RetroCHMP3 blocks budding of enveloped viruses without blocking cytokinesis.

Many enveloped viruses require the endosomal sorting complexes required for transport (ESCRT) pathway to exit infected cells. This highly conserved pathway mediates essential cellular membrane fission events, which restricts the acquisition of adaptive mutations to counteract viral co-option. Here, we describe duplicated and truncated copies of the ESCRT-III factor CHMP3 that block ESCRT-dependent virus budding and arose independently in New World monkeys and mice. When expressed in human cells, these retroCHMP3 proteins potently inhibit release of retroviruses, paramyxoviruses, and filoviruses. Remarkably, retroCHMP3 proteins have evolved to reduce interactions with other ESCRT-III factors and have little effect on cellular ESCRT processes, revealing routes for decoupling cellular ESCRT functions from viral exploitation. The repurposing of duplicated ESCRT-III proteins thus provides a mechanism to generate broad-spectrum viral budding inhibitors without blocking highly conserved essential cellular ESCRT functions.

Structures, Functions, and Dynamics of ESCRT-III/Vps4 Membrane Remodeling and Fission Complexes.

The endosomal sorting complexes required for transport (ESCRT) pathway mediates cellular membrane remodeling and fission reactions. The pathway comprises five core complexes: ALIX, ESCRT-I, ESCRT-II, ESCRT-III, and Vps4. These soluble complexes are typically recruited to target membranes by site-specific adaptors that bind one or both of the early-acting ESCRT factors: ALIX and ESCRT-I/ESCRT-II. These factors, in turn, nucleate assembly of ESCRT-III subunits into membrane-bound filaments that recruit the AAA ATPase Vps4. Together, ESCRT-III filaments and Vps4 remodel and sever membranes. Here, we review recent advances in our understanding of the structures, activities, and mechanisms of the ESCRT-III and Vps4 machinery, including the first high-resolution structures of ESCRT-III filaments, the assembled Vps4 enzyme in complex with an ESCRT-III substrate, the discovery that ESCRT-III/Vps4 complexes can promote both inside-out and outside-in membrane fission reactions, and emerging mechanistic models for ESCRT-mediated membrane fission.

Membrane fission reactions of the mammalian ESCRT pathway.

The endosomal sorting complexes required for transport (ESCRT) pathway was initially defined in yeast genetic screens that identified the factors necessary to sort membrane proteins into intraluminal endosomal vesicles. Subsequent studies have revealed that the mammalian ESCRT pathway also functions in a series of other key cellular processes, including formation of extracellular microvesicles, enveloped virus budding, and the abscission stage of cytokinesis. The core ESCRT machinery comprises Bro1 family proteins and ESCRT-I, ESCRT-II, ESCRT-III, and VPS4 complexes. Site-specific adaptors recruit these soluble factors to assemble on different cellular membranes, where they carry out membrane fission reactions. ESCRT-III proteins form filaments that draw membranes together from the cytoplasmic face, and mechanistic models have been advanced to explain how ESCRT-III filaments and the VPS4 ATPase can work together to catalyze membrane fission.

Immunogenicity and Vaccine Shedding After 1 or 2 Doses of rVSVΔG-ZEBOV-GP Ebola Vaccine (ERVEBO®): Results From a Phase 2, Randomized, Placebo-controlled Trial in Children and Adults.

