HIV‐1 buds from the surface of activated T lymphocytes. In macrophages, however, newly formed HIV‐1 particles amass in the lumen of an intracellular compartment. Here, we demonstrate by live‐cell imaging techniques, by immunocytochemistry and by immuno‐electron microscopy that HIV‐1 structural proteins, particularly the internal structural protein Gag, accumulate at membranes of the late endocytic compartment in a variety of cell types and not just in monocyte/macrophage‐derived cells. Recent biochemical and genetic studies have implicated components of the mammalian vacuolar protein sorting pathway in retroviral budding. Together with those observations, our study suggests that HIV‐1 morphogenesis is thoroughly rooted in the endosomal system.

Specific spatial arrangements of proteins and lipids are central to the coordination of many biological processes. Tetraspanins have been proposed to laterally organize cellular membranes via specific associations with each other and with distinct integrins. Here, we reveal the presence of tetraspanin-enriched microdomains (TEMs) containing the tetraspanins CD9, CD63, CD81, and CD82 at the plasma membrane. Fluorescence and immunoelectron microscopic analyses document that the surface of HeLa cells is covered by several hundred TEMs, each extending over a few hundred nanometers and containing predominantly two or more tetraspanins. Further, we reveal that the human immunodeficiency virus type 1 (HIV-1) Gag protein, which directs viral assembly and release, accumulates at surface TEMs together with the HIV-1 envelope glycoprotein. TSG101 and VPS28, components of the mammalian ESCRT1 (endosomal sorting complex required for transport), which is part of the cellular extravesiculation machinery critical for HIV-1 budding, are also recruited to cell surface TEMs upon virus expression, suggesting that HIV-1 egress can be gated through these newly mapped microdomains.

S pecifi c spatial arrangements of proteins and lipids are central to the coordination of many biological processes. Tetraspanins have been proposed to laterally organize cellular membranes via specifi c associations with each other and with distinct integrins. Here, we reveal the presence of tetraspanin-enriched microdomains (TEMs) containing the tetraspanins CD9, CD63, CD81, and CD82 at the plasma membrane. Fluorescence and immunoelectron microscopic analyses document that the surface of HeLa cells is covered by several hundred TEMs, each extending over a few hundred nanometers and containing predominantly two or more tetraspanins. Further, we reveal that the human immunodefi ciency virus type 1 (HIV-1) Gag protein, which directs viral assembly and release, accumulates at surface TEMs together with the HIV-1 envelope glycoprotein. TSG101 and VPS28, components of the mammalian ESCRT1 (endosomal sorting complex required for transport), which is part of the cellular extravesiculation machinery critical for HIV-1 budding, are also recruited to cell surface TEMs upon virus expression, suggesting that HIV-1 egress can be gated through these newly mapped microdomains.

Human immunodeficiency virus type 1 (HIV-1) Gag is the primary structural protein of the virus and is sufficient for particle formation. We utilized the recently developed biarsenical-labeling method to dynamically observe HIV-1 Gag within live cells by adding a tetracysteine tag (C-C-P-G-C-C) to the C terminus of Gag in both Pr55Gag expression and full-length proviral constructs. Membrane-permeable biarsenical compounds FlAsH and ReAsH covalently bond to this tetracysteine sequence and specifically fluoresce, effectively labeling Gag in the cell. Biarsenical labeling readily and specifically detected a tetracysteine-tagged HIV-1 Gag protein (Gag-TC) in HeLa, Mel JuSo, and Jurkat T cells by deconvolution fluorescence microscopy. Gag-TC was localized primarily at or near the plasma membrane in all cell types examined. Fluorescent two-color analysis of Gag-TC in HeLa cells revealed that nascent Gag was present mostly at the plasma membrane in distinct regions. Intracellular imaging of a Gag-TC myristylation mutant observed a diffuse signal throughout the cell, consistent with the role of myristylation in Gag localization to the plasma membrane. In contrast, mutation of the L-domain core sequence did not appreciably alter the localization of Gag, suggesting that the PTAP L domain functions at the site of budding rather than as a targeting signal. Taken together, our results show that Gag concentrates in specific plasma membrane areas rapidly after translation and demonstrate the utility of biarsenical labeling for visualizing the dynamic localization of Gag.