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The nucleocapsid region of HIV-1 Gag cooperates with the PTAP and LYPXnL late domains to recruit the cellular machinery necessary for viral budding.

Dussupt V, Javid MP, Abou-Jaoudé G, Jadwin JA, de La Cruz J, Nagashima K, Bouamr F - PLoS Pathog. (2009)

Bottom Line: Remarkably, we found that over-expression of Bro1 rescued the release of HIV-1 lacking both L domains.This rescue required the N-terminal region of the NC domain in Gag and the CHMP4 binding site in Bro1.Interestingly, release defects due to mutations in NC that prevented Bro1 mediated rescue of virus egress were rescued by providing a link to the ESCRT machinery via Nedd4.2s over-expression.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
HIV-1 release is mediated through two motifs in the p6 region of Gag, PTAP and LYPX(n)L, which recruit cellular proteins Tsg101 and Alix, respectively. The Nucleocapsid region of Gag (NC), which binds the Bro1 domain of Alix, also plays an important role in HIV-1 release, but the underlying mechanism remains unclear. Here we show that the first 202 residues of the Bro1 domain (Bro(i)) are sufficient to bind Gag. Bro(i) interferes with HIV-1 release in an NC-dependent manner and arrests viral budding at the plasma membrane. Similar interrupted budding structures are seen following over-expression of a fragment containing Bro1 with the adjacent V domain (Bro1-V). Although only Bro1-V contains binding determinants for CHMP4, both Bro(i) and Bro1-V inhibited release via both the PTAP/Tsg101 and the LYPX(n)L/Alix pathways, suggesting that they interfere with a key step in HIV-1 release. Remarkably, we found that over-expression of Bro1 rescued the release of HIV-1 lacking both L domains. This rescue required the N-terminal region of the NC domain in Gag and the CHMP4 binding site in Bro1. Interestingly, release defects due to mutations in NC that prevented Bro1 mediated rescue of virus egress were rescued by providing a link to the ESCRT machinery via Nedd4.2s over-expression. Our data support a model in which NC cooperates with PTAP in the recruitment of cellular proteins necessary for its L domain activity and binds the Bro1-CHMP4 complex required for LYPX(n)L-mediated budding.

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Broi is recruited by Gag to the plasma membrane and interferes with HIV-1 budding.(A) First two upper left panels show a cell expressing HA-Broi (white). The next panel shows a reconstructed 3D view of a stack of Z sections from a cell expressing HA-Broi and Gag-GFP. Lower panels show a single Z section of the same cell showing colocalization of Broi and Gag-GFP at the plasma membrane. Broi (white) was stained with a mouse monoclonal anti-HA antibody and an Alexa 633-conjugated anti-mouse antibody. Nuclei were counterstained with DAPI (blue). F-actin was stained with Alexa 568-conjugated phalloidin (red) to delineate cells. The colocalization channel (yellow) of Broi and Gag-GFP was built using Imaris software. Scale bar = 5 µm. (B, C) Electron micrographs of 293T cells co-transfected with pNL4-3 wt and HA-Broi. (B) Arrested budding structures are indicated with black arrows. Two regions of interest in (a) and (b) show budding structures carrying electron-dense crescent-shaped material at a higher magnification. (C) HIV-1 budding structures tethered to the plasma membrane.
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ppat-1000339-g005: Broi is recruited by Gag to the plasma membrane and interferes with HIV-1 budding.(A) First two upper left panels show a cell expressing HA-Broi (white). The next panel shows a reconstructed 3D view of a stack of Z sections from a cell expressing HA-Broi and Gag-GFP. Lower panels show a single Z section of the same cell showing colocalization of Broi and Gag-GFP at the plasma membrane. Broi (white) was stained with a mouse monoclonal anti-HA antibody and an Alexa 633-conjugated anti-mouse antibody. Nuclei were counterstained with DAPI (blue). F-actin was stained with Alexa 568-conjugated phalloidin (red) to delineate cells. The colocalization channel (yellow) of Broi and Gag-GFP was built using Imaris software. Scale bar = 5 µm. (B, C) Electron micrographs of 293T cells co-transfected with pNL4-3 wt and HA-Broi. (B) Arrested budding structures are indicated with black arrows. Two regions of interest in (a) and (b) show budding structures carrying electron-dense crescent-shaped material at a higher magnification. (C) HIV-1 budding structures tethered to the plasma membrane.

Mentions: Since Broi interacts with Gag, we examined the cellular localization of Broi vis-à-vis HIV-1 Gag. Using confocal microscopy analysis, we found that Broi displayed a diffuse cytoplasmic distribution and localized to intracytoplamic vacuoles (Figure 5A). We next examined the localization of Broi vis-à-vis HIV-1 Gag in the cell and found that both proteins displayed a clear colocalization at the plasma membrane (Figure 5A, lower panels). This result indicated that HIV-1 Gag recruits Broi to the plasma membrane.


