The cellular ALIX protein functions within the ESCRT pathway to facilitate intralumenal endosomal vesicle formation, the abscission stage of cytokinesis, and enveloped virus budding. Here, we report that the C-terminal proline-rich region (PRR) of ALIX folds back against the upstream domains and auto-inhibits V domain binding to viral late domains. Mutations designed to destabilize the closed conformation of the V domain opened the V domain, increased ALIX membrane association, and enhanced virus budding. These observations support a model in which ALIX activation requires dissociation of the autoinhibitory PRR and opening of the V domain arms.Retroviral Gag polyproteins contain short sequence motifs, termed "late domains," that facilitate virus budding by recruiting components of the cellular ESCRT pathway (4, 38). For example, the HIV-1 p6Gag protein contains "PTAP" and "YPXL" late domains (designated by their consensus sequences), that bind directly to the TSG101 and ALIX proteins, respectively (6,9,19,33,39). ALIX, in turn, binds the CHMP4 subunits of the ESCRT-III complex, resulting in recruitment of the VPS4 ATPase, membrane fission, and virus release (10, 27).ALIX contains three distinct structural elements: an N-terminal Bro1 domain, a central V domain, and a C-terminal proline-rich region (PRR). The boomerang-shaped Bro1 domain binds CHMP4 proteins (7,13,20), the V domain comprises two extended three-helix bundles and binds YPXL late domains (7,16,44,45), and the PRR binds a series of other proteins but is predicted to lack a persistent secondary or tertiary structure (7,8,24). Like other ESCRT factors, ALIX must cycle between soluble (inactive) and membrane-associated (active) states. Several lines of evidence suggest that conformational changes accompany (or induce) these transitions. First, recombinant ALIX proteins can form stable monomers and dimers (7, 23), and biochemical evidence suggests that the dimer is the active conformation (5,7,23,29). Second, smallangle X-ray scattering (SAXS) profiles indicate that the two arms of the V domain may open and associate in an antiparallel fashion when the protein dimerizes (29). Third, recent reports show that the PRR can inhibit ALIX binding to conformationally sensitive monoclonal antibodies, CHMP4 proteins, and viral Gag proteins (46-48). However, previous studies have not characterized the structure or conformational transitions of pure, full-length ALIX, because this protein has not been available.Although we were unable to express full-length human ALIX protein in Escherichia coli, we could produce multimilligram quantities of pure recombinant ALIX in insect cells using a baculoviral expression system. Briefly, 2 liters of SF21 cells were infected with a BaculoDirect (Invitrogen) expression vector, which encoded His 6 -ALIX (ALIX residues 1 to 868, WISP10-643). The cells were lysed by sonication 48 h postinfection (300 mM NaCl, 10 mM imidazole, 5% [vol/vol] (Fig. 1A). This procedure typically yielded 5 mg of pure monomeric ALIX, and the protein ide...

Retroviral Gag proteins contain short late-domain motifs that recruit cellular ESCRT pathway proteins to facilitate virus budding. ALIX-binding late domains often contain the core consensus sequence YPX n L (where X n can vary in sequence and length). However, some simian immunodeficiency virus (SIV) Gag proteins lack this consensus sequence, yet still bind ALIX. We mapped divergent, ALIX-binding late domains within the p6 Gag proteins of SIV mac239 ( 40 SREKPYKEVTEDLLHLNSLF 59 ) and SIV agmTan-1 ( 24 AAGAYDPARKLLEQY AKK 41 ). Crystal structures revealed that anchoring tyrosines (in lightface) and nearby hydrophobic residues (underlined) contact the ALIX V domain, revealing how lentiviruses employ a diverse family of late-domain sequences to bind ALIX and promote virus budding.Many enveloped viruses, including retroviruses, recruit proteins of the cellular ESCRT pathway to facilitate budding (reviewed in references 3, 11, and 35). Short sequence motifs, termed late domains, within retroviral Gag polyproteins bind directly to early-acting ESCRT factors, which then recruit and activate the downstream machinery necessary for membrane fission. The three well-characterized late domains are typically denoted by their canonical core amino acid sequences: PTAP late domains bind the ubiquitin E2 variant (UEV) domain of the TSG101 subunit of the ESCRT-I complex, PPXY late domains bind WW domains of NEDD4 family ubiquitin E3 ligases, and YPX n L (where X n can vary in sequence and length) late domains bind the V domain of ALIX (10,20,32,37). In a number of cases, retroviral Gag proteins have been shown to utilize multiple late domains (e.g., see references 3, 4, 7, 11, 16, 31, 33, 34, and 35). We speculate that this phenomenon may be even more prevalent than is currently appreciated because mutations in auxiliary late domains often produce weak or cell-specific phenotypes and because late domains can be difficult to recognize owing to primary sequence divergence. It is therefore of interest to define the range of different sequences that can function as late domains and to learn how sequence variation is tolerated while late-domain function is retained.Strack and colleagues initially reported that ALIX binds core sequences of 35 LYPLTSL 41 and 22 LYPDL 26 within the late domains of human immunodeficiency virus type 1 (HIV-1) p6Gag and equine infectious anemia virus (EIAV) p9 Gag , respectively (32) (anchoring tyrosines are shown in boldface, and nearby hydrophobic residues that contact ALIX are underlined). They also reported that p6Gag proteins from simian immunodeficiency virus SIV mac239 and SIV agmTan-1 can bind and package ALIX into virions, but in those cases the ALIXbinding sites were not fully mapped and were not obvious, because the SIV p6Gag proteins lacked canonical YPX n L ALIX-binding elements. We therefore performed biosensor binding experiments and deletion analyses to quantify and map the ALIX-binding sites. These experiments employed a recombinant ALIX protein that spanned the Bro1 and V domains (residues...

