HIV-1 Gag release from yeast reveals ESCRT interaction with the Gag N-terminal protein region

The HIV-1 protein Gag assembles at the plasma membrane and drives virion budding, assisted by the cellular endosomal complex required for transport (ESCRT) proteins. Two ESCRT proteins, TSG101 and ALIX, bind to the Gag C-terminal p6 peptide. TSG101 binding is important for efficient HIV-1 release, but how ESCRTs contribute to the budding process and how their activity is coordinated with Gag assembly is poorly understood. Yeast, allowing genetic manipulation that is not easily available in human cells, has been used to characterize the cellular ESCRT function. Previous work reported Gag budding from yeast spheroplasts, but Gag release was ESCRT-independent. We developed a yeast model for ESCRT-dependent Gag release. We combined yeast genetics and Gag mutational analysis with Gag-ESCRT binding studies and the characterization of Gag-plasma membrane binding and Gag release. With our system, we identified a previously unknown interaction between ESCRT proteins and the Gag N-terminal protein region. Mutations in the Gag-plasma membrane–binding matrix domain that reduced Gag-ESCRT binding increased Gag-plasma membrane binding and Gag release. ESCRT knockout mutants showed that the release enhancement was an ESCRT-dependent effect. Similarly, matrix mutation enhanced Gag release from human HEK293 cells. Release enhancement partly depended on ALIX binding to p6, although binding site mutation did not impair WT Gag release. Accordingly, the relative affinity for matrix compared with p6 in GST-pulldown experiments was higher for ALIX than for TSG101. We suggest that a transient matrix-ESCRT interaction is replaced when Gag binds to the plasma membrane. This step may activate ESCRT proteins and thereby coordinate ESCRT function with virion assembly.     

ALIX Is Recruited Temporarily into HIV-1 Budding Sites at the End of Gag Assembly

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.     

Human Immunodeficiency Virus Type 1 Nucleocapsid p1 Confers ESCRT Pathway Dependence

To facilitate the release of infectious progeny virions, human immunodeficiency virus type 1 (HIV-1) exploits the Endosomal Sorting Complex Required for Transport (ESCRT) pathway by engaging Tsg101 and ALIX through late assembly (L) domains in the C-terminal p6 domain of Gag. However, the L domains in p6 are known to be dispensable for efficient particle production by certain HIV-1 Gag constructs that have the nucleocapsid (NC) domain replaced by a foreign dimerization domain to substitute for the assembly function of NC. We now show that one such L domain-independent HIV-1 Gag construct (termed Z_WT) that has NC-p1-p6 replaced by a leucine zipper domain is resistant to dominant-negative inhibitors of the ESCRT pathway that block HIV-1 particle production. However, Z_WT became dependent on the presence of an L domain when NC-p1-p6 was restored to its C terminus. Furthermore, when the NC domain was replaced by a leucine zipper, the p1-p6 region, but not p6 alone, conferred sensitivity to inhibition of the ESCRT pathway. In an authentic HIV-1 Gag context, the effect of an inhibitor of the ESCRT pathway on particle production could be alleviated by deleting a portion of the NC domain together with p1. Together, these results indicate that the ESCRT pathway dependence of HIV-1 budding is determined, at least in part, by the NC-p1 region of Gag.Human immunodeficiency virus type 1 (HIV-1) and other retroviruses hijack the cellular Endosomal Sorting Complex Required for Transport (ESCRT) pathway to promote the detachment of virions from the cell surface and from each other (3, 21, 42, 44, 47). The ESCRT pathway was initially identified based on its requirement for the sorting of ubiquitinated cargo into multivesicular bodies (MVB) (50, 51). During MVB biogenesis, the ESCRT pathway drives the membrane deformation and fission events required for the inward vesiculation of the limiting membrane of this organelle (26, 29, 50, 51). More recently, it emerged that the ESCRT pathway is also essential for the normal abscission of daughter cells during the final stage of cell division (10, 43). Most of the components of the ESCRT pathway are involved in the formation of four heteromeric protein complexes termed ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III. Additional components include ALIX, which interacts both with ESCRT-I and ESCRT-III, and the AAA ATPase Vps4, which mediates