BACKGROUND: The rVSVΔG-ZEBOV-GP vaccine (ERVEBO®) is a single-dose, live-attenuated, recombinant vesicular stomatitis virus vaccine indicated for the prevention of Ebola virus disease (EVD) caused by Zaire ebolavirus in individuals 12 months of age and older. METHODS: The Partnership for Research on Ebola VACcination (PREVAC) is a multicenter, phase 2, randomized, double-blind, placebo-controlled trial of 3 vaccine strategies in healthy children (ages 1-17) and adults, with projected 5 years of follow-up (NCT02876328). Using validated assays (GP-ELISA and PRNT), we measured antibody responses after 1-dose rVSVΔG-ZEBOV-GP, 2-dose rVSVΔG-ZEBOV-GP (given on Day 0 and Day 56), or placebo. Furthermore, we quantified vaccine virus shedding in a subset of children's saliva using RT-PCR. RESULTS: In total, 819 children and 783 adults were randomized to receive rVSVΔG-ZEBOV-GP (1 or 2 doses) or placebo. A single dose of rVSVΔG-ZEBOV-GP increased antibody responses by Day 28 that were sustained through Month 12. A second dose of rVSVΔG-ZEBOV-GP given on Day 56 transiently boosted antibody concentrations. In vaccinated children, GP-ELISA titers were superior to placebo and non-inferior to vaccinated adults. Vaccine virus shedding was observed in 31.7% of children, peaking by Day 7, with no shedding observed after Day 28 post-dose 1 or any time post-dose 2. CONCLUSIONS: A single dose of rVSVΔG-ZEBOV-GP induced robust antibody responses in children that was non-inferior to the responses induced in vaccinated adults. Vaccine virus shedding in children was time-limited and only observed after the first dose. Overall, these data support the use of rVSVΔG-ZEBOV-GP for the prevention of EVD in at-risk children. Clinical Trials Registration. The study is registered at ClinicalTrials.gov (NCT02876328), the Pan African Clinical Trials Registry (PACTR201712002760250), and the European Clinical Trials Register (EudraCT number: 2017-001798-18).

Structure of spastin bound to a glutamate-rich peptide implies a hand-over-hand mechanism of substrate translocation.

Many members of the AAA+ ATPase family function as hexamers that unfold their protein substrates. These AAA unfoldases include spastin, which plays a critical role in the architecture of eukaryotic cells by driving the remodeling and severing of microtubules, which are cytoskeletal polymers of tubulin subunits. Here, we demonstrate that a human spastin binds weakly to unmodified peptides from the C-terminal segment of human tubulin α1A/B. A peptide comprising alternating glutamate and tyrosine residues binds more tightly, which is consistent with the known importance of glutamylation for spastin microtubule severing activity. A cryo-EM structure of the spastin-peptide complex at 4.2 Å resolution revealed an asymmetric hexamer in which five spastin subunits adopt a helical, spiral staircase configuration that binds the peptide within the central pore, whereas the sixth subunit of the hexamer is displaced from the peptide/substrate, as if transitioning from one end of the helix to the other. This configuration differs from a recently published structure of spastin from Drosophila melanogaster, which forms a six-subunit spiral without a transitioning subunit. Our structure resembles other recently reported AAA unfoldases, including the meiotic clade relative Vps4, and supports a model in which spastin utilizes a hand-over-hand mechanism of tubulin translocation and microtubule remodeling.

Anxiety among newly-qualified doctors: An eight-year analysis.

Background: Stressed and anxious doctors are more likely to make errors, take time off work and to leave medicine. This study aims to quantify the prevalence of anxiety among newly-qualified Foundation Year 1 doctors (FY1s), identify high risk groups and determine workplace factors associated with anxiety.Methods: We investigated self-reported anxiety among eight cohorts of FY1s between 2010 and 2017. Participants completed an online survey after their first week of work (n = 11,839), with a follow-up survey later in the year (n = 3502). Surveys included questions about the workplace and a validated screening tool for pathological anxiety.Results: Overall, a large proportion of doctors screened positive for pathological anxiety at the start of their FY1 year (27.3%) and after 4 months of work (21.0%). Year-on-year, we found a growing burden of anxiety at the start of FY1 (22.8% in 2010 vs. 29.6% in 2017, p < 0.01) and at follow-up. Anxiety was significantly higher among females (p < 0.01), those aged 21-25 (p < 0.05) and those who did not feel part of a team (p < 0.01).Conclusion: We found a growing burden of anxiety among FY1s associated with a perceived lack of support. We hope our findings will inform interventions to support newly-qualified doctors as they transition into the workplace.

The evolutionary adaptation of the C282Y mutation to culture and climate during the European Neolithic.

