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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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Over-expression of Alix inhibits HIV-1 release.(A) Schematic representation of the domain organization of Alix. Bro1: BCK1-like resistance to osmotic shock 1, V: V-shaped domain, PRD: Proline Rich Domain. Numbers indicate positions of amino acid residues. (B) Virus-release assay showing that over-expression of Alix inhibits virus release. 293T cells were transfected with either wild-type (wt) pNL4-3 plasmid alone or with increasing amounts of HA-Alix. Pelleted virions and cell lysates were analyzed by SDS-PAGE and western blot using the indicated antibodies. Gag intermediate products are indicated with (*) symbols. (C) Relative release efficiency of HIV-1 virions upon over-expression of Alix. The release efficiency in presence of Alix (calculated at the highest amount shown in B, lane 4) is relative to the release efficiency of NL4-3 alone, which was arbitrarily set at 100. Error bars indicate the standard deviation from four separate experiments. (D) Transmission Electron Microscopy (TEM) images of thin-sectioned 293T cells transfected with pNL4-3 wt alone (upper left panel) or pNL4-3 wt + Alix (lower left panel and close-ups of regions of interest a, b, c) showing arrested budding particles.
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ppat-1000339-g001: Over-expression of Alix inhibits HIV-1 release.(A) Schematic representation of the domain organization of Alix. Bro1: BCK1-like resistance to osmotic shock 1, V: V-shaped domain, PRD: Proline Rich Domain. Numbers indicate positions of amino acid residues. (B) Virus-release assay showing that over-expression of Alix inhibits virus release. 293T cells were transfected with either wild-type (wt) pNL4-3 plasmid alone or with increasing amounts of HA-Alix. Pelleted virions and cell lysates were analyzed by SDS-PAGE and western blot using the indicated antibodies. Gag intermediate products are indicated with (*) symbols. (C) Relative release efficiency of HIV-1 virions upon over-expression of Alix. The release efficiency in presence of Alix (calculated at the highest amount shown in B, lane 4) is relative to the release efficiency of NL4-3 alone, which was arbitrarily set at 100. Error bars indicate the standard deviation from four separate experiments. (D) Transmission Electron Microscopy (TEM) images of thin-sectioned 293T cells transfected with pNL4-3 wt alone (upper left panel) or pNL4-3 wt + Alix (lower left panel and close-ups of regions of interest a, b, c) showing arrested budding particles.

Mentions: HIV-1 recruits Tsg101 and Alix to bud from the plasma membrane of infected cells. When the Tsg101-driven pathway is not available (HIV-1 PTAP- mutant), over-expression of Alix has been shown to rescue HIV-1 release defects [44],[47]. Titration analysis indicated that the optimal amount of HA-Alix that stimulated HIV-1 PTAP- was relatively low (Figure S1A, lane 3), whereas in contrast, higher amounts failed to rescue release (Figure S1A, lane 5). Interestingly, amounts of HA-Alix that prevented the rescue of HIV-1 PTAP- release also caused a significant decrease in wt HIV-1 release (Figure S1A, lane 9). Further analysis and quantification of this inhibition, shown in Figure 1, demonstrated that over-expression of increasing amounts of HA-Alix potently inhibited HIV-1 exit, as only ∼15% of virus was detected outside the cell (Figure 1B and 1C). Similar results were obtained when this experiment was performed with the HIV-1 strain BH10 (data not shown). Alix over-expression also decreased Gag processing as a modest accumulation of p25CA and other Gag intermediate processing products was seen (Figure 1B).


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)

Over-expression of Alix inhibits HIV-1 release.(A) Schematic representation of the domain organization of Alix. Bro1: BCK1-like resistance to osmotic shock 1, V: V-shaped domain, PRD: Proline Rich Domain. Numbers indicate positions of amino acid residues. (B) Virus-release assay showing that over-expression of Alix inhibits virus release. 293T cells were transfected with either wild-type (wt) pNL4-3 plasmid alone or with increasing amounts of HA-Alix. Pelleted virions and cell lysates were analyzed by SDS-PAGE and western blot using the indicated antibodies. Gag intermediate products are indicated with (*) symbols. (C) Relative release efficiency of HIV-1 virions upon over-expression of Alix. The release efficiency in presence of Alix (calculated at the highest amount shown in B, lane 4) is relative to the release efficiency of NL4-3 alone, which was arbitrarily set at 100. Error bars indicate the standard deviation from four separate experiments. (D) Transmission Electron Microscopy (TEM) images of thin-sectioned 293T cells transfected with pNL4-3 wt alone (upper left panel) or pNL4-3 wt + Alix (lower left panel and close-ups of regions of interest a, b, c) showing arrested budding particles.
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Related In: Results  -  Collection

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ppat-1000339-g001: Over-expression of Alix inhibits HIV-1 release.(A) Schematic representation of the domain organization of Alix. Bro1: BCK1-like resistance to osmotic shock 1, V: V-shaped domain, PRD: Proline Rich Domain. Numbers indicate positions of amino acid residues. (B) Virus-release assay showing that over-expression of Alix inhibits virus release. 293T cells were transfected with either wild-type (wt) pNL4-3 plasmid alone or with increasing amounts of HA-Alix. Pelleted virions and cell lysates were analyzed by SDS-PAGE and western blot using the indicated antibodies. Gag intermediate products are indicated with (*) symbols. (C) Relative release efficiency of HIV-1 virions upon over-expression of Alix. The release efficiency in presence of Alix (calculated at the highest amount shown in B, lane 4) is relative to the release efficiency of NL4-3 alone, which was arbitrarily set at 100. Error bars indicate the standard deviation from four separate experiments. (D) Transmission Electron Microscopy (TEM) images of thin-sectioned 293T cells transfected with pNL4-3 wt alone (upper left panel) or pNL4-3 wt + Alix (lower left panel and close-ups of regions of interest a, b, c) showing arrested budding particles.
Mentions: HIV-1 recruits Tsg101 and Alix to bud from the plasma membrane of infected cells. When the Tsg101-driven pathway is not available (HIV-1 PTAP- mutant), over-expression of Alix has been shown to rescue HIV-1 release defects [44],[47]. Titration analysis indicated that the optimal amount of HA-Alix that stimulated HIV-1 PTAP- was relatively low (Figure S1A, lane 3), whereas in contrast, higher amounts failed to rescue release (Figure S1A, lane 5). Interestingly, amounts of HA-Alix that prevented the rescue of HIV-1 PTAP- release also caused a significant decrease in wt HIV-1 release (Figure S1A, lane 9). Further analysis and quantification of this inhibition, shown in Figure 1, demonstrated that over-expression of increasing amounts of HA-Alix potently inhibited HIV-1 exit, as only ∼15% of virus was detected outside the cell (Figure 1B and 1C). Similar results were obtained when this experiment was performed with the HIV-1 strain BH10 (data not shown). Alix over-expression also decreased Gag processing as a modest accumulation of p25CA and other Gag intermediate processing products was seen (Figure 1B).

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