the disassembly of ESCRT-III (29, 42).The deformation and scission of endocytic membranes is thought to be mediated by ESCRT-III, which, together with Vps4, constitutes the most conserved element of the pathway (23, 26, 42). Indeed, it was recently shown that purified yeast ESCRT-III induces membrane deformation (52), and in another study three subunits of yeast ESCRT-III were sufficient to promote the formation of intralumenal vesicles in an in vitro assay (61). In mammals, ESCRT-III is formed by the charged MVB proteins (CHMPs), which are structurally related and tightly regulated through autoinhibition (2, 33, 46, 53, 62). The removal of an inhibitory C-terminal domain induces polymerization and association with endosomal membranes and converts CHMPs into potent inhibitors of retroviral budding (34, 46, 53, 60, 62). Alternatively, CHMPs can be converted into strong inhibitors of the ESCRT pathway and of HIV-1 budding through the addition of a bulky tag such as green fluorescent protein (GFP) or red fluorescent protein (RFP) (27, 36, 39, 54). Retroviral budding in general is also strongly inhibited by catalytically inactive Vps4 (22, 41, 55), or upon Vsp4B depletion (31), confirming the crucial role of ESCRT-III.Retroviruses engage the ESCRT pathway through the activity of so-called late assembly (L) domains in Gag. In the case of HIV-1, the primary L domain maps to a conserved PTAP motif in the C-terminal p6 domain of Gag (24, 28) and interacts with the ESCRT-I component Tsg101 (15, 22, 40, 58). HIV-1 p6 also harbors an auxiliary L domain of the LYPx_nL type, which interacts with the V domain of ALIX (20, 35, 39, 54, 59, 63). Interestingly, Tsg101 binding site mutants of HIV-1 can be fully rescued through the overexpression of ALIX, and this rescue depends on the ALIX binding site in p6 (20, 56). In contrast, the overexpression of a specific splice variant of the ubiquitin ligase Nedd4-2 has been shown to rescue the release and infectivity of HIV-1 mutants lacking all known L domains in p6 (12, 57). Nedd4 family ubiquitin ligases had previously been implicated in the function of PPxY-type L domains, which also depend on an intact ESCRT pathway for function (4, 32, 38). However, HIV-1 Gag lacks PPxY motifs, and the WW domains of Nedd4-2, which mediate its interaction with PPxY motifs, are dispensable for the rescue of HIV-1 L domain mutants (57).ALIX also interacts with the nucleocapsid (NC) region of HIV-1 Gag (18, 49), which is located upstream of p6 and the p1 spacer peptide. ALIX binds HIV-1 NC via its Bro1 domain, and the capacity to interact with NC and to stimulate the release of a minimal HIV-1 Gag construct is shared among widely divergent Bro1 domain proteins (48). Based on these findings and the observation that certain mutations in NC cause a phenotype that resembles that of L domain mutants, it has been proposed that NC cooperates with p6 to recruit the machinery required for normal HIV-1 budding (18, 49).NC also plays a role in Gag polyprotein multimerization, and this function of NC depends on its RNA-binding activity (5-8). It has been proposed that the role of the NC-nucleic acid interaction during assembly is to promote the formation of Gag dimers (37), and HIV-1 assembly in the absence of NC can indeed be efficiently rescued by leucine zipper dimerization domains (65). Surprisingly, in this setting the L domains in p6 also became dispensable, since particle production remained efficient even when the entire NC-p1-p6 region of HIV-1 Gag was replaced by a leucine zipper (1, 65). These findings raised the possibility that the reliance of wild-type (WT) HIV-1 Gag on a functional ESCRT pathway is, at least in part, specified by NC-p1-p6. However, it also remained possible that the chimeric Gag constructs engaged the ESCRT pathway in an alternative manner.In the present report, we provide evidence supporting the first of those two possibilities. Particle production became independent of ESCRT when the entire NC-p1-p6 region was replaced by a leucine zipper, and reversion to ESCRT dependence was shown to occur as a result of restoration of p1-p6 but not of p6 alone. Furthermore, although the deletion of p1 alone had little effect in an authentic HIV-1 Gag context, the additional removal of a portion of NC improved particle production in the presence of an inhibitor of the ESCRT pathway. Together, these data imply that the NC-p1 region plays an important role in the ESCRT-dependence of HIV-1 particle production.     

