Activation of the Retroviral Budding Factor ALIX

Zhai, Qianting; Landesman, Michael B.; Chung, Hyo Young; Dierkers, Adam; Jeffries, Cy M.; Trewhella, Jill; Hill, Christopher P.; Sundquist, Wesley I.

doi:10.1128/jvi.02653-10

Cited by 51 publications

(71 citation statements)

References 49 publications

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“…Opening of the V domain would expose different binding sites and consequently modify the function and subcellular localization of Alix (22). This proposed model was recently corroborated by the same authors, who demonstrated that Alix activation/localization requires dissociation of the auto-inhibitory PRR and opening of the V domain arms (43). Therefore, it appears that all the substrates of Ozz identified to date, including Alix, are targeted by this protein in their assembled state and again might require Ozz binding to change conformation and/or to dissociate from their protein partners before their ubiquitination.…”

Section: Discussionsupporting

confidence: 56%

Alix Protein Is Substrate of Ozz-E3 Ligase and Modulates Actin Remodeling in Skeletal Muscle

Bongiovanni

Romancino

Campos

et al. 2012

Journal of Biological Chemistry

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Background: Alix participates in fundamental cellular processes, but how it is regulated remains unknown. Results: Alix is ubiquitinated by the Ozz-E3 ligase and participates in actin cytoskeleton remodeling, filopodia formation, and myoblast migration. Conclusion: Ozz influences Alix conformation and in turn the extent of ubiquitination in Alix. Significance: Ozz-E3 ligase regulates Alix concentration at sites where the actin cytoskeleton undergoes remodeling.

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Section: Discussionsupporting

confidence: 56%

Alix Protein Is Substrate of Ozz-E3 Ligase and Modulates Actin Remodeling in Skeletal Muscle

Bongiovanni

Romancino

Campos

et al. 2012

Journal of Biological Chemistry

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“…6). First, Alix is anchored to sites of assembly via p6-V interactions and Bro1 seems to be unavailable to capture CHMP4, possibly due to a structural masking (45). Next, Bro1 binds NC, possibly in the budding neck, and becomes available to recruit CHMP4 during budding, a sequence of events that fits with the recent visualization of CHMP4 at the membrane once Gag assembly is complete (18).…”

Section: Discussionmentioning

confidence: 51%

Identification of the HIV-1 NC Binding Interface in Alix Bro1 Reveals a Role for RNA

Sette

Dussupt

Bouamr

2012

J Virol

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HIV-1 recruits members of ESCRT, the cell membrane fission machinery that promotes virus exit. HIV-1 Gag protein gains access to ESCRT directly by binding Alix, an ESCRT-associated protein that promotes budding. The Alix Bro1 and V domains bind Gag NC and p6 regions, respectively. Whereas V-p6 binding and function are well characterized, residues in Bro1 that interact with NC and their functional contribution to Alix-mediated HIV-1 budding are unknown. We mapped Bro1 residues that constitute the NC binding interface and found that they are critical for function. Intriguingly, residues involved in interactions on both sides of the Bro1-NC interface are positively charged, suggesting the involvement of a negatively charged cellular factor serving as a bridge. Nuclease treatment eliminated Bro1-NC interactions, revealing the involvement of RNA. These findings establish a direct role for NC in mediating interactions with ESCRT necessary for virus release and report the first evidence of RNA involvement in such recruitments. HIV-1 usurps members of the host cell fission machinery to promote virus release. Two conserved sequences located within the C-terminal p6 domain of Gag, PTAP, and LYPXnL, named late (L) domains, are utilized to fulfill such functions. They bind Tsg101 and Alix, respectively (14,37,40), two host cellular proteins that initiate a set of sequential interactions leading to the recruitment of members of the endosomal sorting complex required for transport (ESCRT) pathway (5, 9, 30). The latter is comprised of three multiprotein complexes, named ESCRT-I, ESCRT-II, and ESCRT-III, that facilitate membrane-modeling events critical for multivesicular body (MVB) generation (2, 3), cytokinesis (7), and autophagy (32).Tsg101 functions in HIV-1 release as part of ESCRT-I (26) and mediates access to members of ESCRT-III, the charged MVB protein CHMP2 and CHMP4 isoforms, as well as the VPS4 ATPase (29,38,41). Whereas interactions that link Tsg101 (and ESCRT-I) to ESCRT-III are still unknown, Alix binds CHMP4 isoforms directly, thus linking Gag to 19,37,39). Although the Tsg101/PTAP pathway is considered predominant in HIV-1 release, the Alix/LYPXnL pathway is also functional in 293T cells and appears to be more efficient in T lymphocytes (11-13, 37, 39). This pathway is also sufficient to drive the release of the equine infectious anemia virus (EIAV) (8, 37), a lentivirus that relies solely on cellular Alix for virus budding.Alix structure revealed two well-ordered domains, the N-terminal boomerang-shaped Bro1 and the central V-shaped domains (12, 21); they interact with the NC and p6 domains of HIV-1 Gag, respectively (10-12, 31, 37). The binding interface between the LYPXnL motif and the V domain and its functional role have been well characterized (12). In contrast, residues in the Alix Bro1 domain that mediate interactions with NC (10, 11, 31) and their role in virus release are not known. We performed a mutational analysis and used a combination of binding and functional assays to map the Bro1-NC interface...