OBJECTIVES: The C282Y allele is the major cause of hemochromatosis as a result of excessive iron absorption. The mutation arose in continental Europe no earlier than 6,000 years ago, coinciding with the arrival of the Neolithic agricultural revolution. Here we hypothesize that this new Neolithic diet, which originated in the sunny warm and dry climates of the Middle East, was carried by migrating farmers into the chilly and damp environments of Europe where iron is a critical micronutrient for effective thermoregulation. We argue that the C282Y allele was an adaptation to this novel environment. MATERIALS AND METHODS: To address our hypothesis, we compiled C282Y allele frequencies, known Neolithic sites in Europe and climatic data on temperature and rainfall for statistical analysis. RESULTS: Our findings indicate that the geographic cline for C282Y frequency in Europe increases as average temperatures decrease below 16°C, a critical threshold for thermoregulation, with rainy days intensifying the trend. DISCUSSION: The results indicate that the deleterious C282Y allele, responsible for most cases of hemochromatosis, may have evolved as a selective advantage to culture and climate during the European Neolithic.

Hemochromatosis: Niche Construction and the Genetic Domino Effect in the European Neolithic.

Hereditary hemochromatosis is caused by a potentially lethal recessive gene (HFE, C282Y allele) that increases iron absorption and reaches polymorphic levels in northern European populations. Because persons carrying the allele absorb iron more readily than do noncarriers, it has often been suggested that HFE is an adaptation to anemia. We hypothesize positive selection for HFE began during or after the European Neolithic with the adoption of an iron-deficient high-grain and dairying diet and consequent anemia, a finding confirmed in Neolithic and later European skeletons. HFE frequency compared with rate of lactase persistence in Eurasia yields a positive linear correlation coefficient of 0.86. We suggest this is just one of many mutations that became common after the adoption of agriculture.

Compliant Intramedullary Stems for Joint Reconstruction.

The longevity of current joint replacements is limited by aseptic loosening, which is the primary cause of non-infectious failure for hip, knee, and ankle arthroplasty. Aseptic loosening is typically caused either by osteolysis from particulate wear, or by high shear stresses at the bone-implant interface from over-constraint. Our objective was to demonstrate feasibility of a compliant intramedullary stem that eliminates over-constraint without generating particulate wear. The compliant stem is built around a compliant mechanism that permits rotation about a single axis. We first established several models to understand the relationship between mechanism geometry and implant performance under a given angular displacement and compressive load. We then used a neural network to identify a design space of geometries that would support an expected 100-year fatigue life inside the body. We additively manufactured one representative mechanism for each of three anatomic locations, and evaluated these prototypes on a KR-210 robot. The neural network predicts maximum stress and torsional stiffness with 2.69% and 4.08% error respectively, relative to finite element analysis data. We identified feasible design spaces for all three of the anatomic locations. Simulated peak stresses for the three stem prototypes were below the fatigue limit. Benchtop performance of all three prototypes was within design specifications. Our results demonstrate the feasibility of designing patient- and joint-specific compliant stems that address the root causes of aseptic loosening. Guided by these results, we expect the use of compliant intramedullary stems in joint reconstruction technology to increase implant lifetime.

ALIX-CHMP4 interactions in the human ESCRT pathway.

The ESCRT pathway facilitates membrane fission events during enveloped virus budding, multivesicular body formation, and cytokinesis. To promote HIV budding and cytokinesis, the ALIX protein must bind and recruit CHMP4 subunits of the ESCRT-III complex, which in turn participate in essential membrane remodeling functions. Here, we report that the Bro1 domain of ALIX binds specifically to C-terminal residues of the human CHMP4 proteins (CHMP4A-C). Crystal structures of the complexes reveal that the CHMP4 C-terminal peptides form amphipathic helices that bind across the conserved concave surface of ALIX(Bro1). ALIX-dependent HIV-1 budding is blocked by mutations in exposed ALIX(Bro1) residues that help contribute to the binding sites for three essential hydrophobic residues that are displayed on one side of the CHMP4 recognition helix (M/L/IxxLxxW). The homologous CHMP1-3 classes of ESCRT-III proteins also have C-terminal amphipathic helices, but, in those cases, the three hydrophobic residues are arrayed with L/I/MxxxLxxL spacing. Thus, the distinct patterns of hydrophobic residues provide a "code" that allows the different ESCRT-III subunits to bind different ESCRT pathway partners, with CHMP1-3 proteins binding MIT domain-containing proteins, such as VPS4 and Vta1/LIP5, and CHMP4 proteins binding Bro1 domain-containing proteins, such as ALIX.

