Biology subjects

Briggs, J. A. G.

Publications and source records attributed to Briggs, J. A. G..

At least 19 recordsLinked to original sources

bioRxiv2026

Molecular mechanism of M1-mediated influenza A virus assembly

Influenza A virus assembly is orchestrated by matrix protein 1 (M1), which engages ribonucleoproteins, mediates glycoprotein incorporation, and scaffolds lipid envelopment, but how these activities are performed and coordinated is unclear. We determined high-resolution structures of M1 within virions, virus-like particles, and in vitro. We found that M1 binds phosphatidylinositol 4,5-bisphosphate at the membrane, oligomerizes through an electrostatic interface, and switches between three alternative conformational states in different regions of the virion. The M1 conformer at the front associates with ribonucleoproteins and initiates budding. In the body of the virion, two conformers alternate to create an extended filament with seam-like discontinuities. Enrichment of the seam-prone conformation at the virion rear induces envelope closure and exposes a binding site for the cytoplasmic tail of neuraminidase (NA) to promote virus release. Conformational switching within a polarized M1 lattice thereby couples organization of the viral components with shaping of the virion architecture.

microbiology↗

bioRxiv2024

Towards community-driven visual proteomics with large-scale cryo-electron tomography of Chlamydomonas reinhardtii

In situ cryo-electron tomography (cryo-ET) has emerged as the method of choice to investigate structures of biomolecules in their native context. However, challenges remain in the efficient production of large-scale cryo-ET datasets, as well as the community sharing of this information-rich data. Here, we applied a cryogenic plasma-based focused ion beam (cryo-PFIB) instrument for high-throughput milling of the green alga Chlamydomonas reinhardtii, a useful model organism for in situ visualization of numerous fundamental cellular processes. Combining cryo-PFIB sample preparation with recent advances in cryo-ET data acquisition and processing, we generated a dataset of 1829 reconstructed and annotated tomograms, which we provide as a community resource to drive method development and inspire biological discovery. To assay the quality of this dataset, we performed subtomogram averaging (STA) of both soluble and membrane-bound complexes ranging in size from >3 MDa to [~]200 kDa, including 80S ribosomes, Rubisco, nucleosomes, microtubules, clathrin, photosystem II, and mitochondrial ATP synthase. The majority of these density maps reached sub-nanometer resolution, demonstrating the potential of this C. reinhardtii dataset, as well as the promise of modern cryo-ET workflows and open data sharing towards visual proteomics.

cell biology↗

bioRxiv2024

The conserved HIV-1 spacer peptide 2 triggers matrix lattice maturation

HIV-1 particles are released in an immature, non-infectious form. Proteolytic cleavage of the main structural polyprotein Gag into functional domains induces rearrangement into mature, infectious virions. In immature virus particles, the Gag membrane binding domain, MA, forms a hexameric protein lattice that undergoes structural transition upon cleavage into a distinct, mature MA lattice. The mechanism of MA lattice maturation is unknown. Here we show that released spacer peptide 2 (SP2), a conserved peptide of unknown function situated [~]300 residues downstream of MA, binds MA to induce structural maturation. By high-resolution in-virus structure determination of MA, we show that MA does not bind lipid into a side pocket as previously thought, but instead binds SP2 as an integral part of the protein-protein interfaces that stabilise the mature lattice. Analysis of Gag cleavage site mutants showed that SP2 release is required for MA maturation, and we demonstrate that SP2 is sufficient to induce maturation of purified MA on lipid layers in vitro. SP2-triggered MA maturation correlated with faster fusion of virus with target cells. Our results reveal a new, unexpected interaction between two HIV-1 components, provide a high-resolution structure of mature MA, establish the trigger of MA structural maturation, and assign function to the SP2 peptide.

