Abstract
The role of actin dynamics in clathrin-mediated endocytosis in mammalian cells is unclear. In this study, we define the role of actin cytoskeleton in Kaposi's sarcoma-associated herpesvirus (KSHV) entry and trafficking in endothelial cells using an immunofluorescence-based assay to visualize viral capsids and the associated cellular components. In contrast to infectivity or reporter assays, this method does not rely on the expression of any viral and reporter genes, but instead directly tracks the accumulation of individual viral particles at the nuclear membrane as an indicator of successful viral entry and trafficking in cells. Inhibitors of endosomal acidification reduced both the percentage of nuclei with viral particles and the total number of viral particles docking at the perinuclear region, indicating endocytosis, rather than plasma membrane fusion, as the primary route for KSHV entry into endothelial cells. Accordingly, a viral envelope protein was only detected on internalized KSHV particles at the early but not late stage of infection. Inhibitors of clathrin- but not caveolae/lipid raft-mediated endocytosis blocked KSHV entry, indicating that clathrin-mediated endocytosis is the major route of KSHV entry into endothelial cells. KSHV particles were colocalized not only with markers of early and recycling endosomes, and lysosomes, but also with actin filaments at the early time points of infection. Consistent with these observations, transferrin, which enters cells by clathrin-mediated endocytosis, was found to be associated with actin filaments together with early and recycling endosomes, and to a lesser degree, with late endosomes and lysosomes. KSHV infection induced dynamic actin cytoskeleton rearrangements. Disruption of the actin cytoskeleton and inhibition of regulators of actin nucleation such as Rho GTPases and Arp2/3 complex profoundly blocked KSHV entry and trafficking. Together, these results indicate an important role for actin dynamics in the internalization and endosomal sorting/trafficking of KSHV and clathrin-mediated endocytosis in endothelial cells.
Highlights
Endocytosis is a constitutive cellular process that results in the internalization of cell surface receptors and ligands, and membrane components, often initiating the activation of signal transduction cascades [1]
We found that Kaposi’s sarcoma-associated herpesvirus (KSHV) uses the clathrin-mediated endocytosis pathway to enter endothelial cells, and this process is regulated by actin dynamics
For herpesviruses that enter the cells via fusion of the viral envelope at the plasma membrane, the viral envelope glycoproteins are undesirable for monitoring virus entry and trafficking [38,39]
Summary
Endocytosis is a constitutive cellular process that results in the internalization of cell surface receptors and ligands, and membrane components, often initiating the activation of signal transduction cascades [1]. The best-described endocytic pathway is clathrin-mediated endocytosis [3]. In this process, the clathrin-coated pits assemble at the plasma membrane and acquire cargo. The plasma membrane proceeds to invaginate and constrict to generate a clathrin-coated vesicle, which is subsequently transported to the interior of the cell, where it loses its clathrin coat and fuses with the early endosome [3]. The orderly transport of endocytic cargo from the cell exterior to the interior is highly regulated, and requires the participation of numerous lipid components and accessory proteins, as well as alterations of fine cellular structures and controlled mechanical force to overcome the physical resistance and propel the vesicle into the cell [4]
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