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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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The NC domain of HIV-1 Gag is the primary target for Broi inhibition.(A) Broi and HIV-1 Gag co-immunoprecipitate in 293T cell lysates. 293T cells were co-transfected with either Gag-Pol (left panel) or GagΔp6-Pol (right panel) constructs along with expression vectors for HA-Alix, HA-Bro1, HA-Broi, or HA-PRD. Cells were lysed in RIPA buffer and clear lysates were incubated with anti-HA antibody-conjugated beads. Both input and immunoprecipitated complexes were ran on SDS-PAGE for western blot analysis using the indicated antibodies. (B) The Bro1 domain of Alix binds NC in vitro. MBP and MBP-NC proteins were expressed in E.coli, immobilized on amylose resin and incubated with lysates from 293T cells expressing HA-Bro1. Captured Bro1 was detected with anti-HA antibody and MBP proteins were visualized by Coomassie blue staining. (C) The release of NL4-3 DelNC/PR- but not NL4-3 PR- is insensitive to the over-expression of Broi. 293T cells were transfected with either pNL4-3 DelNC/PR- (Left panel) or pNL4-3 PR- (right panel) in presence or absence of HA-Broi. Pelleted virions and cell lysates were analyzed by SDS-PAGE and western blot using the indicated antibodies. (D) Alix but not Broi co-immunoprecipitated with HIV-1 Gag DelNC. 293T cells were co-transfected with HIV-1 Gag DelNC/PR- along with either HA-Alix or HA-Broi. Cells were lysed in RIPA buffer and clear lysates were incubated with anti-HA antibody-conjugated beads. Both input and immunoprecipitated complexes were ran on SDS-PAGE for western blot analysis using the indicated antibodies.
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ppat-1000339-g004: The NC domain of HIV-1 Gag is the primary target for Broi inhibition.(A) Broi and HIV-1 Gag co-immunoprecipitate in 293T cell lysates. 293T cells were co-transfected with either Gag-Pol (left panel) or GagΔp6-Pol (right panel) constructs along with expression vectors for HA-Alix, HA-Bro1, HA-Broi, or HA-PRD. Cells were lysed in RIPA buffer and clear lysates were incubated with anti-HA antibody-conjugated beads. Both input and immunoprecipitated complexes were ran on SDS-PAGE for western blot analysis using the indicated antibodies. (B) The Bro1 domain of Alix binds NC in vitro. MBP and MBP-NC proteins were expressed in E.coli, immobilized on amylose resin and incubated with lysates from 293T cells expressing HA-Bro1. Captured Bro1 was detected with anti-HA antibody and MBP proteins were visualized by Coomassie blue staining. (C) The release of NL4-3 DelNC/PR- but not NL4-3 PR- is insensitive to the over-expression of Broi. 293T cells were transfected with either pNL4-3 DelNC/PR- (Left panel) or pNL4-3 PR- (right panel) in presence or absence of HA-Broi. Pelleted virions and cell lysates were analyzed by SDS-PAGE and western blot using the indicated antibodies. (D) Alix but not Broi co-immunoprecipitated with HIV-1 Gag DelNC. 293T cells were co-transfected with HIV-1 Gag DelNC/PR- along with either HA-Alix or HA-Broi. Cells were lysed in RIPA buffer and clear lysates were incubated with anti-HA antibody-conjugated beads. Both input and immunoprecipitated complexes were ran on SDS-PAGE for western blot analysis using the indicated antibodies.

Mentions: The results above indicated that Broi efficiently interfered with HIV-1 release, prompting the question as to whether Broi interacts directly with Gag. To examine this, Alix, Bro1, Broi and PRD were tested for their ability to interact with HIV-1 Gag (Figure 4A, left panel) and a GagΔp6 mutant (right panel) in immunoprecipitation assays. In contrast to PRD, Alix, Bro1 and Broi efficiently pulled-down Gag and the GagΔp6 mutant (Figure 4A). This demonstrated that in the cell, Bro1 binds HIV-1 Gag in a p6-independent manner and that the Broi region is sufficient for this interaction. Further examination by in vitro pull-down assays showed that NC binds the Bro1 domain of Alix (Figure 4B). These data confirmed that Alix contains a novel binding site for Gag located within Bro1, which is in agreement with the recent findings of Popov et al, 2008 [60]. Our results identify the first 202 residues of the Bro1 domain as sufficient for binding HIV-1 Gag.


