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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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HIV-1 virions in MDM accumulate in CD63-containing compartments. Cryosections from MDM infected with HIV-1 Ba-L for 14 d were double labeled for p24 with PAG5 and for CD63 with PAG15. (A) Overview of a large complex intracellular vacuole filled with virions and clusters of small vesicles containing CD63 (white arrowheads). Virions are identified by PAG5 and are frequently labeled with the larger gold particles marking CD63 (e.g., at the large arrows), whereas the small vesicles are labeled only with CD63/PAG15. (B) A smaller vacuole containing virus particles also labeled with CD63/PAG15. Note the electron-dense coat (black arrowheads). (C) Virions budding into a complex vacuole labeled with anti-CD63. Small arrows indicate budding virus particles incorporating CD63 into their membranes. Bars, 100 nm.
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fig5: HIV-1 virions in MDM accumulate in CD63-containing compartments. Cryosections from MDM infected with HIV-1 Ba-L for 14 d were double labeled for p24 with PAG5 and for CD63 with PAG15. (A) Overview of a large complex intracellular vacuole filled with virions and clusters of small vesicles containing CD63 (white arrowheads). Virions are identified by PAG5 and are frequently labeled with the larger gold particles marking CD63 (e.g., at the large arrows), whereas the small vesicles are labeled only with CD63/PAG15. (B) A smaller vacuole containing virus particles also labeled with CD63/PAG15. Note the electron-dense coat (black arrowheads). (C) Virions budding into a complex vacuole labeled with anti-CD63. Small arrows indicate budding virus particles incorporating CD63 into their membranes. Bars, 100 nm.

Mentions: To identify the intracellular vacuoles into which the Env/p24-labeled virus particles were budding, we colabeled ultrathin cryosections with antibodies against various intracellular markers. Recent analyses of HIV-1–infected macrophages indicated that the MHCII protein HLA-DR is incorporated into virions budding into MIICs (Raposo et al., 2002). Because MIICs are a specialized late endosome compartment, we stained infected MDM with antibodies against CD63, a tetraspanin found in late endosomes and MVBs, particularly in the membranes of the internal vesicles. In macrophages, a small amount of CD63 is also found at the plasma membrane. On MDM cryosections, virus-containing vacuoles were strongly labeled with anti-CD63 (Fig. 5). Gold particles identified CD63 within the envelope of individual virions, as well as over small membrane vesicles similar to the internal vesicles of MVBs (Fig. 5 A). This suggests that the compartment into which HIV-1 is budding in MDM is equivalent to the late endosome/MVB compartment. The MDM used in these analyses did not contain many classical MVBs, with numerous internal vesicles, similar to those found in tissue culture cell lines. Instead, anti-CD63 strongly labeled various small membrane vacuoles, vesicles, and tubular profiles that contained few internal vesicles. Virions were seen to bud into these compartments, incorporating CD63 into their membrane envelope as they assembled at the limiting membrane (Fig. 5 C).


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

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

HIV-1 virions in MDM accumulate in CD63-containing compartments. Cryosections from MDM infected with HIV-1 Ba-L for 14 d were double labeled for p24 with PAG5 and for CD63 with PAG15. (A) Overview of a large complex intracellular vacuole filled with virions and clusters of small vesicles containing CD63 (white arrowheads). Virions are identified by PAG5 and are frequently labeled with the larger gold particles marking CD63 (e.g., at the large arrows), whereas the small vesicles are labeled only with CD63/PAG15. (B) A smaller vacuole containing virus particles also labeled with CD63/PAG15. Note the electron-dense coat (black arrowheads). (C) Virions budding into a complex vacuole labeled with anti-CD63. Small arrows indicate budding virus particles incorporating CD63 into their membranes. Bars, 100 nm.
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Related In: Results  -  Collection

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fig5: HIV-1 virions in MDM accumulate in CD63-containing compartments. Cryosections from MDM infected with HIV-1 Ba-L for 14 d were double labeled for p24 with PAG5 and for CD63 with PAG15. (A) Overview of a large complex intracellular vacuole filled with virions and clusters of small vesicles containing CD63 (white arrowheads). Virions are identified by PAG5 and are frequently labeled with the larger gold particles marking CD63 (e.g., at the large arrows), whereas the small vesicles are labeled only with CD63/PAG15. (B) A smaller vacuole containing virus particles also labeled with CD63/PAG15. Note the electron-dense coat (black arrowheads). (C) Virions budding into a complex vacuole labeled with anti-CD63. Small arrows indicate budding virus particles incorporating CD63 into their membranes. Bars, 100 nm.
Mentions: To identify the intracellular vacuoles into which the Env/p24-labeled virus particles were budding, we colabeled ultrathin cryosections with antibodies against various intracellular markers. Recent analyses of HIV-1–infected macrophages indicated that the MHCII protein HLA-DR is incorporated into virions budding into MIICs (Raposo et al., 2002). Because MIICs are a specialized late endosome compartment, we stained infected MDM with antibodies against CD63, a tetraspanin found in late endosomes and MVBs, particularly in the membranes of the internal vesicles. In macrophages, a small amount of CD63 is also found at the plasma membrane. On MDM cryosections, virus-containing vacuoles were strongly labeled with anti-CD63 (Fig. 5). Gold particles identified CD63 within the envelope of individual virions, as well as over small membrane vesicles similar to the internal vesicles of MVBs (Fig. 5 A). This suggests that the compartment into which HIV-1 is budding in MDM is equivalent to the late endosome/MVB compartment. The MDM used in these analyses did not contain many classical MVBs, with numerous internal vesicles, similar to those found in tissue culture cell lines. Instead, anti-CD63 strongly labeled various small membrane vacuoles, vesicles, and tubular profiles that contained few internal vesicles. Virions were seen to bud into these compartments, incorporating CD63 into their membrane envelope as they assembled at the limiting membrane (Fig. 5 C).

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