Membrane Remodeling: ESCRT-III Filaments as Molecular Garrotes.

New reconstitution, biochemical and structural studies are revealing how the core machinery of the ESCRT pathway constricts membranes to promote fission. Equally exciting is the discovery and characterization of conserved ESCRT-like machinery across all three domains of life.

Membrane constriction and thinning by sequential ESCRT-III polymerization.

The endosomal sorting complexes required for transport (ESCRTs) mediate diverse membrane remodeling events. These typically require ESCRT-III proteins to stabilize negatively curved membranes; however, recent work has indicated that certain ESCRT-IIIs also participate in positive-curvature membrane-shaping reactions. ESCRT-IIIs polymerize into membrane-binding filaments, but the structural basis for negative versus positive membrane remodeling by these proteins remains poorly understood. To learn how certain ESCRT-IIIs shape positively curved membranes, we determined structures of human membrane-bound CHMP1B-only, membrane-bound CHMP1B + IST1, and IST1-only filaments by cryo-EM. Our structures show how CHMP1B first polymerizes into a single-stranded helical filament, shaping membranes into moderate-curvature tubules. Subsequently, IST1 assembles a second strand on CHMP1B, further constricting the membrane tube and reducing its diameter nearly to the fission point. Each step of constriction thins the underlying bilayer, lowering the barrier to membrane fission. Our structures reveal how a two-component, sequential polymerization mechanism drives membrane tubulation, constriction and bilayer thinning.

Activation of the endosome-associated ubiquitin isopeptidase AMSH by STAM, a component of the multivesicular body-sorting machinery.

AMSH is an endosomal ubiquitin isopeptidase that can limit EGF receptor downregulation . It directly binds to the SH3 domain of STAM, which is constitutively associated with Hrs, a component of clathrin-coated structures on endosomes. This clathrin coat has been implicated in the recruitment of ubiquitinated growth factor receptors prior to their incorporation into internal vesicles of the multivesicular body (MVB) , through the concerted action of ESCRT complexes I, II, and III . We now show that AMSH is embedded within a network of interactions with components of the MVB-sorting machinery. AMSH and STAM, like Hrs , both bind directly to clathrin. AMSH also interacts with mVps24/CHMP3, a component of ESCRT III complex, and this interaction is reinforced through simultaneous STAM binding. We have explored the effect of interacting components on the in vitro enzymatic activity of AMSH. The enzyme shows specificity for K63- over K48-linked polyubiquitin chains in vitro and is markedly stimulated by coincubation with STAM, indicating that activation of AMSH is coupled to its association with the MVB-sorting machinery. Other interacting factors do not directly stimulate AMSH but may serve to orient the enzyme with respect to substrates on the endosomal membrane.

AMSH is an endosome-associated ubiquitin isopeptidase.

The JAMM (JAB1/MPN/Mov34 metalloenzyme) motif has been proposed to provide the active site for isopeptidase activity associated with the Rpn11/POH1 subunit of the 19S-proteasome and the Csn5-subunit of the signalosome. We have looked for similar activity in associated molecule with the SH3 domain of STAM (AMSH), a JAMM domain-containing protein that associates with the SH3-domain of STAM, a protein, which regulates receptor sorting at the endosome. We demonstrate isopeptidase activity against K48-linked tetraubiquitin and K63-linked polyubiquitin chains to generate di-ubiquitin and free ubiquitin, respectively. An inactivating mutation (D348A) in AMSH leads to accumulation of ubiquitin on endosomes and the concomitant stabilization of a ubiquitinated form of STAM, which requires an intact ubiquitin interaction motif (UIM) within STAM. Short interfering RNA knockdown of AMSH enhances the degradation rate of EGF receptor (EGFR) following acute stimulation and ubiquitinated EGFR provides a substrate for AMSH in vitro. We propose that AMSH is a deubiquitinating enzyme with functions at the endosome, which oppose the ubiquitin-dependent sorting of receptors to lysosomes.

