Limits...
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.

Show MeSH

Related in: MedlinePlus

Overview of anti-p24 and -p17 staining on HIV-infected MDM. Semi-thin cryosections of MDM infected with HIV-1 Ba-L and labeled with anti-p24 (A–D), or infected with HIV-1 SF162 and labeled with anti-p17 (E–G), were stained with Alexa® Fluor 488 goat anti–mouse IgG. Cells had been infected for 7 (A), 12 (B), 15 (C), and 20 d (D); or 14 (E), 21 (F), and 30 d (G). Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172706&req=5

fig2: Overview of anti-p24 and -p17 staining on HIV-infected MDM. Semi-thin cryosections of MDM infected with HIV-1 Ba-L and labeled with anti-p24 (A–D), or infected with HIV-1 SF162 and labeled with anti-p17 (E–G), were stained with Alexa® Fluor 488 goat anti–mouse IgG. Cells had been infected for 7 (A), 12 (B), 15 (C), and 20 d (D); or 14 (E), 21 (F), and 30 d (G). Bars, 10 μm.

Mentions: To study virus assembly, MDM infected with HIV-1 Ba-L for various times were prepared for cryosection immunolabeling, and semi-thin sections (0.5 μm) were analyzed for expression of viral antigens by fluorescence microscopy. Viral capsid (p24) and matrix (p17) antigens were detected in a large proportion of the cells at all times. At 7 to 20 d, staining with the anti-p24 antibody, which also detects the Gag polyprotein precursor p55, was seen associated with intracellular granules (Fig. 2, A–D), and only rarely were small spots or patches of fluorescence detected at or close to the cell surface. Similar results were obtained with MDM infected with another macrophage-tropic HIV-1 strain (SF162) for 14, 21, or 30 d. Labeling with an anti-matrix (p17) mAb identified this viral antigen in intracellular spots and clusters (Fig. 2, E–G), but not at the cell surface. Thus, HIV-1 appears to accumulate predominantly intracellularly in MDM, in agreement with previous reports (Orenstein et al., 1988; Raposo et al., 2002). Similar results were obtained with MDM infected with the SL-2 and JR-FL strains of HIV-1, though the proportion of infected cells in these cultures was lower.


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

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

Overview of anti-p24 and -p17 staining on HIV-infected MDM. Semi-thin cryosections of MDM infected with HIV-1 Ba-L and labeled with anti-p24 (A–D), or infected with HIV-1 SF162 and labeled with anti-p17 (E–G), were stained with Alexa® Fluor 488 goat anti–mouse IgG. Cells had been infected for 7 (A), 12 (B), 15 (C), and 20 d (D); or 14 (E), 21 (F), and 30 d (G). Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Overview of anti-p24 and -p17 staining on HIV-infected MDM. Semi-thin cryosections of MDM infected with HIV-1 Ba-L and labeled with anti-p24 (A–D), or infected with HIV-1 SF162 and labeled with anti-p17 (E–G), were stained with Alexa® Fluor 488 goat anti–mouse IgG. Cells had been infected for 7 (A), 12 (B), 15 (C), and 20 d (D); or 14 (E), 21 (F), and 30 d (G). Bars, 10 μm.
Mentions: To study virus assembly, MDM infected with HIV-1 Ba-L for various times were prepared for cryosection immunolabeling, and semi-thin sections (0.5 μm) were analyzed for expression of viral antigens by fluorescence microscopy. Viral capsid (p24) and matrix (p17) antigens were detected in a large proportion of the cells at all times. At 7 to 20 d, staining with the anti-p24 antibody, which also detects the Gag polyprotein precursor p55, was seen associated with intracellular granules (Fig. 2, A–D), and only rarely were small spots or patches of fluorescence detected at or close to the cell surface. Similar results were obtained with MDM infected with another macrophage-tropic HIV-1 strain (SF162) for 14, 21, or 30 d. Labeling with an anti-matrix (p17) mAb identified this viral antigen in intracellular spots and clusters (Fig. 2, E–G), but not at the cell surface. Thus, HIV-1 appears to accumulate predominantly intracellularly in MDM, in agreement with previous reports (Orenstein et al., 1988; Raposo et al., 2002). Similar results were obtained with MDM infected with the SL-2 and JR-FL strains of HIV-1, though the proportion of infected cells in these cultures was lower.

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