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      HIV-1 requires Arf6-mediated membrane dynamics to efficiently enter and infect T lymphocytes

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          Abstract

          As Arf6 is key to coordinating plasma membrane trafficking and regulates cellular invasion by several microorganisms, the authors studied Arf6 function during early HIV-1 infection. The data suggest that HIV-1 requires Arf6-driven plasma membrane dynamics and depends on GTP/GDP activity to efficiently fuse, enter, and infect CD4+ T lymphocytes.

          Abstract

          As the initial barrier to viral entry, the plasma membrane along with the membrane trafficking machinery and cytoskeleton are of fundamental importance in the viral cycle. However, little is known about the contribution of plasma membrane dynamics during early human immunodeficiency virus type 1 (HIV-1) infection. Considering that ADP ribosylation factor 6 (Arf6) regulates cellular invasion via several microorganisms by coordinating membrane trafficking, our aim was to study the function of Arf6-mediated membrane dynamics on HIV-1 entry and infection of T lymphocytes. We observed that an alteration of the Arf6–guanosine 5′-diphosphate/guanosine 5′-triphosphate (GTP/GDP) cycle, by GDP-bound or GTP-bound inactive mutants or by specific Arf6 silencing, inhibited HIV-1 envelope–induced membrane fusion, entry, and infection of T lymphocytes and permissive cells, regardless of viral tropism. Furthermore, cell-to-cell HIV-1 transmission of primary human CD4 + T lymphocytes was inhibited by Arf6 knockdown. Total internal reflection fluorescence microscopy showed that Arf6 mutants provoked the accumulation of phosphatidylinositol-(4,5)-biphosphate–associated structures on the plasma membrane of permissive cells, without affecting CD4-viral attachment but impeding CD4-dependent HIV-1 entry. Arf6 silencing or its mutants did not affect fusion, entry, and infection of vesicular stomatitis virus G–pseudotyped viruses or ligand-induced CXCR4 or CCR5 endocytosis, both clathrin-dependent processes. Therefore we propose that efficient early HIV-1 infection of CD4 + T lymphocytes requires Arf6-coordinated plasma membrane dynamics that promote viral fusion and entry.

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          Most cited references77

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          Virus Entry: Open Sesame

          Detailed information about the replication cycle of viruses and their interactions with host organisms is required to develop strategies to stop them. Cell biology studies, live-cell imaging, and systems biology have started to illuminate the multiple and subtly different pathways that animal viruses use to enter host cells. These insights are revolutionizing our understanding of endocytosis and the movement of vesicles within cells. In addition, such insights reveal new targets for attacking viruses before they can usurp the host-cell machinery for replication.
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            HIV enters cells via endocytosis and dynamin-dependent fusion with endosomes.

            Enveloped viruses that rely on a low pH-dependent step for entry initiate infection by fusing with acidic endosomes, whereas the entry sites for pH-independent viruses, such as HIV-1, have not been defined. These viruses have long been assumed to fuse directly with the plasma membrane. Here we used population-based measurements of the viral content delivery into the cytosol and time-resolved imaging of single viruses to demonstrate that complete HIV-1 fusion occurred in endosomes. In contrast, viral fusion with the plasma membrane did not progress beyond the lipid mixing step. HIV-1 underwent receptor-mediated internalization long before endosomal fusion, thus minimizing the surface exposure of conserved viral epitopes during fusion and reducing the efficacy of inhibitors targeting these epitopes. We also show that, strikingly, endosomal fusion is sensitive to a dynamin inhibitor, dynasore. These findings imply that HIV-1 infects cells via endocytosis and envelope glycoprotein- and dynamin-dependent fusion with intracellular compartments.
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              Recruitment of HIV and its receptors to dendritic cell-T cell junctions.

              Monocyte-derived dendritic cells (MDDCs) can efficiently bind and transfer HIV infectivity without themselves becoming infected. Using live-cell microscopy, we found that HIV was recruited to sites of cell contact in MDDCs. Analysis of conjugates between MDDCs and T cells revealed that, in the absence of antigen-specific signaling, the HIV receptors CD4, CCR5, and CXCR4 on the T cell were recruited to the interface while the MDDCs concentrated HIV to the same region. We propose that contact between dendritic cells and T cells facilitates transmission of HIV by locally concentrating virus, receptor, and coreceptor during the formation of an infectious synapse.
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                Author and article information

                Contributors
                Role: Monitoring Editor
                Journal
                Mol Biol Cell
                molbiolcell
                mbc
                Mol. Bio. Cell
                Molecular Biology of the Cell
                The American Society for Cell Biology
                1059-1524
                1939-4586
                15 April 2011
                : 22
                : 8
                : 1148-1166
                Affiliations
                [1] aLaboratorio de Inmunología Celular y Viral, Laboratorio de Neurosecreción, Unidad de Farmacología, Departamento de Medicina Física y Farmacología, Facultad de Medicina, Instituto de Tecnologías Biomédicas, Universidad de La Laguna, Campus de Ofra s/n, Tenerife 38071, Spain
                [2] bFundació irsiCaixa-HIVACAT, Institut de Recerca en Ciències de la Salut Germans Trias i Pujol, Hospital Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona 08916, Barcelona, Catalonia, Spain
                University of Geneva
                Author notes
                Address correspondence to: Agustín Valenzuela-Fernández ( avalenzu@ 123456ull.es ).

                *These authors contributed equally to this work.

                Article
                E10-08-0722
                10.1091/mbc.E10-08-0722
                3078069
                21346189
                58f4b4e6-178b-4719-b60e-0d4569b35939
                © 2011 García-Expósito et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License ( http://creativecommons.org/licenses/by-nc-sa/3.0).

                “ASCB®,“ “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology.

                History
                : 25 August 2010
                : 01 February 2011
                : 10 February 2011
                Categories
                Articles
                Cell Biology of Disease

                Molecular biology
                Molecular biology

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