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“…2C). It has been proposed that the Cterminal proline-rich domain (PRD) of ALIX maintains it in an autoinhibited conformation (43,44). When the C-terminal PRD of ALIX was deleted, recruitment was strongly enhanced (Fig.…”

Section: Resultsmentioning

confidence: 99%

In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters

Carlson

Hurley

2012

Proc. Natl. Acad. Sci. U.S.A.

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Most membrane-enveloped viruses depend on host proteins of the endosomal sorting complex required for transport (ESCRT) machinery for their release. HIV-1 is the prototypic ESCRT-dependent virus. The direct interactions between HIV-1 and the early ESCRT factors TSG101 and ALIX have been mapped in detail. However, the full pathway of ESCRT recruitment to HIV-1 budding sites, which culminates with the assembly of the late-acting CHMP4, CHMP3, CHMP2, and CHMP1 subunits, is less completely understood. Here, we report the biochemical reconstitution of ESCRT recruitment to viral assembly sites, using purified proteins and giant unilamellar vesicles. The myristylated full-length Gag protein of HIV-1 was purified to monodispersity. Myr-Gag forms clusters on giant unilamellar vesicle membranes containing the plasma membrane lipid PIð4,5ÞP 2 . These Gag clusters package a fluorescent oligonucleotide, and recruit early ESCRT complexes ESCRT-I or ALIX with the appropriate dependence on the Gag PTAP and LYPðXÞ n L motifs. ALIX directly recruits the key ESCRT-III subunit CHMP4. ESCRT-I can only recruit CHMP4 when ESCRT-II and CHMP6 are present as intermediary factors. Downstream of CHMP4, CHMP3 and CHMP2 assemble synergistically, with the presence of both subunits required for efficient recruitment. The very late-acting factor CHMP1 is not recruited unless the pathway is completed through CHMP3 and CHMP2. These findings define the minimal sets of components needed to complete ESCRT assembly at HIV-1 budding sites, and provide a starting point for in vitro structural and biophysical dissection of the system. confocal microscopy | host-pathogen interaction | membrane traffic | virus budding M ost membrane-enveloped viruses utilize host proteins of the endosomal sorting complex required for transport (ESCRT) machinery for their release (1-3). This dependence was first described for HIV-1, where mutation of an ESCRTbinding motif (late domain) in the viral protein Gag was shown to stall virus bud release at a late stage in virus assembly (4, 5). Late domains have subsequently been found in other retroviruses and numerous other virus families including, e.g., flavi-, filo-and rhabdoviruses (6). Indeed, the only well-characterized case of ESCRT-independent release of a membrane-enveloped virus is influenza virus (7). In normal cell physiology, ESCRTs function in cytokinesis, formation of intralumenal vesicles in multivesicular endosomes, and vesicle release from the plasma membrane (8-10). These seemingly disparate processes all involve membrane budding away from the cytoplasm, and ESCRTs are the only described protein machinery to perform vesicle formation with this topology, which is analogous to virus release.The initial events in HIV-1 ESCRT recruitment are well characterized. The structural protein of HIV-1, Gag, binds earlyacting ESCRT factors through two late-domain motifs in its C-terminal p6 domain: a PTAP sequence that interacts with the TSG101 subunit of ESCRT-I (11-16) and a LYPðXÞ n L motif that interacts with the ESCRT...

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Activation of the Retroviral Budding Factor ALIX

Cited by 51 publications

References 49 publications

Alix Protein Is Substrate of Ozz-E3 Ligase and Modulates Actin Remodeling in Skeletal Muscle

Alix Protein Is Substrate of Ozz-E3 Ligase and Modulates Actin Remodeling in Skeletal Muscle

Identification of the HIV-1 NC Binding Interface in Alix Bro1 Reveals a Role for RNA

In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters

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