Structure of the Bro1 domain protein BROX and functional analyses of the ALIX Bro1 domain in HIV-1 budding

Background

Bro1 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 NC^Gag 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 Findings

We 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/Significance

Our 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.     

Divergent Bro1 Domains Share the Capacity To Bind Human Immunodeficiency Virus Type 1 Nucleocapsid and To Enhance Virus-Like Particle Production

To promote the release of infectious virions, human immunodeficiency virus type 1 (HIV-1) exploits the endosomal sorting complex required for transport (ESCRT) pathway by engaging Tsg101 and ALIX through late assembly (L) domains in p6 Gag. An LYPx_nL motif in p6 serves as docking site for the central V domain of ALIX and is required for its ability to stimulate HIV-1 budding. Additionally, the nucleocapsid (NC) domain of Gag binds to the N-terminal Bro1 domain of ALIX, which connects ALIX to the membrane-deforming ESCRT-III complex via its CHMP4 subunits. Since the isolated Bro1 domain of ALIX is sufficient to markedly stimulate virus-like particle (VLP) production in a minimal Gag rescue assay, we examined whether the Bro1 domains of other human proteins possess a similar activity. We now show that the Bro1 domain-only protein Brox and the isolated Bro1 domains of HD-PTP and rhophilin all bind to HIV-1 NC. Furthermore, all shared the capacity to stimulate VLP production by a minimal HIV-1 Gag molecule, and Brox in particular was as potent as the Bro1 domain of ALIX in this assay. Unexpectedly, Brox retained significant activity even if its CHMP4 binding site was disrupted. Thus, the ability to assist in VLP production may be an intrinsic property of the boomerang-shaped Bro1 domain.Retroviruses engage an endosomal budding machinery via so-called late assembly (L) domains in Gag to promote virus budding at the plasma membrane (4, 17, 33). In the case of human immunodeficiency virus type 1 (HIV-1), the C-terminal p6 domain of Gag harbors a conserved P(T/S)AP motif, which binds to the host protein Tsg101 and functions as the primary L domain (18, 29, 44). Additionally, HIV-1 p6 contains an auxiliary L domain of the LYPx_nL type, which serves as a docking site for ALIX (28, 41, 45). Tsg101 and ALIX are both components of a protein network that is required for the biogenesis of multivesicular bodies (MVB) (22, 38). These compartments are formed through the budding of vesicles from the limiting membrane of endosomes into their lumen, a process that is topologically equivalent to virus budding at the plasma membrane. Recently, it emerged that the protein network essential for MVB formation also functions in cytokinesis, which requires a membrane fission event of similar topology (7, 32).Most of the components of the protein network that mediates these events are subunits of heteromeric endosomal sorting complexes required for transport (ESCRT) (3, 22, 38). For instance, Tsg101 is a subunit of the heterotetrameric ESCRT-I complex (22, 38). ESCRT-I and the downstream ESCRT-II are stable complexes, whereas ESCRT-III assembles only upon membrane binding (38). ESCRT-III is formed by the structurally related human CHMP proteins, which exist in an autoinhibited monomeric conformation in the cytosol (40, 46). A conformational change from a closed to an open conformation is thus likely required for the activation of CHMP proteins and the assembly of ESCRT-III. Interestingly, the uncontrolled activation of CHMP proteins through the removal of autoinhibitory C-terminal sequences results in the potent inhibition of HIV-1 budding, indicating a central role for ESCRT-III in retroviral release (46).ALIX consists of a boomerang-shaped N-terminal Bro1 domain, a central ligand binding domain that is shaped like a V, and a C-terminal proline-rich region (16). While ALIX is essential for equine anemia virus budding, its role in HIV-1 budding is less critical than that of Tsg101 (8, 16, 28, 41). However, ALIX can clearly support efficient HIV-1 budding, because its overexpression potently rescues the release defect of Tsg101 binding site mutants (16, 43). This effect of ALIX depends on the interaction between its central V domain and the LYPx_nL motif in HIV-1 p6 (16, 43), confirming that this motif constitutes a functional L domain.The Bro1 domain of ALIX interacts tightly with ESCRT-III subunit CHMP4B and less avidly with CHMP4A and CHMP4C (25, 28, 41, 45). The ability of ALIX to rescue HIV-1 L domain mutants depends on the interaction between its Bro1 domain and CHMP4, indicating that CHMP4 is of particular importance in viral budding (16, 43). Interestingly, human CHMP4A assembles into membrane-attached filaments if overexpressed in mammalian cells, and these filaments can be induced to form circular arrays that drive the formation of buds and tubules with the same topology as that of a retroviral bud (21). Also, the single yeast ortholog of the mammalian CHMP4 proteins forms homo-oligomeric filaments on endosomes that appear to drive MVB sorting and biogenesis (42).By binding to membranes with its convex surface, the Bro1 domain of ALIX could also contribute directly to the generation of negative curvature required for budding away from the cytosol. In support of this notion, we recently observed that the isolated Bro1 domain of ALIX can potently enhance the formation of virus-like particles (VLP) by a minimal HIV-1 Gag construct that retains the primary L domain but lacks certain assembly domains and thus is presumably defective in its ability to deform membranes (37). We also observed that the Bro1 domain of ALIX physically interacts with the nucleocapsid (NC) region of HIV-1 Gag and that mutations in NC that interfere with the interaction induce a phenotype that resembles that of L domain mutants (37).Despite limited sequence homology between human ALIX and a yeast counterpart, the structures of their Bro1 domains are largely superimposable (16, 26), suggesting that all Bro1 domains have a shape that would be compatible with a membrane-deforming function. We therefore asked whether the ability to stimulate VLP production is unique to the Bro1 domain of ALIX or a property of Bro1 domains in general. We now show that widely divergent Bro1 domains share the ability to associate with HIV-1 Gag in an NC-dependent manner and to enhance VLP production by a minimal Gag molecule. In particular, a human Bro1 domain-only protein termed Brox (23) was as potent as the ALIX Bro1 domain in stimulating VLP production, and even forms of Brox that did not bind to CHMP4 retained significant activity. We thus propose that Bro1 domains are inherently capable of promoting budding events away from the cytosol.     

NEDD4L overexpression rescues the release and infectivity of human immunodeficiency virus type 1 constructs lacking PTAP and YPXL late domains

The cellular ESCRT pathway functions in membrane remodeling events that accompany endosomal protein sorting, cytokinesis, and enveloped RNA virus budding. In the last case, short sequence motifs (termed late domains) within human immunodeficiency virus type 1 (HIV-1) p6(Gag) bind and recruit two ESCRT pathway proteins, TSG101 and ALIX, to facilitate virus budding. We now report that overexpression of the HECT ubiquitin E3 ligase, NEDD4L/NEDD4-2, stimulated the release of HIV-1 constructs that lacked TSG101- and ALIX-binding late domains, increasing infectious titers >20-fold. Furthermore, depletion of endogenous NEDD4L inhibited the release of these crippled viruses and led to cytokinesis defects. Stimulation of virus budding was dependent upon the ubiquitin ligase activity of NEDD4L and required only the minimal HIV-1 Gag assembly regions, demonstrating that Gag has ubiquitin-dependent, cis-acting late domain activities located outside of the p6 region. NEDD4L stimulation also required TSG101 and resulted in ubiquitylation of several ESCRT-I subunits, including TSG101. Finally, we found that TSG101/ESCRT-I was required for efficient release of Mason-Pfizer monkey virus, which buds primarily by using a PPXY late domain to recruit NEDD4-like proteins. These observations suggest that NEDD4L and possibly other NEDD4-like proteins can ubiquitylate and activate ESCRT-I to function in virus budding.     

In vitro neurotoxicity of salsolinol is attenuated by the presynaptic protein α-synuclein

Background

Salsolinol (SALSO), a product from the reaction of dopamine (DA) with acetaldehyde, is found increased in dopaminergic neurons of Parkinson's disease (PD) patients. The administration of SALSO in rats causes myenteric neurodegeneration followed by the formation of deposits of the protein α-synuclein (aS), whose aggregation is intimately associated to PD.