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)

The NC domain of HIV-1 Gag is the primary target for Broi inhibition.(A) Broi and HIV-1 Gag co-immunoprecipitate in 293T cell lysates. 293T cells were co-transfected with either Gag-Pol (left panel) or GagΔp6-Pol (right panel) constructs along with expression vectors for HA-Alix, HA-Bro1, HA-Broi, or HA-PRD. Cells were lysed in RIPA buffer and clear lysates were incubated with anti-HA antibody-conjugated beads. Both input and immunoprecipitated complexes were ran on SDS-PAGE for western blot analysis using the indicated antibodies. (B) The Bro1 domain of Alix binds NC in vitro. MBP and MBP-NC proteins were expressed in E.coli, immobilized on amylose resin and incubated with lysates from 293T cells expressing HA-Bro1. Captured Bro1 was detected with anti-HA antibody and MBP proteins were visualized by Coomassie blue staining. (C) The release of NL4-3 DelNC/PR- but not NL4-3 PR- is insensitive to the over-expression of Broi. 293T cells were transfected with either pNL4-3 DelNC/PR- (Left panel) or pNL4-3 PR- (right panel) in presence or absence of HA-Broi. Pelleted virions and cell lysates were analyzed by SDS-PAGE and western blot using the indicated antibodies. (D) Alix but not Broi co-immunoprecipitated with HIV-1 Gag DelNC. 293T cells were co-transfected with HIV-1 Gag DelNC/PR- along with either HA-Alix or HA-Broi. Cells were lysed in RIPA buffer and clear lysates were incubated with anti-HA antibody-conjugated beads. Both input and immunoprecipitated complexes were ran on SDS-PAGE for western blot analysis using the indicated antibodies.
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getmorefigures.php?uid=PMC2651531&req=5

ppat-1000339-g004: The NC domain of HIV-1 Gag is the primary target for Broi inhibition.(A) Broi and HIV-1 Gag co-immunoprecipitate in 293T cell lysates. 293T cells were co-transfected with either Gag-Pol (left panel) or GagΔp6-Pol (right panel) constructs along with expression vectors for HA-Alix, HA-Bro1, HA-Broi, or HA-PRD. Cells were lysed in RIPA buffer and clear lysates were incubated with anti-HA antibody-conjugated beads. Both input and immunoprecipitated complexes were ran on SDS-PAGE for western blot analysis using the indicated antibodies. (B) The Bro1 domain of Alix binds NC in vitro. MBP and MBP-NC proteins were expressed in E.coli, immobilized on amylose resin and incubated with lysates from 293T cells expressing HA-Bro1. Captured Bro1 was detected with anti-HA antibody and MBP proteins were visualized by Coomassie blue staining. (C) The release of NL4-3 DelNC/PR- but not NL4-3 PR- is insensitive to the over-expression of Broi. 293T cells were transfected with either pNL4-3 DelNC/PR- (Left panel) or pNL4-3 PR- (right panel) in presence or absence of HA-Broi. Pelleted virions and cell lysates were analyzed by SDS-PAGE and western blot using the indicated antibodies. (D) Alix but not Broi co-immunoprecipitated with HIV-1 Gag DelNC. 293T cells were co-transfected with HIV-1 Gag DelNC/PR- along with either HA-Alix or HA-Broi. Cells were lysed in RIPA buffer and clear lysates were incubated with anti-HA antibody-conjugated beads. Both input and immunoprecipitated complexes were ran on SDS-PAGE for western blot analysis using the indicated antibodies.
Mentions: The results above indicated that Broi efficiently interfered with HIV-1 release, prompting the question as to whether Broi interacts directly with Gag. To examine this, Alix, Bro1, Broi and PRD were tested for their ability to interact with HIV-1 Gag (Figure 4A, left panel) and a GagΔp6 mutant (right panel) in immunoprecipitation assays. In contrast to PRD, Alix, Bro1 and Broi efficiently pulled-down Gag and the GagΔp6 mutant (Figure 4A). This demonstrated that in the cell, Bro1 binds HIV-1 Gag in a p6-independent manner and that the Broi region is sufficient for this interaction. Further examination by in vitro pull-down assays showed that NC binds the Bro1 domain of Alix (Figure 4B). These data confirmed that Alix contains a novel binding site for Gag located within Bro1, which is in agreement with the recent findings of Popov et al, 2008 [60]. Our results identify the first 202 residues of the Bro1 domain as sufficient for binding HIV-1 Gag.

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