microbiology↗

bioRxiv2024

A physical model for M1-mediated influenza A virus assembly

Influenza A virus particles assemble at the plasma membrane of infected cells. During assembly all components of the virus come together in a coordinated manner to deform the membrane into a protrusion eventually forming a new, membrane-enveloped virus. Here we integrate recent molecular insights of this process, particularly concerning the structure of the matrix protein 1 (M1), within a theoretical framework describing the mechanics of virus assembly. Our model describes M1 polymerization and membrane protrusion formation, explaining why it is efficient for M1 to form long strands assembling into helices in filamentous virions. Eventually, we find how the architecture of M1 helices is controlled by physical properties of viral proteins and the host cell membrane. Finally, by considering the growth force and speed of viral filaments, we propose that the helical geometry of M1 strands might have evolved to optimize for fast and efficient virus assembly and growth. SignificanceInfluenza A virus remains a major threat to public health. Its most abundant viral protein, matrix protein 1 (M1), forms an endoskeleton underneath the viral membrane, but how this endoskeleton contributes to the virus lifecycle is poorly understood. Combining cryo-electron tomography data and structural data with theoretical predictions, we explain how the energetically favorable polymerization of M1 into helical strands mediates the membrane deformations that permit the virus to exit infected cells. Our analysis of M1s variable architecture provides insights into adaptive strategies of the virus for efficient growth under variable local conditions. The quantitative framework developed in this study could be extrapolated to other enveloped viruses and generally applied to protein-driven membrane deformations.

biophysics↗

bioRxiv2024

Ultrastructural Dynamics of Dopaminergic Presynaptic Release Sites revealed by Cryo-correlative Light and Electron Microscopy

Dopaminergic neurons are fundamental in governing motivation, movement, and many aspects of cognition. The targeted modulation of dopaminergic signaling serves as a cornerstone in developing therapeutic interventions for conditions such as Parkinsons disease, schizophrenia, and addiction. Despite the pivotal role of dopaminergic neurons, the ultrastructure and associated dynamics of dopaminergic synapses remain poorly understood. Here, we develop and utilize a cryo-correlative light and electron microscopy process chain to investigate the micro- to nanoscale architecture and organelle content of dopaminergic presynaptic release sites. Using cryo electron tomography, we identify several protein complexes crucial to dopaminergic function and we utilize subtomogram averaging to resolve in situ assemblies of the TRiC/CCT chaperone and vacuolar-type ATPase. Lastly, we find that pharmacological treatments using either dopamine or the dopamine D2 receptor antagonist, haloperidol, bidirectionally modulate vesicular content, mitochondrial size and calcium phosphate deposition. These findings contribute to our general understanding of the composition and ultrastructural dynamics of dopaminergic presynaptic release sites and provide a methodological platform for further studies of the structure and cell biology of dopaminergic neurons and their responses.

neuroscience↗

bioRxiv2023

Molecular dynamics simulations of HIV-1 matrix-membrane interactions at different stages of viral maturation

Although the structural rearrangement of the membrane-bound matrix (MA) protein trimers upon HIV-1 maturation has been reported, the consequences of MA maturation on the MA-lipid interactions are not well understood. Long-timescale molecular dynamics (MD) simulations of the MA multimeric assemblies of immature and mature virus particles with our realistic asymmetric membrane model have explored MA-lipid interactions and lateral organization of lipids around MA complexes. The number of stable MA-PS and MA-PIP2 interactions at the trimeric interface of the mature MA complex is observed to be greater compared to that of the immature MA complex. Our simulations identified an alternative PIP2 binding site in the immature MA complex where the multivalent headgroup of a PIP2 lipid with a greater negative charge binds to multiple basic amino acid residues such as, ARG3 residues of both the MA monomers at the trimeric interface, and HBR residues (LYS29, LYS31) of one of the MA monomers. Our enhanced sampling simulations have explored the conformational space of phospholipids at different binding sites of the trimeric interface of MA complexes. Unlike the immature MA complex, the 2 acyl tail of two PIP2 lipids at the trimeric interface of the mature MA complex is observed to sample stable binding pockets of MA consisting of helix4 residues. Together, our results provide molecular-level insights into the interactions of MA trimeric complexes with membrane and different lipid conformations at the specific binding sites of MA protein before and after viral maturation. SignificanceHIV-1 maturation facilitates the conversion of a newly formed immature virus particle to a mature infectious virion and initiates a new round of infection. The contributions of specific protein-lipid interactions in the HIV-1 assembly process are well recognized, however, the interactions of matrix protein lattice with the membrane before and after HIV-1 maturation are yet to be fully understood. Based on our simulated data, supported by prior experimental observations, the present study provides a molecular-level understanding of possible altered binding mode of PIP2 lipids after viral maturation. Identification of protein-lipid specific interactions, and lipid sorting data obtained from our long-time and large-scale atomistic MD simulations advance the understanding of the HIV-1 matrix and membrane maturation.