Human immunodeficiency virus type 1 Gag engages the Bro1 domain of ALIX/AIP1 through the nucleocapsid

Human immunodeficiency virus type 1 (HIV-1) and other retroviruses harbor short peptide motifs in Gag that promote the release of infectious virions. These motifs, known as late assembly (L) domains, recruit a cellular budding machinery that is required for the formation of multivesicular bodies (MVBs). The primary L domain of HIV-1 maps to a PTAP motif in the p6 region of Gag and engages the MVB pathway by binding to Tsg101. Additionally, HIV-1 p6 harbors an auxiliary L domain that binds to the V domain of ALIX, another component of the MVB pathway. We now show that ALIX also binds to the nucleocapsid (NC) domain of HIV-1 Gag and that ALIX and its isolated Bro1 domain can be specifically packaged into viral particles via NC. The interaction with ALIX depended on the zinc fingers of NC, which mediate the specific packaging of genomic viral RNA, but was not disrupted by nuclease treatment. We also observed that HIV-1 zinc finger mutants were defective for particle production and exhibited a similar defect in Gag processing as a PTAP deletion mutant. The effects of the zinc finger and PTAP mutations were not additive, suggesting a functional relationship between NC and p6. However, in contrast to the PTAP deletion mutant, the double mutants could not be rescued by overexpressing ALIX, further supporting the notion that NC plays a role in virus release.     

A lack of peptide binding and decreased thermostability suggests that the CASKIN2 scaffolding protein SH3 domain may be vestigial

Background

CASKIN2 is a neuronal signaling scaffolding protein comprised of multiple ankyrin repeats, two SAM domains, and one SH3 domain. The CASKIN2 SH3 domain for an NMR structural determination because its peptide-binding cleft appeared to deviate from the repertoire of aromatic enriched amino acids that typically bind polyproline-rich sequences.

Results

The structure demonstrated that two non-canonical basic amino acids (K290/R319) in the binding cleft were accommodated well in the SH3 fold. An K290Y/R319W double mutant restoring the typical aromatic amino acids found in the binding cleft resulted in a 20 °C relative increase in the thermal stability. Considering the reduced stability, we speculated that the CASKIN2 SH3 could be a nonfunctional remnant in this scaffolding protein.

Conclusions

While the NMR structure demonstrates that the CASKIN2 SH3 domain is folded, its cleft has suffered two substitutions that prevent it from binding typical polyproline ligands. This observation led us to additionally survey and describe other SH3 domains in the Protein Data Bank that may have similarly lost their ability to promote protein-protein interactions.

     