A Tunable Microfluidic Device Enables Cargo Encapsulation by Cell- or Organelle-Sized Lipid Vesicles Comprising Asymmetric Lipid Bilayers.

Cellular membranes play host to a wide variety of morphologically and chemically complex processes. Although model membranes, like liposomes, are already widely used to reconstitute and study these processes, better tools are needed for making model bilayers that faithfully mimic cellular membranes. Existing methods for fabricating cell-sized (µm) or organelle-sized (tens to hundreds of nanometers) lipid vesicles have distinctly different requirements. Of particular note for biology, it remains challenging for any technique to efficiently encapsulate fragile cargo molecules or to generate liposomes with stable, asymmetric lipid leaflets within the bilayer. Here a tunable microfluidic device and protocol for fabricating liposomes with desired diameters ranging from ≈10 µm to ≈100 nm are described. Lipid vesicle size is templated by the simple inclusion of a polycarbonate filter within the microfluidic system and tuned with flow rate. It is shown that the vesicles made with this device are stable, unilamellar, lipid asymmetric, and capable of supporting transmembrane protein assembly, peripheral membrane protein binding, as well as soluble cargo encapsulation (including designer nanocages for biotechnology applications). These fabricated vesicles provide a new platform for studying the biophysically rich processes found within lipid-lipid and lipid-protein systems typically associated with cellular membranes.

Endosomal dynamics of Met determine signaling output.

Proteasomal activity is required for Met receptor degradation after acute stimulation with hepatocyte growth factor (HGF). Inhibition of proteasomal activity with lactacystin leads to a block in the endocytic trafficking of Met such that the receptor fails to reach late endosomes/lysosomes, where degradation by acid-dependent proteases takes place (). In this article, we have biochemically determined Met internalization rates from the cell surface and shown that lactacystin does not inhibit the initial HGF-dependent internalization step of Met. Instead, it promotes the recycling pathway from early endosomes at the expense of sorting to late endosomes, thereby ensuring rapid return of internalized Met to the cell surface. We have used this perturbation of Met endosomal sorting by lactacystin to examine the consequences for HGF-dependent signaling outputs. In control cells HGF-dependent receptor autophosphorylation reaches a maximal level over 5-10 min but then attenuates over the ensuing 50 min. Furthermore, Met dephosphorylation can be kinetically dissociated from Met degradation. In lactacystin-treated cells, we observe a failure of Met dephosphorylation as well as Met degradation. Elements of the mitogen-activated protein kinase cascade, downstream of receptor activation, show a normal kinetic profile of phosphorylation, indicating that the mitogen-activated protein kinase pathway can attenuate in the face of sustained receptor activation. The HGF-dependent phosphorylation of a receptor substrate that is localized to clathrin-coated regions of sorting endosomes, Hrs, is dramatically reduced by lactacystin treatment. Reduction of cellular Hrs levels by short interfering RNA modestly retards Met degradation and markedly prevents the attenuation of Met phosphorylation. HGF-dependent Hrs phosphorylation and Met dephosphorylation may provide signatures for retention of the receptor in coated regions of the endosome implicated in sorting to lysosomes.

Structure and membrane remodeling activity of ESCRT-III helical polymers.

The endosomal sorting complexes required for transport (ESCRT) proteins mediate fundamental membrane remodeling events that require stabilizing negative membrane curvature. These include endosomal intralumenal vesicle formation, HIV budding, nuclear envelope closure, and cytokinetic abscission. ESCRT-III subunits perform key roles in these processes by changing conformation and polymerizing into membrane-remodeling filaments. Here, we report the 4 angstrom resolution cryogenic electron microscopy reconstruction of a one-start, double-stranded helical copolymer composed of two different human ESCRT-III subunits, charged multivesicular body protein 1B (CHMP1B) and increased sodium tolerance 1 (IST1). The inner strand comprises "open" CHMP1B subunits that interlock in an elaborate domain-swapped architecture and is encircled by an outer strand of "closed" IST1 subunits. Unlike other ESCRT-III proteins, CHMP1B and IST1 polymers form external coats on positively curved membranes in vitro and in vivo. Our analysis suggests how common ESCRT-III filament architectures could stabilize different degrees and directions of membrane curvature.