Polymerization of Gag on the inner leaflet of the plasma membrane drives the assembly of Human Immunodeficiency Virus 1 (HIV-1). Gag recruits components of the endosomal sorting complexes required for transport (ESCRT) to facilitate membrane fission and virion release. ESCRT assembly is initiated by recruitment of ALIX and TSG101/ESCRT-I, which bind directly to the viral Gag protein and then recruit the downstream ESCRT-III and VPS4 factors to complete the budding process. In contrast to previous models, we show that ALIX is recruited transiently at the end of Gag assembly, and that most ALIX molecules are recycled into the cytosol as the virus buds, although a subset remains within the virion. Our experiments imply that ALIX is recruited to the neck of the assembling virion and is mostly recycled after virion release.

BackgroundBro1 domains are elongated, banana-shaped domains that were first identified in the yeast ESCRT pathway protein, Bro1p. Humans express three Bro1 domain-containing proteins: ALIX, BROX, and HD-PTP, which function in association with the ESCRT pathway to help mediate intraluminal vesicle formation at multivesicular bodies, the abscission stage of cytokinesis, and/or enveloped virus budding. Human Bro1 domains share the ability to bind the CHMP4 subset of ESCRT-III proteins, associate with the HIV-1 NCGag protein, and stimulate the budding of viral Gag proteins. The curved Bro1 domain structure has also been proposed to mediate membrane bending. To date, crystal structures have only been available for the related Bro1 domains from the Bro1p and ALIX proteins, and structures of additional family members should therefore aid in the identification of key structural and functional elements.Methodology/Principal FindingsWe report the crystal structure of the human BROX protein, which comprises a single Bro1 domain. The Bro1 domains from BROX, Bro1p and ALIX adopt similar overall structures and share two common exposed hydrophobic surfaces. Surface 1 is located on the concave face and forms the CHMP4 binding site, whereas Surface 2 is located at the narrow end of the domain. The structures differ in that only ALIX has an extended loop that projects away from the convex face to expose the hydrophobic Phe105 side chain at its tip. Functional studies demonstrated that mutations in Surface 1, Surface 2, or Phe105 all impair the ability of ALIX to stimulate HIV-1 budding.Conclusions/SignificanceOur studies reveal similarities in the overall folds and hydrophobic protein interaction sites of different Bro1 domains, and show that a unique extended loop contributes to the ability of ALIX to function in HIV-1 budding.

Vesicular stomatitis virus (VSV) is a prototypic negative sense single-stranded RNA virus. The bullet-shape appearance of the virion results from tightly wound helical turns of the nucleoprotein encapsidated RNA template (N-RNA) around a central cavity. Transcription and replication require polymerase complexes, which include a catalytic subunit L and a template-binding subunit P. L and P are inferred to be in the cavity, however lacking direct observation, their exact position has remained unclear. Using super-resolution fluorescence imaging and atomic force microscopy (AFM) on single VSV virions, we show that L and P are packaged asymmetrically towards the blunt end of the virus. The number of L and P proteins varies between individual virions and they occupy 57 ± 12 nm of the 150 nm central cavity of the virus. Our finding positions the polymerases at the opposite end of the genome with respect to the only transcriptional promoter.