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Infectious HIV-1 assembles in late endosomes in primary macrophages.

Pelchen-Matthews A, Kramer B, Marsh M - J. Cell Biol. (2003)

Bottom Line: Only antibodies against antigens found in late endosomes precipitated infectious virus, whereas antibodies against proteins located primarily on the cell surface did not.Our data indicate that most of the infectious HIV produced by primary macrophages is assembled on late endocytic membranes and acquires antigens characteristic of this compartment.This notion has significant implications for understanding the biology of HIV and its cell-cell transmission.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Unit, Medical Research (MRC) Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK.

ABSTRACT
Although human immunodeficiency virus type 1 (HIV-1) is generally thought to assemble at the plasma membrane of infected cells, virions have been observed in intracellular compartments in macrophages. Here, we investigated virus assembly in HIV-1-infected primary human monocyte-derived macrophages (MDM). Electron microscopy of cryosections showed virus particles, identified by their morphology and positive labeling with antibodies to the viral p17, p24, and envelope proteins, in intracellular vacuoles. Immunolabeling demonstrated that these compartments contained the late endosomal marker CD63, which was enriched on vesicles within these structures and incorporated into the envelope of budding virions. The virus-containing vacuoles were also labeled with antibodies against LAMP-1, CD81, and CD82, which were also incorporated into the viral envelope. To assess the cellular source of infectious viruses derived from MDM, virus-containing media from infected cells were precipitated with specific antibodies. Only antibodies against antigens found in late endosomes precipitated infectious virus, whereas antibodies against proteins located primarily on the cell surface did not. Our data indicate that most of the infectious HIV produced by primary macrophages is assembled on late endocytic membranes and acquires antigens characteristic of this compartment. This notion has significant implications for understanding the biology of HIV and its cell-cell transmission.

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Exocytosis of virions on infected MDM. Plasma membrane invaginations probably resulting from the fusion of intracellular virus-containing vacuoles with the plasma membrane. Cryosection in A was double labeled for LAMP-1/PAG5 and p17/PAG10, whereas B was triple labeled for p17/PAG5, CD63/PAG10, and LAMP-1/PAG15. Although these invaginations are continuous with the cell surface, they show labeling for LAMP-1 (white arrowheads) and CD63. Virions are interspersed with many small vesicles (black arrows) that are strongly labeled for CD63 (B, black arrows). Also, note the thick electron-dense coats on some parts of these invaginations (black arrowheads). Bars, 100 nm.
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fig9: Exocytosis of virions on infected MDM. Plasma membrane invaginations probably resulting from the fusion of intracellular virus-containing vacuoles with the plasma membrane. Cryosection in A was double labeled for LAMP-1/PAG5 and p17/PAG10, whereas B was triple labeled for p17/PAG5, CD63/PAG10, and LAMP-1/PAG15. Although these invaginations are continuous with the cell surface, they show labeling for LAMP-1 (white arrowheads) and CD63. Virions are interspersed with many small vesicles (black arrows) that are strongly labeled for CD63 (B, black arrows). Also, note the thick electron-dense coats on some parts of these invaginations (black arrowheads). Bars, 100 nm.

Mentions: In hematopoietic cells, late endocytic compartments can function as secretory organelles (Blott and Griffiths, 2002). Moreover, MIICs have been proposed to fuse with the plasma membrane, either directly or via membrane tubules (Raposo et al., 1996; Boes et al., 2002; Chow et al., 2002). Our EM observations of infected MDM suggested that such a mechanism might provide a route for virus release. Virions were not generally found on the surface of the infected cells, but occasionally accumulations of virions were seen in invaginations or deep pockets of the plasma membrane (Fig. 9). The virions in these invaginations were often interspersed with small vesicles resembling exosomes, the MVB-derived vesicles released by certain hematopoietic cells when their MVBs fuse with the plasma membrane (Stoorvogel et al., 2002; Thery et al., 2002), which could be labeled with antibodies against LAMP-1 and CD63 (Fig. 9). This suggests that the plasma membrane pockets represent exocytic profiles resulting from the fusion of the virus-containing vacuoles with the cell surface. Thus, in human MDM, infectious HIV-1 is produced in intracellular compartments with features of late endosomes or MVBs, and these viruses can be released to the extracellular medium by secretion.


Infectious HIV-1 assembles in late endosomes in primary macrophages.

