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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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Time course of HIV-1 Ba-L production in MDM. Human MDM were infected with 9.3 × 105 focus-forming units (FFU) of HIV-1 Ba-L. Supernatants were collected daily and analyzed for (A) p24 content (○) or reverse transcriptase (•) and (B) infectivity on NP-2 CD4/CCR5 indicator cells. Activities are corrected for the dilution effect of feeding.
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fig1: Time course of HIV-1 Ba-L production in MDM. Human MDM were infected with 9.3 × 105 focus-forming units (FFU) of HIV-1 Ba-L. Supernatants were collected daily and analyzed for (A) p24 content (○) or reverse transcriptase (•) and (B) infectivity on NP-2 CD4/CCR5 indicator cells. Activities are corrected for the dilution effect of feeding.

Mentions: To assess the time course of the infections, supernatants were collected from MDM cultures infected with HIV-1 Ba-L, and the amount of released virus was determined by measuring levels of viral capsid, p24, or reverse transcriptase activity (Fig. 1 A). The infectivity of the released viruses was tested in a focus-forming assay after infection of susceptible NP-2 target cells (Fig. 1 B). Virus could be detected in the MDM medium as early as 3 d after infection and accumulated rapidly over the following days. The amount of infectious virus released reached a maximum at 8 d after infection and was maintained thereafter. Measurement of p24 concentration and reverse transcriptase activity demonstrated that the cells continued to release virus for a further two weeks.


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

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

Time course of HIV-1 Ba-L production in MDM. Human MDM were infected with 9.3 × 105 focus-forming units (FFU) of HIV-1 Ba-L. Supernatants were collected daily and analyzed for (A) p24 content (○) or reverse transcriptase (•) and (B) infectivity on NP-2 CD4/CCR5 indicator cells. Activities are corrected for the dilution effect of feeding.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Time course of HIV-1 Ba-L production in MDM. Human MDM were infected with 9.3 × 105 focus-forming units (FFU) of HIV-1 Ba-L. Supernatants were collected daily and analyzed for (A) p24 content (○) or reverse transcriptase (•) and (B) infectivity on NP-2 CD4/CCR5 indicator cells. Activities are corrected for the dilution effect of feeding.
Mentions: To assess the time course of the infections, supernatants were collected from MDM cultures infected with HIV-1 Ba-L, and the amount of released virus was determined by measuring levels of viral capsid, p24, or reverse transcriptase activity (Fig. 1 A). The infectivity of the released viruses was tested in a focus-forming assay after infection of susceptible NP-2 target cells (Fig. 1 B). Virus could be detected in the MDM medium as early as 3 d after infection and accumulated rapidly over the following days. The amount of infectious virus released reached a maximum at 8 d after infection and was maintained thereafter. Measurement of p24 concentration and reverse transcriptase activity demonstrated that the cells continued to release virus for a further two weeks.

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