biophysics↗

bioRxiv2023

PNMA2 forms non-enveloped virus-like capsids that trigger paraneoplastic neurological syndrome

The paraneoplastic Ma antigen (PNMA) genes are associated with cancer-induced paraneoplastic syndromes that present with neurological symptoms and autoantibody production. How PNMA proteins trigger a severe autoimmune disease is unclear. PNMA genes are predominately expressed in the central nervous system with little known functions but are ectopically expressed in some tumors. Here, we show that PNMA2 is derived from a Ty3 retrotransposon that encodes a protein which forms virus-like capsids released from cells as non-enveloped particles. Recombinant PNMA2 capsids injected into mice induce a robust autoimmune reaction with significant generation of autoantibodies that preferentially bind external "spike" PNMA2 capsid epitopes, while capsid-assembly-defective PNMA2 protein is not immunogenic. PNMA2 autoantibodies present in cerebrospinal fluid of patients with anti-Ma2 paraneoplastic neurologic disease show similar preferential binding to PNMA2 "spike" capsid epitopes. These observations suggest that PNMA2 capsids released from tumors trigger an autoimmune response that underlies Ma2 paraneoplastic neurological syndrome.

neuroscience↗

bioRxiv2022

Structural insights into HIV-1 polyanion-dependent capsid lattice formation revealed by singleparticle cryo-EM

The HIV-1 capsid houses the viral genome and interacts extensively with host cell proteins throughout the viral life cycle. It is composed of capsid protein (CA), which assembles into a conical fullerene lattice composed of roughly 200 CA hexamers and 12 CA pentamers. Previous structural analyses of individual CA hexamers and pentamers have provided valuable insight into capsid structure and function, but high-resolution information about these assemblies in the broader context of the capsid lattice is lacking. In this study, we combined cryo-electron tomography and single particle analysis cryo-electron microscopy to determine high-resolution structures of continuous regions of the capsid lattice containing both hexamers and pentamers. We also developed a new method of in vitro lattice assembly that enabled us to directly study the lattice under a wider range of conditions than has previously been possible. Using this approach, we identified a critical role for inositol hexakisphosphate (IP6) in pentamer formation and determined the structure of the CA lattice bound to the capsid-targeting antiretroviral drug GS-6207 (Lenacapvir). Our work reveals new structural details of the mature HIV-1 CA lattice and establishes the combination of lattice templating and single particle analysis as a robust strategy for studying retroviral capsid structure and capsid interactions with host proteins and antiviral compounds. Significance statementThe mature HIV-1 capsid is composed of the capsid (CA) protein arranged in a conical lattice of hexamers and pentamers. Numerous structures of individual CA hexamers and pentamers alone have been published, but the molecular details of these assemblies in a more global, lattice-wide context are lacking. Here, we present high-resolution cryo-electron microscopy structures of continuous regions of the capsid lattice containing both hexamers and pentamers. We also describe key differences in the assembly and structures of these oligomers that have important implications for understanding retroviral maturation and for ongoing efforts to pharmacologically target the HIV-1 capsid.

biochemistry↗

bioRxiv2022

Two structural switches in HIV-1 capsid regulate capsid curvature and host factor binding