Pelchen-Matthews A, Kramer B, Marsh M - J. Cell Biol. (2003)

Exocytosis of virions on infected MDM. Plasma membrane invaginations probably resulting from the fusion of intracellular virus-containing vacuoles with the plasma membrane. Cryosection in A was double labeled for LAMP-1/PAG5 and p17/PAG10, whereas B was triple labeled for p17/PAG5, CD63/PAG10, and LAMP-1/PAG15. Although these invaginations are continuous with the cell surface, they show labeling for LAMP-1 (white arrowheads) and CD63. Virions are interspersed with many small vesicles (black arrows) that are strongly labeled for CD63 (B, black arrows). Also, note the thick electron-dense coats on some parts of these invaginations (black arrowheads). Bars, 100 nm.
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Related In: Results  -  Collection

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fig9: Exocytosis of virions on infected MDM. Plasma membrane invaginations probably resulting from the fusion of intracellular virus-containing vacuoles with the plasma membrane. Cryosection in A was double labeled for LAMP-1/PAG5 and p17/PAG10, whereas B was triple labeled for p17/PAG5, CD63/PAG10, and LAMP-1/PAG15. Although these invaginations are continuous with the cell surface, they show labeling for LAMP-1 (white arrowheads) and CD63. Virions are interspersed with many small vesicles (black arrows) that are strongly labeled for CD63 (B, black arrows). Also, note the thick electron-dense coats on some parts of these invaginations (black arrowheads). Bars, 100 nm.
Mentions: In hematopoietic cells, late endocytic compartments can function as secretory organelles (Blott and Griffiths, 2002). Moreover, MIICs have been proposed to fuse with the plasma membrane, either directly or via membrane tubules (Raposo et al., 1996; Boes et al., 2002; Chow et al., 2002). Our EM observations of infected MDM suggested that such a mechanism might provide a route for virus release. Virions were not generally found on the surface of the infected cells, but occasionally accumulations of virions were seen in invaginations or deep pockets of the plasma membrane (Fig. 9). The virions in these invaginations were often interspersed with small vesicles resembling exosomes, the MVB-derived vesicles released by certain hematopoietic cells when their MVBs fuse with the plasma membrane (Stoorvogel et al., 2002; Thery et al., 2002), which could be labeled with antibodies against LAMP-1 and CD63 (Fig. 9). This suggests that the plasma membrane pockets represent exocytic profiles resulting from the fusion of the virus-containing vacuoles with the cell surface. Thus, in human MDM, infectious HIV-1 is produced in intracellular compartments with features of late endosomes or MVBs, and these viruses can be released to the extracellular medium by secretion.

Bottom Line: Only antibodies against antigens found in late endosomes precipitated infectious virus, whereas antibodies against proteins located primarily on the cell surface did not.Our data indicate that most of the infectious HIV produced by primary macrophages is assembled on late endocytic membranes and acquires antigens characteristic of this compartment.This notion has significant implications for understanding the biology of HIV and its cell-cell transmission.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Unit, Medical Research (MRC) Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK.

ABSTRACT
Although human immunodeficiency virus type 1 (HIV-1) is generally thought to assemble at the plasma membrane of infected cells, virions have been observed in intracellular compartments in macrophages. Here, we investigated virus assembly in HIV-1-infected primary human monocyte-derived macrophages (MDM). Electron microscopy of cryosections showed virus particles, identified by their morphology and positive labeling with antibodies to the viral p17, p24, and envelope proteins, in intracellular vacuoles. Immunolabeling demonstrated that these compartments contained the late endosomal marker CD63, which was enriched on vesicles within these structures and incorporated into the envelope of budding virions. The virus-containing vacuoles were also labeled with antibodies against LAMP-1, CD81, and CD82, which were also incorporated into the viral envelope. To assess the cellular source of infectious viruses derived from MDM, virus-containing media from infected cells were precipitated with specific antibodies. Only antibodies against antigens found in late endosomes precipitated infectious virus, whereas antibodies against proteins located primarily on the cell surface did not. Our data indicate that most of the infectious HIV produced by primary macrophages is assembled on late endocytic membranes and acquires antigens characteristic of this compartment. This notion has significant implications for understanding the biology of HIV and its cell-cell transmission.

Show MeSH
Related in: MedlinePlus