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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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Detection of HIV-1 envelope on intracellular HIV-1 virions in MDM. Cryosections from MDM infected with HIV-1 Ba-L for 14 d were stained with anti-Env 2G12 and PAG10 (A) or double labeled for p24 with PAG5 and Env with PAG10 (B). The inset in A shows a detailed view with gold particles over the viral membrane on equatorially sectioned virions, or all over virions cut more tangentially. Bars, 100 nm.
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fig4: Detection of HIV-1 envelope on intracellular HIV-1 virions in MDM. Cryosections from MDM infected with HIV-1 Ba-L for 14 d were stained with anti-Env 2G12 and PAG10 (A) or double labeled for p24 with PAG5 and Env with PAG10 (B). The inset in A shows a detailed view with gold particles over the viral membrane on equatorially sectioned virions, or all over virions cut more tangentially. Bars, 100 nm.

Mentions: When cryosections of HIV-1 Ba-L–infected MDM were stained with an antibody against the gp120 component of HIV-1 Env (2G12), strong labeling was observed over intracellular particles morphologically identical to those labeled for p24 and/or p17 (Fig. 4 A). These particles were labeled with, on average, five gold particles per virion, suggesting that they had incorporated significant levels of Env. Similarly, viruses could be labeled with another anti-Env antibody (b12), though the labeling was not as strong (unpublished data). Double staining confirmed that Env was enriched on p24-containing particles (Fig. 4 B), consistent with the possibility that these particles are infectious viruses. On the 2G12-stained cryosections, the intracellular virus particles were by far the most strongly labeled structures. However, some labeling was seen on other structures including various tubules, vacuoles, or membrane cisternae, often found in the vicinity of the Golgi apparatus/TGN. Only low levels of labeling were observed at the plasma membrane.


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

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

Detection of HIV-1 envelope on intracellular HIV-1 virions in MDM. Cryosections from MDM infected with HIV-1 Ba-L for 14 d were stained with anti-Env 2G12 and PAG10 (A) or double labeled for p24 with PAG5 and Env with PAG10 (B). The inset in A shows a detailed view with gold particles over the viral membrane on equatorially sectioned virions, or all over virions cut more tangentially. Bars, 100 nm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172706&req=5

fig4: Detection of HIV-1 envelope on intracellular HIV-1 virions in MDM. Cryosections from MDM infected with HIV-1 Ba-L for 14 d were stained with anti-Env 2G12 and PAG10 (A) or double labeled for p24 with PAG5 and Env with PAG10 (B). The inset in A shows a detailed view with gold particles over the viral membrane on equatorially sectioned virions, or all over virions cut more tangentially. Bars, 100 nm.
Mentions: When cryosections of HIV-1 Ba-L–infected MDM were stained with an antibody against the gp120 component of HIV-1 Env (2G12), strong labeling was observed over intracellular particles morphologically identical to those labeled for p24 and/or p17 (Fig. 4 A). These particles were labeled with, on average, five gold particles per virion, suggesting that they had incorporated significant levels of Env. Similarly, viruses could be labeled with another anti-Env antibody (b12), though the labeling was not as strong (unpublished data). Double staining confirmed that Env was enriched on p24-containing particles (Fig. 4 B), consistent with the possibility that these particles are infectious viruses. On the 2G12-stained cryosections, the intracellular virus particles were by far the most strongly labeled structures. However, some labeling was seen on other structures including various tubules, vacuoles, or membrane cisternae, often found in the vicinity of the Golgi apparatus/TGN. Only low levels of labeling were observed at the plasma membrane.

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