The nucleocapsid region of HIV-1 Gag cooperates with the PTAP and LYPXnL late domains to recruit the cellular machinery necessary for viral budding.

Dussupt V, Javid MP, Abou-Jaoudé G, Jadwin JA, de La Cruz J, Nagashima K, Bouamr F - PLoS Pathog. (2009)

Broi is recruited by Gag to the plasma membrane and interferes with HIV-1 budding.(A) First two upper left panels show a cell expressing HA-Broi (white). The next panel shows a reconstructed 3D view of a stack of Z sections from a cell expressing HA-Broi and Gag-GFP. Lower panels show a single Z section of the same cell showing colocalization of Broi and Gag-GFP at the plasma membrane. Broi (white) was stained with a mouse monoclonal anti-HA antibody and an Alexa 633-conjugated anti-mouse antibody. Nuclei were counterstained with DAPI (blue). F-actin was stained with Alexa 568-conjugated phalloidin (red) to delineate cells. The colocalization channel (yellow) of Broi and Gag-GFP was built using Imaris software. Scale bar = 5 µm. (B, C) Electron micrographs of 293T cells co-transfected with pNL4-3 wt and HA-Broi. (B) Arrested budding structures are indicated with black arrows. Two regions of interest in (a) and (b) show budding structures carrying electron-dense crescent-shaped material at a higher magnification. (C) HIV-1 budding structures tethered to the plasma membrane.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2651531&req=5

ppat-1000339-g005: Broi is recruited by Gag to the plasma membrane and interferes with HIV-1 budding.(A) First two upper left panels show a cell expressing HA-Broi (white). The next panel shows a reconstructed 3D view of a stack of Z sections from a cell expressing HA-Broi and Gag-GFP. Lower panels show a single Z section of the same cell showing colocalization of Broi and Gag-GFP at the plasma membrane. Broi (white) was stained with a mouse monoclonal anti-HA antibody and an Alexa 633-conjugated anti-mouse antibody. Nuclei were counterstained with DAPI (blue). F-actin was stained with Alexa 568-conjugated phalloidin (red) to delineate cells. The colocalization channel (yellow) of Broi and Gag-GFP was built using Imaris software. Scale bar = 5 µm. (B, C) Electron micrographs of 293T cells co-transfected with pNL4-3 wt and HA-Broi. (B) Arrested budding structures are indicated with black arrows. Two regions of interest in (a) and (b) show budding structures carrying electron-dense crescent-shaped material at a higher magnification. (C) HIV-1 budding structures tethered to the plasma membrane.
Mentions: Since Broi interacts with Gag, we examined the cellular localization of Broi vis-à-vis HIV-1 Gag. Using confocal microscopy analysis, we found that Broi displayed a diffuse cytoplasmic distribution and localized to intracytoplamic vacuoles (Figure 5A). We next examined the localization of Broi vis-à-vis HIV-1 Gag in the cell and found that both proteins displayed a clear colocalization at the plasma membrane (Figure 5A, lower panels). This result indicated that HIV-1 Gag recruits Broi to the plasma membrane.

Bottom Line: Remarkably, we found that over-expression of Bro1 rescued the release of HIV-1 lacking both L domains.This rescue required the N-terminal region of the NC domain in Gag and the CHMP4 binding site in Bro1.Interestingly, release defects due to mutations in NC that prevented Bro1 mediated rescue of virus egress were rescued by providing a link to the ESCRT machinery via Nedd4.2s over-expression.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
HIV-1 release is mediated through two motifs in the p6 region of Gag, PTAP and LYPX(n)L, which recruit cellular proteins Tsg101 and Alix, respectively. The Nucleocapsid region of Gag (NC), which binds the Bro1 domain of Alix, also plays an important role in HIV-1 release, but the underlying mechanism remains unclear. Here we show that the first 202 residues of the Bro1 domain (Bro(i)) are sufficient to bind Gag. Bro(i) interferes with HIV-1 release in an NC-dependent manner and arrests viral budding at the plasma membrane. Similar interrupted budding structures are seen following over-expression of a fragment containing Bro1 with the adjacent V domain (Bro1-V). Although only Bro1-V contains binding determinants for CHMP4, both Bro(i) and Bro1-V inhibited release via both the PTAP/Tsg101 and the LYPX(n)L/Alix pathways, suggesting that they interfere with a key step in HIV-1 release. Remarkably, we found that over-expression of Bro1 rescued the release of HIV-1 lacking both L domains. This rescue required the N-terminal region of the NC domain in Gag and the CHMP4 binding site in Bro1. Interestingly, release defects due to mutations in NC that prevented Bro1 mediated rescue of virus egress were rescued by providing a link to the ESCRT machinery via Nedd4.2s over-expression. Our data support a model in which NC cooperates with PTAP in the recruitment of cellular proteins necessary for its L domain activity and binds the Bro1-CHMP4 complex required for LYPX(n)L-mediated budding.

Show MeSH
Related in: MedlinePlus