanism used by bluetongue virus-1 during infection of BHK cells.
abstract: Acid dependent infection of Hela and Vero cells by BTV-10 occurs from within early-endosomes following virus uptake by clathrin-mediated endocytosis (Forzan et al., 2007: J Virol 81: 4819-4827). Here we report that BTV-1 infection of BHK cells is also dependent on a low endosomal pH; however, virus entry and infection were not inhibited by dominant-negative mutants of Eps15, AP180 or the 'aa' splice variant of dynamin-2, which were shown to inhibit clathrin-mediated endocytosis. In addition, infection was not inhibited by depletion of cellular cholesterol, which suggests that virus entry is not mediated by a lipid-raft dependent process such as caveolae-mediated endocytosis. Although virus entry and infection were not inhibited by the dominant-negative dynamin-2 mutant, entry was inhibited by the general dynamin inhibitor, dynasore, indicating that virus entry is dynamin dependent. During entry, BTV-1 co-localised with LAMP-1 but not with transferrin, suggesting that virus is delivered to late-endosomal compartments without first passing through early-endosomes. BTV-1 entry and infection were inhibited by EIPA and cytochalasin-D, known macropinocytosis inhibitors, and during entry virus co-localised with dextran, a known marker for macropinocytosis/fluid-phase uptake. Our results extend earlier observations with BTV-10, and show that BTV-1 can infect BHK cells via an entry mechanism that is clathrin and cholesterol-independent, but requires dynamin, and shares certain characteristics in common with macropinocytosis.
authors.affiliation: Pirbright Laboratory, Institute for Animal Health, Woking, United Kingdom.
authors.forename: Sarah
authors.initials: S
authors.lastname: Gold
authors.forename: Paul
authors.initials: P
authors.lastname: Monaghan
authors.forename: Peter
authors.initials: P
authors.lastname: Mertens
authors.forename: Terry
authors.initials: T
authors.lastname: Jackson
mesh_terms: D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001820:Bluetongue virus / Q000502:physiology*; D002460:Cell Line; D002784:Cholesterol / Q000502:physiology; D002966:Clathrin / Q000502:physiology*; D006224:Cricetinae; D015638:Cytochalasin D / Q000494:pharmacology; D004705:Endocytosis; D006863:Hydrogen-Ion Concentration; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010873:Pinocytosis / Q000187:drug effects
chemical_list: D002966:Clathrin; D015638:Cytochalasin D; D000584:Amiloride; D002784:Cholesterol; C039614:ethylisopropylamiloride