The mature HIV-1 capsid protects the viral genome and interacts with host proteins to travel from the cell periphery into the nucleus. To achieve this, the capsid protein, CA, constructs conical capsids from a lattice of hexamers and pentamers, and engages in and then relinquishes multiple interactions with cellular proteins in an orchestrated fashion. Cellular host factors including Nup153, CPSF6 and Sec24C engage the same pocket within CA hexamers. How CA assembles pentamers and hexamers of different curvatures, how CA oligomerization states or curvature might modulate host-protein interactions, and how binding of multiple co-factors to a single site is coordinated, all remain to be elucidated. Here, we have resolved the structure of the mature HIV-1 CA pentamer and hexamer from conical CA-IP6 polyhedra to high resolution. We have determined structures of hexamers in the context of multiple lattice curvatures and number of pentamer contacts. Comparison of these structures, bound or not to host protein peptides, revealed two structural switches within HIV-1 CA that modulate peptide binding according to CA lattice curvature and whether CA is hexameric or pentameric. These observations suggest that the conical HIV-1 capsid has different host-protein binding properties at different positions on its surface, which may facilitate cell entry and represent an evolutionary advantage of conical morphology. Significance statementHIV-1 particles contain a characteristic, conical capsid that shields the genome from the cellular immune system and recruits cellular proteins to direct the capsid to the nucleus. The cone forms from hexamers of CA protein, and twelve pentamers that accommodate curvature. We obtained detailed 3D models of pentamers and hexamers at positions on capsid surfaces with different curvatures. We find two places in CA that switch conformation according to the local capsid curvature and whether CA is in a pentamer or hexamer. We also obtained models of CA bound to peptides from cellular proteins. The data show how switches in CA help it form a cone shape, and interact differently with cellular proteins at different positions on the cone surface.

microbiology↗

bioRxiv2022

FCHO controls AP2's critical endocytic roles through a PtdIns4,5P2 membrane-dependent switch

Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo-adaptor AP2 requires membrane-localised FCHO. Here, live-cell eTIRF-SIM shows that FCHO marks sites of clathrin- coated pit (CCP) initiation, which mature into uniform sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Eps15 ring. We dissect the network of interactions between the FCHO interdomain-linker and AP2, which concentrates, orients, tethers and partially destabilizes closed AP2 at the plasma membrane. AP2s subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with PtdIns4,5P2, clathrin, cargo and CME accessory factors. FCHO can now relocate toward a CCPs outer edge to engage and activate further AP2s to drive CCP growth/maturation. 125 character summaryFCHO primes AP2 for CCV incorporation, a process that triggers FCHO release to enable activation/recruitment of further AP2s

cell biology↗

bioRxiv2021

Strain and crack propagation of HIV-1 capsids during uncoating

Viral replication in HIV-1 relies on a fullerene-shaped capsid to transport genetic material deep into the nucleus of an infected cell. Capsid stability is linked to the presence of cofactors, including inositol hexakisphosphate (IP6) that bind to pores found in the capsid. Using extensive all-atom molecular dynamics simulations of HIV-1 cores imaged from cryo-electron tomography (cryo-ET) in intact virions, which contain IP6 and a ribonucleoprotein complex, we find markedly striated patterns of strain on capsid lattices. The presence of these cofactors also increases rigidity of the capsid. Conformational analysis of capsid (CA) proteins show CA accommodates strain by locally flexing away from structures resolved using x-ray crystallography and cryo-electron microscopy. Then, cryo-ET of HIV-1 cores undergoing endogenous reverse transcription demonstrate that lattice strain increases in the capsid prior to mechanical failure and that the capsid ruptures by crack propagation along regions of high strain. These results uncover HIV-1 capsid properties involved in their critical disassembly process. Significance statementThe mature capsids of HIV-1 are transiently stable complexes that self-assemble around the viral genome during maturation, and uncoat to release preintegration complexes that archive a double-stranded DNA copy of the virus in the host cell genome. However, a detailed view of how HIV cores rupture remains lacking. Here, we elucidate the physical properties involved in capsid rupture using a combination of large-scale all-atom molecular dynamics simulations and cryo-electron tomography. We find that intrinsic strain on the capsid forms highly correlated patterns along the capsid surface, along which cracks propagate. Capsid rigidity also increases with high strain. Our findings provide fundamental insight into viral capsid uncoating.

