Abstract
Purpose:
We have earlier shown that human adenovirus type D37 (HAdV-D37) used caveolae mediated entry into human corneal fibroblasts (HCF) causing keratitis. Our earlier reports also suggested a central role for activated Src in viral entry and infection. Dynamins are associated with multiple cellular processes including endocytosis of caveolae, fission and release of transport vesicles, and regulating the dynamics of microtubules. Dynamin has also been shown to be important in the entry of other viruses. Here we studied the role of dynamin-2 in the intracellular trafficking of HAdV-D37 in HCF.
Methods:
HCF were pretreated with DMSO, bafilomycin A1, or cytochalasin B for 30min and then infected with HAdV-D37 for 1h. Cells were also transfected with siRNA against dynamin-2 or scRNA prior to infection. Cells were Trizol treated for RNA isolation or lysed for subsequent real time-PCR or protein studies, respectively. For microtubule visualization, cells grown on slide chambers were treated with DMSO or nocodazole for 1hr, and then infected with Cy3-labeled HAdV-D37. Cells were then fixed in 4% paraformaldehyde, washed in PBS 2% BSA, permeabilized in 0.1% Triton X-100, blocked in 2% BSA-PBS, incubated in anti-tubulin antibodies for 1hr at room temperature, washed again, and mounted for confocal microscopy.<br />
Results:
Neither the endosomal acidification inhibitor, bafilomycin A1 or the actin polymerization inhibitor, cytochalasin B had an effect on adenoviral infection of HCF. However, the microtubule inhibitor, nocodazole repressed viral gene expression in a dose dependent manner, consistent with reduced trafficking of virus to the nucleus. Dynamin-2 siRNA increased Src activation, microtubule accumulation, and viral gene expression. Upon dynamin-2 overexpression, Src activation and virus trafficking in corneal cells were both reduced.<br />
Conclusions:
Dynamin-2 knock down results in stability and accumulation of microtubules, which in HCF, enhances HAdV-D37 trafficking. These results are consistent with a negative role for dynamin-2 in HAdV-D37 infection and suggests that microtubule stability is a potential target for anti-adenoviral therapy in the cornea.