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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-containing vacuoles in MDM labeled with antibodies to LAMP-1. (A) Cryosections from MDM infected with HIV-1 Ba-L for 7 d were labeled with a rabbit antiserum to LAMP-1 and PAG10. Note gold labeling associated with the large virus-containing vacuole, including gold particles on individual virions (white arrowheads). However, lysosomal membranes nearby contain significantly more gold particles (arrows). (B) Cryosection double labeled with rabbit anti-LAMP-1/PAG5 and anti-p17/PAG10. The large virus- containing vacuole contains many p17-labeled virions, whereas a few 5 nm gold particles identify associated LAMP-1 (e.g., at the white arrowheads). More strongly LAMP-1–labeled membranes are observed nearby (arrow). (C) A large virus vacuole triple labeled with anti-p17/PAG5, anti-CD63/PAG10, and rabbit anti-LAMP-1/PAG15. Many virions are labeled with the anti-CD63/PAG10 (black arrowheads), and some also contain a few LAMP-1/PAG15 particles, some of which are identified by white arrowheads. The rare, small internal vesicles contain just CD63 (small arrows). CD63 and LAMP-1–labeled membranes are also observed nearby (large arrows). Bars, 100 nm.
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fig6: Virus-containing vacuoles in MDM labeled with antibodies to LAMP-1. (A) Cryosections from MDM infected with HIV-1 Ba-L for 7 d were labeled with a rabbit antiserum to LAMP-1 and PAG10. Note gold labeling associated with the large virus-containing vacuole, including gold particles on individual virions (white arrowheads). However, lysosomal membranes nearby contain significantly more gold particles (arrows). (B) Cryosection double labeled with rabbit anti-LAMP-1/PAG5 and anti-p17/PAG10. The large virus- containing vacuole contains many p17-labeled virions, whereas a few 5 nm gold particles identify associated LAMP-1 (e.g., at the white arrowheads). More strongly LAMP-1–labeled membranes are observed nearby (arrow). (C) A large virus vacuole triple labeled with anti-p17/PAG5, anti-CD63/PAG10, and rabbit anti-LAMP-1/PAG15. Many virions are labeled with the anti-CD63/PAG10 (black arrowheads), and some also contain a few LAMP-1/PAG15 particles, some of which are identified by white arrowheads. The rare, small internal vesicles contain just CD63 (small arrows). CD63 and LAMP-1–labeled membranes are also observed nearby (large arrows). Bars, 100 nm.

Mentions: We also stained MDM with antibodies against lysosomal markers. Although antibodies against LAMP-1, LAMP-2, or the macrophage-specific lysosomal glycoprotein CD68/macrosialin occasionally identified electron-dense lysosomes, the majority of the labeling was found over small vacuoles and membrane tubules, some of which contained internal vesicles or other structures similar to those labeled with anti-CD63. The anti-LAMP-1 antibody showed strong labeling on cryosections and was seen over the virus-containing vacuoles (Fig. 6), occasionally even over viral envelopes (Fig. 6, A and C). However, labeling of the virus-containing vacuoles was weak compared with virus-negative, LAMP-1–labeled membrane tubules and vesicles that were frequently found nearby (Fig. 6, A and B). This was clear in triple labeling, where sections were costained for viral p17, CD63, and LAMP-1 (Fig. 6 C). Although high levels of CD63 were associated with virus vacuoles and found over the virions, the level of LAMP-1 labeling over this compartment was low. This suggests that the CD63-containing virus vacuoles represent a late endosome compartment, which differs from the LAMP-1–enriched membranes nearby. Other lysosomal markers such as LAMP-2 or CD68 were expressed at much lower levels than LAMP-1 in these MDM. Nonetheless, these markers were also occasionally found associated with virus-containing vacuoles (Fig. 7 A; unpublished data).


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

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

Virus-containing vacuoles in MDM labeled with antibodies to LAMP-1. (A) Cryosections from MDM infected with HIV-1 Ba-L for 7 d were labeled with a rabbit antiserum to LAMP-1 and PAG10. Note gold labeling associated with the large virus-containing vacuole, including gold particles on individual virions (white arrowheads). However, lysosomal membranes nearby contain significantly more gold particles (arrows). (B) Cryosection double labeled with rabbit anti-LAMP-1/PAG5 and anti-p17/PAG10. The large virus- containing vacuole contains many p17-labeled virions, whereas a few 5 nm gold particles identify associated LAMP-1 (e.g., at the white arrowheads). More strongly LAMP-1–labeled membranes are observed nearby (arrow). (C) A large virus vacuole triple labeled with anti-p17/PAG5, anti-CD63/PAG10, and rabbit anti-LAMP-1/PAG15. Many virions are labeled with the anti-CD63/PAG10 (black arrowheads), and some also contain a few LAMP-1/PAG15 particles, some of which are identified by white arrowheads. The rare, small internal vesicles contain just CD63 (small arrows). CD63 and LAMP-1–labeled membranes are also observed nearby (large arrows). Bars, 100 nm.
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fig6: Virus-containing vacuoles in MDM labeled with antibodies to LAMP-1. (A) Cryosections from MDM infected with HIV-1 Ba-L for 7 d were labeled with a rabbit antiserum to LAMP-1 and PAG10. Note gold labeling associated with the large virus-containing vacuole, including gold particles on individual virions (white arrowheads). However, lysosomal membranes nearby contain significantly more gold particles (arrows). (B) Cryosection double labeled with rabbit anti-LAMP-1/PAG5 and anti-p17/PAG10. The large virus- containing vacuole contains many p17-labeled virions, whereas a few 5 nm gold particles identify associated LAMP-1 (e.g., at the white arrowheads). More strongly LAMP-1–labeled membranes are observed nearby (arrow). (C) A large virus vacuole triple labeled with anti-p17/PAG5, anti-CD63/PAG10, and rabbit anti-LAMP-1/PAG15. Many virions are labeled with the anti-CD63/PAG10 (black arrowheads), and some also contain a few LAMP-1/PAG15 particles, some of which are identified by white arrowheads. The rare, small internal vesicles contain just CD63 (small arrows). CD63 and LAMP-1–labeled membranes are also observed nearby (large arrows). Bars, 100 nm.
Mentions: We also stained MDM with antibodies against lysosomal markers. Although antibodies against LAMP-1, LAMP-2, or the macrophage-specific lysosomal glycoprotein CD68/macrosialin occasionally identified electron-dense lysosomes, the majority of the labeling was found over small vacuoles and membrane tubules, some of which contained internal vesicles or other structures similar to those labeled with anti-CD63. The anti-LAMP-1 antibody showed strong labeling on cryosections and was seen over the virus-containing vacuoles (Fig. 6), occasionally even over viral envelopes (Fig. 6, A and C). However, labeling of the virus-containing vacuoles was weak compared with virus-negative, LAMP-1–labeled membrane tubules and vesicles that were frequently found nearby (Fig. 6, A and B). This was clear in triple labeling, where sections were costained for viral p17, CD63, and LAMP-1 (Fig. 6 C). Although high levels of CD63 were associated with virus vacuoles and found over the virions, the level of LAMP-1 labeling over this compartment was low. This suggests that the CD63-containing virus vacuoles represent a late endosome compartment, which differs from the LAMP-1–enriched membranes nearby. Other lysosomal markers such as LAMP-2 or CD68 were expressed at much lower levels than LAMP-1 in these MDM. Nonetheless, these markers were also occasionally found associated with virus-containing vacuoles (Fig. 7 A; unpublished data).

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