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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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Intracellular HIV-1 in MDM. Cryosections from MDM infected with HIV-1 Ba-L were stained with anti-p17 (A and A′) or anti-p24 antibodies (B and B′) and 10 nm PAG. Alternatively, sections were double labeled for p24 (PAG 5 nm) and p17 (PAG 10 nm) (C and C′). Virus particles were found primarily in intracellular vacuoles at all times, i.e., in these cells infected for 7 (B′), 12 (A′), 14 (A and B), or 20 d (C and C′). Arrows identify budding or immature virions in A′, B′, or C′. D shows a cell infected with HIV-1 SF162 for 14 d, stained with anti-p24 and PAG10. Note the small unlabeled internal vesicles (C and D, white arrowheads) and the flat coat on some of these vacuoles (B and D, black arrowheads). Bars, 100 nm.
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fig3: Intracellular HIV-1 in MDM. Cryosections from MDM infected with HIV-1 Ba-L were stained with anti-p17 (A and A′) or anti-p24 antibodies (B and B′) and 10 nm PAG. Alternatively, sections were double labeled for p24 (PAG 5 nm) and p17 (PAG 10 nm) (C and C′). Virus particles were found primarily in intracellular vacuoles at all times, i.e., in these cells infected for 7 (B′), 12 (A′), 14 (A and B), or 20 d (C and C′). Arrows identify budding or immature virions in A′, B′, or C′. D shows a cell infected with HIV-1 SF162 for 14 d, stained with anti-p24 and PAG10. Note the small unlabeled internal vesicles (C and D, white arrowheads) and the flat coat on some of these vacuoles (B and D, black arrowheads). Bars, 100 nm.

Mentions: To study the intracellular viral antigens in more detail, ultrathin cryosections (∼50 nm) of infected MDM were labeled with antibodies against p17 or p24 and protein A gold (PAG) and observed by EM. These antibodies stained electron-dense 100–110-nm membrane-containing particles, often with truncated cone-shaped cores typical of mature HIV-1 (Fig. 3). Anti-p17 antibody labeling was often seen close to the membrane of these particles (Fig. 3 A), but was absent over budding figures or particles with the appearance of immature virions, consistent with this antibody recognizing only the cleaved Gag protein (Fig. 3 A′; Ferns et al., 1987). Anti-p24 showed strong labeling over particles with the appearance of both immature and mature virions, with up to 20–30 gold particles per virus particle (Fig. 3, B and B′). The different distributions of the p17 and p24 antigen were confirmed on sections double labeled with these antibodies (Fig. 3, C and C′).


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

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

Intracellular HIV-1 in MDM. Cryosections from MDM infected with HIV-1 Ba-L were stained with anti-p17 (A and A′) or anti-p24 antibodies (B and B′) and 10 nm PAG. Alternatively, sections were double labeled for p24 (PAG 5 nm) and p17 (PAG 10 nm) (C and C′). Virus particles were found primarily in intracellular vacuoles at all times, i.e., in these cells infected for 7 (B′), 12 (A′), 14 (A and B), or 20 d (C and C′). Arrows identify budding or immature virions in A′, B′, or C′. D shows a cell infected with HIV-1 SF162 for 14 d, stained with anti-p24 and PAG10. Note the small unlabeled internal vesicles (C and D, white arrowheads) and the flat coat on some of these vacuoles (B and D, black arrowheads). Bars, 100 nm.
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fig3: Intracellular HIV-1 in MDM. Cryosections from MDM infected with HIV-1 Ba-L were stained with anti-p17 (A and A′) or anti-p24 antibodies (B and B′) and 10 nm PAG. Alternatively, sections were double labeled for p24 (PAG 5 nm) and p17 (PAG 10 nm) (C and C′). Virus particles were found primarily in intracellular vacuoles at all times, i.e., in these cells infected for 7 (B′), 12 (A′), 14 (A and B), or 20 d (C and C′). Arrows identify budding or immature virions in A′, B′, or C′. D shows a cell infected with HIV-1 SF162 for 14 d, stained with anti-p24 and PAG10. Note the small unlabeled internal vesicles (C and D, white arrowheads) and the flat coat on some of these vacuoles (B and D, black arrowheads). Bars, 100 nm.
Mentions: To study the intracellular viral antigens in more detail, ultrathin cryosections (∼50 nm) of infected MDM were labeled with antibodies against p17 or p24 and protein A gold (PAG) and observed by EM. These antibodies stained electron-dense 100–110-nm membrane-containing particles, often with truncated cone-shaped cores typical of mature HIV-1 (Fig. 3). Anti-p17 antibody labeling was often seen close to the membrane of these particles (Fig. 3 A), but was absent over budding figures or particles with the appearance of immature virions, consistent with this antibody recognizing only the cleaved Gag protein (Fig. 3 A′; Ferns et al., 1987). Anti-p24 showed strong labeling over particles with the appearance of both immature and mature virions, with up to 20–30 gold particles per virus particle (Fig. 3, B and B′). The different distributions of the p17 and p24 antigen were confirmed on sections double labeled with these antibodies (Fig. 3, C and 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