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2007_HIV_assebly_budding_spread_in_T_cells_take_place_in_TEMs

2007_HIV_assebly_budding_spread_in_T_cells_take_place_in_TEMs

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J OURNAL OF V IROLOGY , Aug. 2007, p. 7873–7884 Vol. 81, No. 15 0022-538X/07/$08.00 0 doi:10.1128/JVI.01845-06 Copyright © 2007, American Society for Microbiology. All Rights Reserved. Human Immunodeficiency Virus Type 1 Assembly, Budding, and Cell-Cell Spread in T Cells Take Place in Tetraspanin-Enriched Plasma Membrane Domains Clare Jolly* and Quentin J. Sattentau The Sir William Dunn School of Pathology, The University of Oxford, Oxford OX1 3RE, United Kingdom Received 24 August 2006/Accepted 15 May 2007 Human immunodeficiency virus type-1 (HIV-1) egress from infected CD4 T cells is thought to be via assembly and budding at the plasma membrane and may involve components of the T-cell secretory apparatus, including tetraspanins. However, many studies on HIV-1 assembly have examined the trafficking of viral proteins in isolation, and most have used immortalized epithelial, fibroblastic, or hematopoietic cell lines that may not necessarily reflect natural infection of susceptible T cells. Here we have used immunofluorescence and cryoimmunoelectron microscopy (CEM) to examine protein transport during HIV-1 assembly in productively infected Jurkat CD4 T cells and primary CD4 T cells. The HIV-1 envelope glycoprotein (Env) and the core protein (Gag) colocalize strongly with CD63 and CD81 and less strongly with CD9, whereas no colocalization was seen between Env or Gag and the late endosome/lysosomal marker Lamp2. CEM revealed incorporation of CD63 and CD81 but not Lamp2 into virions budding at the plasma membrane, and this was supported by immunoprecipitation studies, confirming that HIV-1 egress in T cells is trafficked via tetraspanin-enriched membrane domains (TEMs) that are distinct from lysosomal compartments. CD63, CD81, and, to a lesser extent, CD9 were recruited to the virological synapse (VS), and antibodies against these tetraspanins reduced VS formation. We propose that HIV-1 promotes virus assembly and cell-cell transfer in T cells by targeting plasma membrane TEMs. The major targets of human immunodeficiency virus type 1 (HIV-1) infection in vivo are CD4 T cells and macrophages. In both cell types, infection culminates in release of progeny virions by budding and release; however, the sites at which HIV-1 assembles and buds differ between these two cell types. In CD4 T cells, it is generally accepted that HIV-1 buds from the plasma membrane (13, 15, 16, 23, 27, 38, 39, 41, 43, 49). In contrast, the principal site of viral assembly in macrophages has been proposed to be intracellular vesicles potentially cor- responding to multivesicular bodies (MVBs), where virus may remain infectious for subsequent exocytosis (31, 36, 42, 44, 51, 54), although this has been challenged (30, 63). The molecular pathways regulating the trafficking of HIV-1 proteins have been intensely studied over recent years and have provided many important insights into virus assembly. In infected T cells, both HIV-1 Env and Gag are targeted to GM-1-rich, lipid raft-like plasma membrane, in part by palmi- toylation and myristoylation signals (3, 7, 55, 56). At the plasma membrane, HIV-1 Env associates with the outer leaf-
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