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      Actin Dynamics Regulate Multiple Endosomal Steps during Kaposi's Sarcoma-Associated Herpesvirus Entry and Trafficking in Endothelial Cells

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      1 , 2 , 1 , 2 , 3 , 4 , 5 , *
      PLoS Pathogens
      Public Library of Science

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          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.

          Author Summary

          Endocytosis, an essential biological process mediating cellular internalization events, is often exploited by pathogens for their entry into target cells. The role of actin cytoskeleton in clathrin-mediated endocytosis in mammalian cells remains unclear. Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus linked to the development of Kaposi's sarcoma, an endothelial malignancy commonly found in AIDS patients, and several other malignancies. In this study, we found that KSHV uses the clathrin-mediated endocytosis pathway to enter endothelial cells, and this process is regulated by actin dynamics. We found KSHV particles in early and recycling endosomes, and lysosomes, which are docked on actin filaments at the early time points of viral infection. Similarly, transferrin, which enters cells by clathrin-mediated endocytosis, is associated with actin filaments together with early and recycling endosomes, and, to a lesser degree, with late endosomes and lysosomes. 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 in endothelial cells. Together, these results define an important role for actin dynamics in multiple endosomal steps during KSHV infection and clathrin-mediated endocytosis in endothelial cells.

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          Most cited references75

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          Regulated portals of entry into the cell.

          The plasma membrane is the interface between cells and their harsh environment. Uptake of nutrients and all communication among cells and between cells and their environment occurs through this interface. 'Endocytosis' encompasses several diverse mechanisms by which cells internalize macromolecules and particles into transport vesicles derived from the plasma membrane. It controls entry into the cell and has a crucial role in development, the immune response, neurotransmission, intercellular communication, signal transduction, and cellular and organismal homeostasis. As the complexity of molecular interactions governing endocytosis are revealed, it has become increasingly clear that it is tightly coordinated and coupled with overall cell physiology and thus, must be viewed in a broader context than simple vesicular trafficking.
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            Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation

            The clathrin-coated pit lattice is held onto the plasma membrane by an integral membrane protein that binds the clathrin AP-2 subunit with high affinity. In vitro studies have suggested that this protein controls the assembly of the pit because membrane bound AP-2 is required for lattice assembly. If so, the AP-2 binding site must be a resident protein of the coated pit and recycle with other receptors that enter cells through this pathway. Proper recycling, however, would require the switching off of AP-2 binding to allow the binding site to travel through the endocytic pathway unencumbered. Evidence for this hypothesis has been revealed by the cationic amphiphilic class of drugs (CAD), which have previously been found to inhibit receptor recycling. Incubation of human fibroblasts in the presence of these drugs caused clathrin lattices to assemble on endosomal membranes and at the same time prevented coated pit assembly at the cell surface. These effects suggest that CADs reverse an on/off switch that controls AP-2 binding to membranes. We conclude that cells have a mechanism for switching on and off AP-2 binding during the endocytic cycle.
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              ERM proteins and merlin: integrators at the cell cortex.

              A fundamental property of many plasma-membrane proteins is their association with the underlying cytoskeleton to determine cell shape, and to participate in adhesion, motility and other plasma-membrane processes, including endocytosis and exocytosis. The ezrin-radixin-moesin (ERM) proteins are crucial components that provide a regulated linkage between membrane proteins and the cortical cytoskeleton, and also participate in signal-transduction pathways. The closely related tumour suppressor merlin shares many properties with ERM proteins, yet also provides a distinct and essential function.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                July 2009
                July 2009
                10 July 2009
                : 5
                : 7
                : e1000512
                Affiliations
                [1 ]Tumor Virology Program, Greehey Children's Cancer Research Institute, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America
                [2 ]Department of Pediatrics, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America
                [3 ]Department of Microbiology and Immunology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America
                [4 ]Cancer Therapy and Research Center, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America
                [5 ]Tumor Virology Group, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
                University of Southern California School of Medicine, United States of America
                Author notes

                Conceived and designed the experiments: SJG. Performed the experiments: WG SJG. Analyzed the data: WG SJG. Wrote the paper: WG SJG.

                Article
                08-PLPA-RA-1499R3
                10.1371/journal.ppat.1000512
                2702172
                19593382
                7a36eb62-1ea8-431e-8aa4-79660ddbba46
                Greene, Gao. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 24 November 2008
                : 15 June 2009
                Page count
                Pages: 18
                Categories
                Research Article
                Cell Biology/Cytoskeleton
                Cell Biology/Membranes and Sorting
                Virology/Host Invasion and Cell Entry
                Virology/Viruses and Cancer

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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