Burning cellular bridges: Two pathways to the big breakup

During cytokinetic abscission, the endosomal sorting complex required for transport (ESCRT) proteins are recruited to the midbody and direct the severing of the intercellular bridge. In this issue, Christ et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201507009) demonstrate that two separate but redundant pathways exist to recruit ESCRT-III proteins to the midbody.Over the past 140 years, eukaryotic cell division has been extensively studied and is now understood to be an elaborate, tightly regulated set of events that culminates in the formation of two distinct daughter cells. The M phase of animal cells is characterized by a profound structural reorganization, regulated by a cohort of mitotic kinases and performed by mitosis-specific cytoskeletal structures, including the spindle apparatus and the cytokinetic midbody (Scholey et al., 2003). The completion of cytokinesis, called abscission, involves the severing of the intercellular bridge on both sides of the midbody. In 2007, two landmark studies demonstrated that several ESCRT proteins localize to the midbody and are required for the completion of cytokinesis (Carlton and Martin-Serrano, 2007; Morita et al., 2007). Aside from abscission, the ESCRTs participate in the formation of multivesicular endosomes (MVEs), function in plasma membrane repair, and participate in numerous other cellular processes (Katzmann et al., 2002; Morita and Sundquist, 2004; Hurley, 2015).The canonical model for ESCRT function at MVEs involves the hierarchical recruitment of ESCRT proteins in four unique complexes: ESCRT-0 through ESCRT-III. The ESCRTs cluster cargos and deform membrane, and current models suggest that ESCRT-III subunits polymerize to form filaments that spiral down into the neck of a nascent intralumenal vesicle (Schuh and Audhya, 2014). With the assistance of the VPS4 AAA ATPase, ESCRT filaments are remodeled to facilitate vesicle fission (Shen et al., 2014). Though MVE maturation utilizes all four ESCRT complexes, cytokinetic abscission has been previously thought to require only ESCRT-I, ESCRT-III, and the ESCRT-associated ALG2-interacting factor ALIX (Morita et al., 2010). ALIX interacts with both the ESCRT-I protein TSG101 and all three ESCRT-III CHMP4 isoforms and has been postulated to act as an ESCRT-II bypass for linking ESCRT-I and ESCRT-III in abscission (Schuh and Audhya, 2014). However, the precise mechanism underlying the recruitment of the ESCRT-III complex to the midbody during cytokinesis has remained ambiguous.In this issue, Christ et al. address how the ESCRT-III component CHMP4B (Vps32 in other metazoan systems) is recruited to the midbody and demonstrate the necessity of the ESCRT-II complex in this process. They observed that recruitment of CHMP4B to the midbody was abrogated when they codepleted ALIX and the ESCRT-I component TSG101 in cultured HeLa cells, but that CHMP4B did accumulate when only one of these components was depleted. These data indicate that CHMP4B can be recruited to the midbody via TSG101 or ALIX, but that the two proteins are unlikely to perform this function as a complex, suggesting that CHMP4B recruitment to the midbody involves two independent pathways.After immunofluorescence staining of fixed cells, Christ et al. (2016) found that the endogenous ESCRT-III protein CHMP6 and the ESCRT-II protein EAP20 (VPS20 and VPS25 in other systems, respectively) localize to the midbody, consistent with a previous overexpression study (Thoresen et al., 2014). They additionally performed several depletion experiments to establish that ESCRT-II recruits CHMP6 without affecting TSG101 localization, demonstrating that CHMP6 acts downstream of ESCRT-I and ESCRT-II. This shows that ESCRT-I recruits ESCRT-III to the cytokinetic midbody the same way it does at the MVE.Christ et al. (2016) also show that CHMP4B can still be recruited normally when the ESCRT-II component EAP30 (VPS22 in other systems) is depleted, but not when EAP30 is codepleted with ALIX, strongly suggesting that ALIX-dependent accumulation of CHMP4B does not involve CHMP6 and, more generally, that there are two pathways that can each recruit CHMP4B to the midbody: an ESCRT-I–ESCRT-II–CHMP6 pathway and an ALIX-dependent pathway. It will be important for future work to consider the partial redundancy between these two pathways when assaying the dispensability of early acting ESCRT complexes in cellular processes.In addition, Christ et al. (2016) observed that ALIX depletion led to furrow regression and binucleation in dividing cells with chromatin spanning the intercellular bridge, the same phenotype observed in cells expressing a CHMP4C construct lacking the ALIX interaction domain. Further, they showed that CHMP4C localization to the midbody is abrogated after ALIX depletion but is unaffected by TSG101 knockdowns, strongly implicating ALIX in CHMP4C recruitment independently of ESCRT-I.Our overall understanding of the regulation of abscission still remains elementary (Fig. 1). In addition to the roles of the ESCRT machinery, the chromosomal passenger complex (CPC) regulates the timing of cytokinesis and abscission via interactions with the Polo-like kinase PLK1, the mitotic kinesin-like protein MKLP1, and CEP55, a key component of the midbody that associates directly with both ESCRT-I and ALIX (Schuh and Audhya, 2014). One current model is that the CPC promotes the formation of a ternary complex consisting of CHMP4C, ANCHR, and VPS4 and prevents premature action by VPS4 in response to chromatin trapped in the midbody (Thoresen et al., 2014). It has also been suggested that CHMP4C phosphorylation by the enzymatic core of the CPC, the Aurora B kinase, directs CHMP4C localization to the midbody and its retention of VPS4 (Carlton et al., 2012). With the new findings by Christ et al. (2016), the relationship between Aurora B–mediated phosphorylation of CHMP4C and its ability to bind ALIX must now be further explored. Additionally, because ALIX appears to be the primary factor that recruits CHMP4C to the midbody, it may represent a novel therapeutic target for activation or bypass of the NoCut abscission checkpoint.Open in a separate windowFigure 1.Model for the recruitment of CHMP4B and CHMP4C to the midbody and their roles in regulating the timing of abscission. PLK-1 phosphorylation of CEP55 inhibits its binding to MKLP1. At the end of anaphase, PLK1 is degraded and MKLP1 recruits CEP55 to the midbody. CEP55 recruits TSG101 and ALIX to the midbody, and Christ et al. (2016) demonstrate that there are two pathways that lead to the subsequent recruitment of CHMP4B: one through ESCRT-I–ESCRT-II–CHMP6 and the second directly through ALIX. ALIX also recruits CHMP4C, which, upon phosphorylation by the CPC, is hypothesized to form a ternary complex with ANCHR and VPS4. Formation of this complex prevents VPS4 from facilitating the completion of abscission until all chromatin is cleared from the intercellular bridge.In contrast to the necessity of ALIX during cytokinetic abscission, its role during MVE formation and ubiquitin-dependent cargo degradation remains debatable. Depletion studies suggest that ALIX is dispensable for the lysosomal sorting of several cargoes (Bowers et al., 2006). However, ALIX is capable of targeting to late endosomal membranes through its interaction with lysobisphosphatidic acid, and some data suggest that ALIX can promote ESCRT-III filament assembly at MVEs (Matsuo et al., 2004; Pires et al., 2009; Bissig and Gruenberg, 2014). In the future, it will be essential to elucidate the mechanisms by which ALIX and CHMP6 direct the nucleation of CHMP4B/ESCRT-III spiral filaments and to determine whether the membrane landscapes of the MVE and the cytokinetic bridge differ in a manner that promotes one pathway over the other. As cryoelectron microscopy–based approaches in cells and reconstituted systems advance, the answer to these questions may become more accessible.     

Interaction of the anticancer p28 peptide with p53-DBD as studied by fluorescence,FRET, docking and MD simulations

Background

The p28 peptide, derived from the blue copper protein Azurin, exerts an anticancer action due to interaction with the tumor suppressor p53, likely interfering with its down-regulators. Knowledge of both the kinetics and topological details of the interaction, could greatly help to understand the peptide anticancer mechanism.

