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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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Virus precipitation with antibodies against cellular proteins. Cell-free supernatants of MDM-derived HIV-1 Ba-L were precipitated with the antibodies indicated. Unprecipitated supernatants were analyzed for remaining infectious virus by a focus-forming assay on NP-2 CD4/CCR5 cells (A). Pellets were analyzed for precipitated viral protein by p24 ELISA (B).
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fig8: Virus precipitation with antibodies against cellular proteins. Cell-free supernatants of MDM-derived HIV-1 Ba-L were precipitated with the antibodies indicated. Unprecipitated supernatants were analyzed for remaining infectious virus by a focus-forming assay on NP-2 CD4/CCR5 cells (A). Pellets were analyzed for precipitated viral protein by p24 ELISA (B).

Mentions: Nonspecific precipitation of infectious virus was low, as the anti-VSV-G antibody produced only a small (<10%) reduction in the number of infectious focus-forming units (FFU; unpublished data). By contrast, precipitation with the anti-Env antibody completely eliminated infectivity in the supernatant. The antibody against the cellular protein HLA-DR also precipitated a significant amount of infectious virus, in agreement with previous reports that this protein is incorporated into the HIV-1 envelope (Esser et al., 2001; Raposo et al., 2002). Of the antibodies against tetraspanin markers, anti-CD63 showed the most efficient removal of infectious virus from the supernatant; this was observed with two different anti-CD63 antibodies (Table I). The other tetraspanin antibodies, anti-CD81, -CD82, -CD53, and -CD151 also precipitated some of the infectious virus, though much less efficiently, suggesting that not all virus particles incorporated sufficient levels of these molecules into their envelopes for immunoadsorption to occur (Fig. 8 A). Precipitation with antibodies against LAMP proteins showed that LAMP-1 was incorporated into some of the infectious virus, but LAMP-2 was not detectable (Table I). Antibodies against well-characterized cell surface proteins, including GPI-anchored proteins (CD14, CD52, and CD55) and various adhesion proteins (CD11a and CD169), did not precipitate significant amounts of infectious virus. However, some HIV infectivity was removed by antibodies to the GPI-anchored protein CD59, and by CD54 (ICAM-1; Fig. 8 A and Table I).


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

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

Virus precipitation with antibodies against cellular proteins. Cell-free supernatants of MDM-derived HIV-1 Ba-L were precipitated with the antibodies indicated. Unprecipitated supernatants were analyzed for remaining infectious virus by a focus-forming assay on NP-2 CD4/CCR5 cells (A). Pellets were analyzed for precipitated viral protein by p24 ELISA (B).
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Virus precipitation with antibodies against cellular proteins. Cell-free supernatants of MDM-derived HIV-1 Ba-L were precipitated with the antibodies indicated. Unprecipitated supernatants were analyzed for remaining infectious virus by a focus-forming assay on NP-2 CD4/CCR5 cells (A). Pellets were analyzed for precipitated viral protein by p24 ELISA (B).
Mentions: Nonspecific precipitation of infectious virus was low, as the anti-VSV-G antibody produced only a small (<10%) reduction in the number of infectious focus-forming units (FFU; unpublished data). By contrast, precipitation with the anti-Env antibody completely eliminated infectivity in the supernatant. The antibody against the cellular protein HLA-DR also precipitated a significant amount of infectious virus, in agreement with previous reports that this protein is incorporated into the HIV-1 envelope (Esser et al., 2001; Raposo et al., 2002). Of the antibodies against tetraspanin markers, anti-CD63 showed the most efficient removal of infectious virus from the supernatant; this was observed with two different anti-CD63 antibodies (Table I). The other tetraspanin antibodies, anti-CD81, -CD82, -CD53, and -CD151 also precipitated some of the infectious virus, though much less efficiently, suggesting that not all virus particles incorporated sufficient levels of these molecules into their envelopes for immunoadsorption to occur (Fig. 8 A). Precipitation with antibodies against LAMP proteins showed that LAMP-1 was incorporated into some of the infectious virus, but LAMP-2 was not detectable (Table I). Antibodies against well-characterized cell surface proteins, including GPI-anchored proteins (CD14, CD52, and CD55) and various adhesion proteins (CD11a and CD169), did not precipitate significant amounts of infectious virus. However, some HIV infectivity was removed by antibodies to the GPI-anchored protein CD59, and by CD54 (ICAM-1; Fig. 8 A and Table I).

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