biophysics↗

bioRxiv2021

Structures and function of locked conformations of SARS-CoV-2 spike

The spike (S) protein of SARS-CoV-2 has been observed in three distinct pre-fusion conformations: locked, closed and open. Of these, the function of the locked conformation remains poorly understood. Here we engineered a SARS-CoV-2 S protein construct "S-R/x3" to arrest SARS-CoV-2 spikes in the locked conformation by a disulfide bond. Using this construct we determined high-resolution structures confirming that the x3 disulfide bond has the ability to stabilize the otherwise transient locked conformations. Structural analyses reveal that wild-type SARS-CoV-2 spike can adopt two distinct locked-1 and locked-2 conformations. For the D614G spike, based on which all variants of concern were evolved, only the locked-2 conformation was observed. Analysis of the structures suggests that rigidified domain D in the locked conformations interacts with the hinge to domain C and thereby restrains RBD movement. Structural change in domain D correlates with spike conformational change. We propose that the locked-1 and locked-2 conformations of S are present in the acidic high-lipid cellular compartments during virus assembly and egress. In this model, release of the virion into the neutral pH extracellular space would favour transition to the closed or open conformations. The dynamics of this transition can be altered by mutations that modulate domain D structure, as is the case for the D614G mutation, leading to changes in viral fitness. The S-R/x3 construct provides a tool for the further structural and functional characterization of the locked conformations of S, as well as how sequence changes might alter S assembly and regulation of receptor binding domain dynamics.

microbiology↗

bioRxiv2021

The molecular infrastructure of glutamatergic synapses in the mammalian forebrain

Glutamatergic synapses form the vast majority of connections within neuronal circuits, but how these subcellular structures are molecularly organized within the mammalian brain is poorly understood. Conventional electron microscopy using chemically fixed, metal- stained tissue has identified a proteinaceous, membrane-associated thickening called the postsynaptic density (PSD). Here, we combined mouse genetics and cryo-electron tomography to determine the 3D molecular architecture of fresh isolated and anatomically intact synapses in the adult forebrain. The native glutamatergic synapse did not consistently show a higher density of proteins at the postsynaptic membrane thought to be characteristic of the PSD. Instead, a synaptoplasm consisting of cytoskeletal elements, macromolecular complexes and membrane-bound organelles extended throughout the pre- and post- synaptic compartments. Snapshots of active processes gave insights into membrane remodeling processes. Clusters of up to 60 ionotropic glutamate receptors were positioned inside and outside the synaptic cleft. Together, these information-rich tomographic maps present a detailed molecular framework for the coordinated activity of synapses in the adult mammalian brain.

neuroscience↗

bioRxiv2021

SARS-CoV-2 spike protein arrested in the closed state induces potent neutralizing responses

The majority of SARS-CoV-2 vaccines in use or in advanced clinical development are based on the viral spike protein (S) as their immunogen. S is present on virions as pre-fusion trimers in which the receptor binding domain (RBD) is stochastically open or closed. Neutralizing antibodies have been described that act against both open and closed conformations. The long-term success of vaccination strategies will depend upon inducing antibodies that provide long-lasting broad immunity against evolving, circulating SARS-CoV-2 strains, while avoiding the risk of antibody dependent enhancement as observed with other Coronavirus vaccines. Here we have assessed the results of immunization in a mouse model using an S protein trimer that is arrested in the closed state to prevent exposure of the receptor binding site and therefore interaction with the receptor. We compared this with a range of other modified S protein constructs, including representatives used in current vaccines. We found that all trimeric S proteins induce a long-lived, strongly neutralizing antibody response as well as T-cell responses. Notably, the protein binding properties of sera induced by the closed spike differed from those induced by standard S protein constructs. Closed S proteins induced more potent neutralising responses than expected based on the degree to which they inhibit interactions between the RBD and ACE2. These observations suggest that closed spikes recruit different, but equally potent, virus-inhibiting immune responses than open spikes, and that this is likely to include neutralizing antibodies against conformational epitopes present in the closed conformation. Together with their improved stability and storage properties we suggest that closed spikes may be a valuable component of refined, next-generation vaccines.

