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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 N-terminal region of Bro1 inhibits the release of HIV-1.(A) Schematic representation of constructs used in the experiment. Numbers indicate positions of amino acid residues. (B, C, D) 293T cells were transfected with either pNL4-3 plasmid alone or with increasing amounts of the indicated dominant-negative fragments of Alix. Pelleted virions and cell lysates were analyzed by SDS-PAGE and western blot using the indicated antibodies. (B) Over-expression of Bro1-V but not delBroV inhibited virus-release. (C) The Bro1-VF676D mutant retains inhibitory effect on HIV-1 release. (D) Broi, but not Bro1, inhibits HIV-1 release. (E) Relative release efficiency of HIV-1 virions upon over-expression of each of the four N-terminal fragments of Alix. Error bars indicate the standard deviation from three separate experiments.
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ppat-1000339-g002: The N-terminal region of Bro1 inhibits the release of HIV-1.(A) Schematic representation of constructs used in the experiment. Numbers indicate positions of amino acid residues. (B, C, D) 293T cells were transfected with either pNL4-3 plasmid alone or with increasing amounts of the indicated dominant-negative fragments of Alix. Pelleted virions and cell lysates were analyzed by SDS-PAGE and western blot using the indicated antibodies. (B) Over-expression of Bro1-V but not delBroV inhibited virus-release. (C) The Bro1-VF676D mutant retains inhibitory effect on HIV-1 release. (D) Broi, but not Bro1, inhibits HIV-1 release. (E) Relative release efficiency of HIV-1 virions upon over-expression of each of the four N-terminal fragments of Alix. Error bars indicate the standard deviation from three separate experiments.

Mentions: We tested fragments of the Alix protein to determine the role of the various domains in the inhibition the protein exerted on HIV-1 release. We first removed the C-terminal PRD to exclude the involvement of the numerous cellular partners known to bind this region, including Tsg101. We then focused on fragments containing the N-terminal Bro1 and the central V domain (Figure 2A) because these domains bind ESCRT-III and Gag, respectively, and were shown to play key roles in Alix function during virus release [44],[47],[60],[65]. Over-expression of Bro1-V efficiently inhibited HIV-1 release but had no detectable effect on Gag accumulation in the cell (Figure 2B). Removal of the N-terminal half of the Bro1 domain reversed the inhibitory effect of the Bro1-V fragment (Figure 2B), implying that binding via the V domain is not sufficient for inhibition in this context. To examine whether the V domain binding to Gag is involved in the Bro1-V block of HIV-1, we next tested the effect of the Bro1-VF676D mutant that no longer binds the LYPXnL motif. We found that Bro1-VF676D retained potent inhibition of HIV-1 release (Figure 2C), indicating that the Gag binding site in the V domain is dispensable for interference with viral exit in this context and suggesting that Bro1-V blocked HIV-1 production via regions located within the Bro1 domain. To map these inhibitory regions, we over-expressed the entire Bro1 domain and found that surprisingly, it had no inhibitory effect on HIV-1. In contrast, expression of a shorter fragment encompassing the first 202 residues of the Bro1 domain (Broi) caused a potent inhibition of HIV-1 release (Figure 2D). Broi had little effect on Gag accumulation in the cell (Figure 2D). These findings identify the N-terminal 202 residues of the Bro1 domain as the region that drives inhibition of HIV-1 release (Figure 2E).


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 N-terminal region of Bro1 inhibits the release of HIV-1.(A) Schematic representation of constructs used in the experiment. Numbers indicate positions of amino acid residues. (B, C, D) 293T cells were transfected with either pNL4-3 plasmid alone or with increasing amounts of the indicated dominant-negative fragments of Alix. Pelleted virions and cell lysates were analyzed by SDS-PAGE and western blot using the indicated antibodies. (B) Over-expression of Bro1-V but not delBroV inhibited virus-release. (C) The Bro1-VF676D mutant retains inhibitory effect on HIV-1 release. (D) Broi, but not Bro1, inhibits HIV-1 release. (E) Relative release efficiency of HIV-1 virions upon over-expression of each of the four N-terminal fragments of Alix. Error bars indicate the standard deviation from three separate experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2651531&req=5

ppat-1000339-g002: The N-terminal region of Bro1 inhibits the release of HIV-1.(A) Schematic representation of constructs used in the experiment. Numbers indicate positions of amino acid residues. (B, C, D) 293T cells were transfected with either pNL4-3 plasmid alone or with increasing amounts of the indicated dominant-negative fragments of Alix. Pelleted virions and cell lysates were analyzed by SDS-PAGE and western blot using the indicated antibodies. (B) Over-expression of Bro1-V but not delBroV inhibited virus-release. (C) The Bro1-VF676D mutant retains inhibitory effect on HIV-1 release. (D) Broi, but not Bro1, inhibits HIV-1 release. (E) Relative release efficiency of HIV-1 virions upon over-expression of each of the four N-terminal fragments of Alix. Error bars indicate the standard deviation from three separate experiments.
Mentions: We tested fragments of the Alix protein to determine the role of the various domains in the inhibition the protein exerted on HIV-1 release. We first removed the C-terminal PRD to exclude the involvement of the numerous cellular partners known to bind this region, including Tsg101. We then focused on fragments containing the N-terminal Bro1 and the central V domain (Figure 2A) because these domains bind ESCRT-III and Gag, respectively, and were shown to play key roles in Alix function during virus release [44],[47],[60],[65]. Over-expression of Bro1-V efficiently inhibited HIV-1 release but had no detectable effect on Gag accumulation in the cell (Figure 2B). Removal of the N-terminal half of the Bro1 domain reversed the inhibitory effect of the Bro1-V fragment (Figure 2B), implying that binding via the V domain is not sufficient for inhibition in this context. To examine whether the V domain binding to Gag is involved in the Bro1-V block of HIV-1, we next tested the effect of the Bro1-VF676D mutant that no longer binds the LYPXnL motif. We found that Bro1-VF676D retained potent inhibition of HIV-1 release (Figure 2C), indicating that the Gag binding site in the V domain is dispensable for interference with viral exit in this context and suggesting that Bro1-V blocked HIV-1 production via regions located within the Bro1 domain. To map these inhibitory regions, we over-expressed the entire Bro1 domain and found that surprisingly, it had no inhibitory effect on HIV-1. In contrast, expression of a shorter fragment encompassing the first 202 residues of the Bro1 domain (Broi) caused a potent inhibition of HIV-1 release (Figure 2D). Broi had little effect on Gag accumulation in the cell (Figure 2D). These findings identify the N-terminal 202 residues of the Bro1 domain as the region that drives inhibition of HIV-1 release (Figure 2E).

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