An LYPSL Late Domain in the Gag Protein Contributes to the Efficient Release and Replication of Rous Sarcoma Virus

The efficient release of newly assembled retrovirus particles from the plasma membrane requires the recruitment of a network of cellular proteins (ESCRT machinery) normally involved in the biogenesis of multivesicular bodies and in cytokinesis. Retroviruses and other enveloped viruses recruit the ESCRT machinery through three classes of short amino acid consensus sequences termed late domains: PT/SAP, PPXY, and LYPX_nL. The major late domain of Rous sarcoma virus (RSV) has been mapped to a PPPY motif in Gag that binds members of the Nedd4 family of ubiquitin ligases. RSV Gag also contains a second putative late domain motif, LYPSL, positioned 5 amino acids downstream of PPPY. LYPX_nL motifs have been shown to support budding in other retroviruses by binding the ESCRT adaptor protein Alix. To investigate a possible role of the LYPSL motif in RSV budding, we constructed PPPY and LYPSL mutants in the context of an infectious virus and then analyzed the budding rates, spreading profiles, and budding morphology. The data imply that the LYPSL motif acts as a secondary late domain and that its role in budding is amplified in the absence of a fully functional PPPY motif. The LYPXL motif proved to be a stronger late domain when an aspartic acid was substituted for the native serine, recapitulating the properties of the LYPDL late domain of equine infectious anemia virus. The overexpression of human Alix in the absence of a fully functional PPPY late domain partially rescued both the viral budding rate and viral replication, supporting a model in which the RSV LYPSL motif mediates budding through an interaction with the ESCRT adaptor protein Alix.Retroviruses acquire their lipid envelopes from the plasma membrane as they bud from the cell. Although the structural protein Gag is both necessary and sufficient for the assembly of virus-like particles (VLPs), the membrane scission step of virus egress requires the recruitment of a network of cellular proteins normally involved in two analogous cellular membrane fission events, the budding of cargo-containing vesicles into multivesicular bodies (MVBs) (for review, see references 1, 5, 11, and 50) and the separation of two daughter cells during cytokinesis (3, 4). This cellular network of proteins, collectively called the ESCRT (endosomal sorting complex required for transport) machinery, includes four sequentially recruited high-molecular-weight protein complexes (ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III) and is essential for the transport of transmembrane cargo proteins to the lysosome for degradation via an MVB intermediate.In addition to the multiprotein ESCRT complexes, other proteins are required to promote the budding of vesicles into the MVB. Ubiquitin ligases (such as Nedd4) monoubiquitinate both ESCRT components and transmembrane cargo proteins, tagging them for the MVB pathway. Adaptor proteins connect cargo proteins to ESCRT complexes or ESCRT complexes to each other. Ultimately, the final membrane fission event of vesicle budding is mediated by an AAA ATPase (Vps4).Retroviruses as well as other enveloped viruses use three amino acid consensus sequences, PPXY, PT/SAP, and LYPX_nL, as docking sites for the components of the cellular ESCRT machinery. The deletion or mutation of these sequences, termed late domains, results in the failure of the virus to recruit the budding machinery to the site of assembly and thereby results in a block at the late stage of virus release in which fully assembled but immature virus particles remain attached to the plasma membrane. The PPXY late domain interacts with the WW domains of the Nedd4 family of ubiquitin ligases. Multiple ESCRT components bind to monoubiquitin tags on both cargo and ESCRT proteins. The PT/SAP late domain binds the ESCRT-I complex component, Tsg101 (tumor susceptibility gene 101). The LYPX_nL late domain interacts with an adaptor protein of the ESCRT pathway, Alix (ALG-2-interacting protein X; also called AIP1) (reviewed in reference 12). Alix interacts with both Tsg101 of the ESCRT-I complex and CMHP4 of the ESCRT-III complex. A possible fourth class of late domains for the paramyxovirus SV5 was reported previously (47). The late domain function in this case has been mapped to an FPIV sequence in the M (matrix) protein. To date, this motif has yet to be shown to be important for the budding of any other virus, and an FPIV-interacting cellular protein has yet to be identified.Often, retroviruses rely on multiple late domains for efficient budding (2, 13, 16, 29, 30). For example, in addition to its PT/SAP motif in human immunodeficiency virus type 1 (HIV-1) p6, which binds Tsg101 (6, 14, 34, 52), HIV-1 also harbors an Alix-binding LYPX_nL motif that functions in budding (13, 33, 34, 48, 52). Mutation of this LYPX_nL motif results in only a modest reduction in HIV-1 budding (10). However, the effects of mutations in the LYPX_nL motif become more obvious in the context of a minimal Gag in which the globular domain of MA and the N-terminal domain of CA are absent (48). Furthermore, the role of this motif also seems to vary among cell types. For example, the deletion of this motif decreases HIV-1 particle production 2- to 3-fold in COS-7 cells (15) but has no consequence for HeLa cells (7). The relationship of the two viral late domains to each other is unknown. It is possible that they are partially redundant, are cooperative (since they act at slightly different steps in the ESCRT pathway), or are cell type specific. It has been observed that the mutation of one late domain has a larger effect on budding than the mutation of the other, implying a hierarchy of function. For example, in HIV-1, PTAP acts as the dominant late domain and LYPX_nL acts as a secondary late domain. Equine infectious anemia virus (EIAV) seems to be an exception in that it relies only on a single LYPDL motif for late domain function.Like other retroviruses, the avian alpharetrovirus Rous sarcoma virus (RSV) requires the ESCRT pathway for release, as evidenced by the observation that a dominant-negative mutant of the ATPase Vps4, which is required for the final step of the ESCRT pathway that releases the ESCRT-III complex, inhibits RSV budding in a dose-dependent manner (37). Mutational analysis mapped the RSV late domain to the PPPY motif in the small spacer peptide p2b of Gag (41, 54, 56). This PPPY motif was previously shown to interact with chicken members of the Nedd4 family of ubiquitin ligases (21, 51). RSV Gag also harbors an LYPSL late domain consensus motif 5 amino acids downstream from PPPY in the p10 domain, which could potentially promote budding via an interaction with Alix.Alix, a 97-kDa adaptor protein with diverse functions, is composed of an N-terminal Bro1 domain, a central V domain, and a C-terminal proline-rich region (10, 22, 26, 58). The proline-rich region is assumed to be unstructured and binds Tsg101 and endophilins. The Bro1 domain, which binds CHMP4, is curved and resembles a banana shape. CHMP4 binding is functionally important for promoting HIV-1 budding (10). It was suggested previously that its convex face may allow Alix to sense negative curvatures in membranes (17, 22). At least for HIV-1, the Alix Bro1 domain also interacts with the Gag NC domain (42, 43). The central V domain of Alix, which is named for its shape, has a conserved hydrophobic pocket on the second arm near the apex of the V that is responsible for the binding of the LYPX_nL late domains of HIV-1 and EIAV (10, 26, 58).In the present study, we investigated the role of the LYPSL motif in RSV budding and replication. We report here that not only the PPPY motif but also the LYPSL motif act as late domains. The contribution of the LYPSL motif to the budding rate and spreading rate is secondary to that of the PPPY motif but increases in the absence of a fully functional PPPY motif. The Alix overexpression-mediated rescue of PPPY mutants supports a model in which the LYPSL late domain functions through an interaction with Alix.     

