Gold, Sarah Jane; (2010) Cell entry of bluetongue virus. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Pharmacological and dominant-negative (Dn) inhibitors of endocytosis were used to investigate the cell entry pathway used by BTV-1 in mammalian cells. Infection, but not cell entry was inhibited in BHK, Vero and BPAEC cells, by reagents that raise endosomal pH suggesting that virus must be delivered to acidic compartments for infection. Entry and infection of BHK cells was not inhibited by three different dominant-negative inhibitors (Dn Eps15, AP180C and Dn Dynamin-2) of clathrin-mediated endocytosis, and during entry, virus did not colocalise with transferrin-positive early-endosomes. The failure of Dn Dynamin-2 to inhibit BTV-1 infection also excludes a role for caveolae during entry. This conclusion was supported by showing that Dn Caveolin- 1 and depletion of cellular cholesterol using methyl-β-cyclodextrin or nystatin did not inhibit infection. The observations that methyl-β-cyclodextrin and nystatin did not inhibit infection also rule out a role for other cholesterol/lipid raft-dependent pathways in entry. However, entry was inhibited by the dynamin inhibitor, dynasore, indicating that entry is dependent on a form of dynamin. During entry, BTV-1 was co-localised with dextran, a marker for macropinocytosis. EIPA, a known macropinocytosis inhibitor, blocked entry of both BTV-1 and dextran but not transferrin. Disruption of the actin cytoskeleton using cytochalasin D and latrunculin A inhibited infection further suggesting a role for macropinocytosis in entry. Dn Rab 34 which has been shown to inhibit macropinocytosis in some cell types also inhibited BTV-1 infection. During the first 2 hours of entry, BTV-1 was increasingly co-localised with LAMP-1 indicating that virus is delivered to late-endosomes and/or lysosomes by-passing early endosomes, a characteristic also shared by macropinocytosis. In summary, BTV-1 entry and infection of BHK cells can occur independently of cholesterol, and clathrin- and caveolae-mediated endocytosis, but depends on a specific form of dynamin, and shares characteristics in common with macropinocytosis.

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