Molecular mechanism of selenoprotein P synthesis

Background

Selenoprotein synthesis requires the reinterpretation of a UGA stop codon as one that encodes selenocysteine (Sec), a process that requires a set of dedicated translation factors. Among the mammalian selenoproteins, Selenoprotein P (SELENOP) is unique as it contains a selenocysteine-rich domain that requires multiple Sec incorporation events.

Basic residues in the nucleocapsid domain of Gag are critical for late events of HIV-1 budding

The p6 region of HIV-1 Gag contains two late (L) domains, PTAP and LYPXnL, that bind the cellular proteins Tsg101 and Alix, respectively. These interactions are thought to recruit members of the host fission machinery (ESCRT) to facilitate HIV-1 release. Here we report a new role for the p6-adjacent nucleocapsid (NC) domain in HIV-1 release. The mutation of basic residues in NC caused a pronounced decrease in virus release from 293T cells, although NC mutant Gag proteins retained the ability to interact with cellular membranes and RNAs. Remarkably, electron microscopy analyses of these mutants revealed arrested budding particles at the plasma membrane, analogous to those seen following the disruption of the PTAP motif. This result indicated that the basic residues in NC are important for virus budding. When analyzed in physiologically more relevant T-cell lines (Jurkat and CEM), NC mutant viruses remained tethered to the plasma membrane or to each other by a membranous stalk, suggesting membrane fission impairment. Remarkably, NC mutant release defects were alleviated by the coexpression of a Gag protein carrying a wild-type (WT) NC domain but devoid of all L domain motifs and by providing alternative access to the ESCRT pathway, through the in trans expression of the ubiquitin ligase Nedd4.2s. Since NC mutant Gag proteins retained the interaction with Tsg101, we concluded that NC mutant budding arrests might have resulted from the inability of Gag to recruit or utilize members of the host ESCRT machinery that act downstream of Tsg101. Together, these data support a model in which NC plays a critical role in HIV-1 budding.     

Endosomal Trafficking of HIV-1 Gag and Genomic RNAs Regulates Viral Egress

HIV-1 Gag can assemble and generate virions at the plasma membrane, but it is also present in endosomes where its role remains incompletely characterized. Here, we show that HIV-1 RNAs and Gag are transported on endosomal vesicles positive for TiVamp, a v-SNARE involved in fusion events with the plasma membrane. Inhibition of endosomal traffic did not prevent viral release. However, inhibiting lysosomal degradation induced an accumulation of Gag in endosomes and increased viral production 7-fold, indicating that transport of Gag to lysosomes negatively regulates budding. This also suggested that endosomal Gag-RNA complexes could access retrograde pathways to the cell surface and indeed, depleting cells of TiVamp-reduced viral production. Moreover, inhibition of endosomal transport prevented the accumulation of Gag at sites of cellular contact. HIV-1 Gag could thus generate virions using two pathways, either directly from the plasma membrane or through an endosome-dependent route. Endosomal Gag-RNA complexes may be delivered at specific sites to facilitate cell-to-cell viral transmission.The production of infectious retroviral particles is an ordered process that includes many steps (for review see Refs. 1–3). In particular, three major viral components, Gag, the envelope, and genomic RNAs have to traffic inside the cell to reach their assembly site. Viral biogenesis is driven by the polyprotein Gag, which is able to make viral-like particles when expressed alone (4). Upon release, HIV-1⁴ Gag is processed by the viral protease into matrix (MA(p17)), capsid (CA(p24)), nucleocapsid (NC(p7)), p6, and smaller peptides SP1 and SP2. Gag contains several domains that are essential for viral assembly: a membrane binding domain (M) in MA; a Gag-Gag interaction domain in CA; an assembly domain (I) in NC; and a late domain (L) in p6, which recruits the cellular budding machinery. Genomic RNAs are specifically recognized by NC, and they play fundamental roles in viral biogenesis by acting as a scaffold for Gag multimerization (5).It has been demonstrated that retroviruses bud by hijacking the endosomal machinery that sorts proteins into internal vesicles of multivesicular bodies (for review, see Refs. 6, 7). Indeed, these vesicles bud with the same topology as viral particles. Proteins sorted into this pathway are usually destined for degradation in lysosomes, but some can also recycle to the plasma membrane (for review see Refs. 8, 9). They are also frequently ubiquitinated on their cytoplasmic domain (10, 11), allowing their recognition by ESCRT complexes. ESCRT-0 and ESCRT-I recognize ubiquitinated cargo present at the surface of endosomes and recruit other ESCRT complexes (12–14). ESCRT-III is believed to function directly in the formation of multivesicular body intralumenal vesicles (12), even though its mechanism of action is currently not understood. Remarkably, Gag L domains interact directly with components of the multivesicular body-sorting machinery (for review see Ref. 15). HIV-1 Gag uses a PTAP motif to bind Tsg101, a component of ESCRT-I (16–19), and a YPLTSL motif to interact with Alix, a protein linked to ESCRT-I and -III (20–22). Finally, various ubiquitin ligases are also required directly or indirectly during HIV-1 biogenesis (23, 24; for review see Ref. 25).In many cell lines, Gag is found both at the plasma membrane and in endosomes. This has led to the hypothesis that there are several assembly sites for HIV-1 (1, 3). First, Gag can initiate and complete assembly at the plasma membrane. This is thought to occur predominantly in T lymphocytes, and this process is supported by several lines of evidences: (i) disruption of endosomal trafficking with drugs does not prevent viral production (26, 27); (ii) ESCRT complexes can be recruited at the plasma membrane, at sites where Gag accumulates (28–30); (iii) Gag can be seen multimerizing and budding from the plasma membrane in live cells (31). Second, Gag could initiate assembly in endosomes, and then traffic to the cell surface to be released. This is mainly supported by the presence of Gag in endosomes in several cell lines (32–34), including T cells and more strikingly macrophages (32, 35, 36–39). However, we are currently lacking functional experiments addressing the role of this endosomal pool of Gag, and it is still not clear to what extent it contributes to the production of viral particles. Nevertheless, the presence of Gag in endosomes might facilitate recruitment of ESCRT complexes (34, 40), packaging of viral genomic RNAs (32, 41), and incorporation of the envelope (42). It may also be important for polarized budding (43, 44) and to create a viral reservoir in infected cells (45, 46).Despite great progress, the traffic of HIV-1 components is still not fully elucidated. In particular, the transport of the genomic RNAs is poorly understood. In this study, we have used single molecule techniques to investigate the trafficking of HIV-1 RNAs in fixed and live cells, and we show that they are transported on endosomal vesicles. We also obtained functional evidence that Gag and viral RNAs can use at least two trafficking pathways to produce virions, one going directly from the plasma membrane and another one passing through endosomes.     

Amphipathic helical peptides hamper protein-protein interactions of the intrinsically disordered chromatin nuclear protein 1 (NUPR1)

Background

NUPR1 is a multifunctional intrinsically disordered protein (IDP) involved, among other functions, in chromatin remodelling, and development of pancreatic ductal adenocarcinoma (PDAC). It interacts with several biomolecules through hydrophobic patches around residues Ala33 and Thr68. The drug trifluoperazine (TFP), which hampers PDAC development in xenografted mice, also binds to those regions. Because of the large size of the hot-spot interface of NUPR1, small molecules could not be adequate to modulate its functions.

X-ray spectroscopy and imaging of selenium in living systems

Background

Selenium is an essential element with a rich and varied chemistry in living organisms. It plays a variety of important roles ranging from being essential in enzymes that are critical for redox homeostasis to acting as a deterrent for herbivory in hyperaccumulating plants. Despite its importance there are many open questions, especially related to its chemistry in situ within living organisms.

Inhibition of amyloid fibril formation in the variable domain of λ6 light chain mutant Wil caused by the interaction between its unfolded state and epigallocatechin-3-O-gallate

Background

Light chains are abnormally overexpressed from disordered monoclonal B-cells and form amyloid fibrils, which are then deposited on the affected organ, leading to a form of systemic amyloidosis known as AL (Amyloid Light chain) amyloidosis. A green tea catechin, epigallocatechin-3-O-gallate (EGCG), which is thought to inhibit various amyloidoses, is a potent inhibitor of amyloid fibril formation in AL amyloidosis.

Decoding the intrinsic mechanism that prohibits ALIX interaction with ESCRT and viral proteins

The adaptor protein ALIX [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] links retroviruses to ESCRT (endosomal sorting complex required for transport) machinery during retroviral budding. This function of ALIX requires its interaction with the ESCRT-III component CHMP4 (charged multivesicular body protein 4) at the N-terminal Bro1 domain and retroviral Gag proteins at the middle V domain. Since cytoplasmic or recombinant ALIX is unable to interact with CHMP4 or retroviral Gag proteins under non-denaturing conditions, we constructed ALIX truncations and mutations to define the intrinsic mechanism through which ALIX interactions with these partner proteins are prohibited. Our results demonstrate that an intramolecular interaction between Patch 2 in the Bro1 domain and the TSG101 (tumour susceptibility gene 101 protein)-docking site in the proline-rich domain locks ALIX into a closed conformation that renders ALIX unable to interact with CHMP4 and retroviral Gag proteins. Relieving the intramolecular interaction of ALIX, by ectopically expressing a binding partner for one of the intramolecular interaction sites or by deleting one of these sites, promotes ALIX interaction with these partner proteins and facilitates ALIX association with the membrane. Ectopic expression of a GFP (green fluorescent protein)-ALIX mutant with a constitutively open conformation, but not the wild-type protein, increases EIAV (equine infectious anaemia virus) budding from HEK (human embryonic kidney)-293 cells. These findings predict that relieving the autoinhibitory intramolecular interaction of ALIX is a critical step for ALIX to participate in retroviral budding.