immunology↗

bioRxiv2020

Architecture and mechanism of metazoan retromer:SNX3 tubular coat assembly

Retromer is a master regulator of cargo retrieval from endosomes, which is critical for many cellular processes including signalling, immunity, neuroprotection and virus infection. To function in different trafficking routes, retromer core (VPS26/VPS29/VPS35) assembles with a range of sorting nexins to generate tubular carriers and incorporate assorted cargoes. We elucidate the structural basis of membrane remodelling and coupled cargo recognition by assembling metazoan and fungal retromer core trimers on cargo-containing membranes with sorting nexin adaptor SNX3 and determining their structures using cryo-electron tomography. Assembly leads to formation of tubular carriers in the absence of canonical membrane curvature drivers. Interfaces in the retromer coat provide a structural explanation for Parkinsons disease-linked mutations. We demonstrate that retromer core trimer forms an invariant, evolutionarily-conserved scaffold that can incorporate different auxiliary membrane adaptors by changing its mode of membrane recruitment, so modulating membrane bending and cargo incorporation and thereby allowing retromer to traffic assorted cargoes along different cellular transport routes.

molecular biology↗

bioRxiv2020

Immature HIV-1 assembles from Gag dimers leaving partial hexamers at lattice edges as substrates for proteolytic maturation.

The CA (capsid) domain of immature HIV-1 Gag and the adjacent spacer peptide 1 (SP1) play a key role in viral assembly by forming a lattice of CA hexamers, which adapts to viral envelope curvature by incorporating small lattice defects and a large gap at the site of budding. This lattice is stabilized by intra- and inter-hexameric CA-CA interactions, which are important in regulating viral assembly and maturation. We applied subtomogram averaging and classification to determine the structure of CA at lattice edges and found that they form partial hexamers. These structures reveal the network of interactions formed by CA-SP1at the lattice edge. We also performed atomistic molecular dynamics simulations of CA-CA interactions stabilizing the immature lattice and of partial CA-SP1 helical bundles. Free energy calculations reveal increased propensity for helix-to-coil transitions in partial hexamers compared to complete six-helix bundles. Taken together, these results suggest that the CA dimer is the basic unit of lattice assembly, that partial hexamers exist at lattice edges, that these are in a helix-coil dynamic equilibrium and that partial helical bundles are more likely to unfold, representing potential sites for HIV-1 maturation initiation. Significance StatementHIV-1 particle assembly is driven by the viral Gag protein, which oligomerizes into an hexameric array on the inner surface of the viral envelope, forming a truncated spherical lattice containing large and small gaps. Gag is then cut by the viral protease, disassembles and rearranges to form the mature, infectious virus. Here, we present structures and molecular dynamics simulations of the edges of the immature Gag lattice. Our analysis shows that Gag dimers are the basic assembly unit of the HIV-1 particle, that lattice edges are partial hexamers, and that partial hexamers are prone to structural changes allowing protease to cut Gag. These findings provide insights into assembly of the immature virus, its structure, and how it disassembles during maturation.

microbiology↗

bioRxiv2020

Maturation of the matrix and viral membrane of HIV-1

Gag - the main structural protein of HIV-1 - is recruited to the plasma membrane for virus assembly by its matrix (MA) domain. Gag is subsequently cleaved into its component domains, causing structural maturation to repurpose the virion for cell entry. We determined the structure and arrangement of MA within immature and mature HIV-1, providing a basis to understand MAs role in virus assembly. Unexpectedly, we found that MA rearranges during maturation, to form a new, hexameric lattice in which the acyl chain of a phospholipid extends out of the membrane to bind a pocket in MA. Our data suggest that proteolytic maturation of HIV-1 not only achieves assembly of the viral capsid surrounding the genome, but extends to repurpose the membrane-bound MA lattice for an entry or post-entry function, and causes partial removal of 2,500 acyl chains from the viral membrane.

microbiology↗

bioRxiv2020

Structures, conformations and distributions of SARS-CoV-2 spike protein trimers on intact virions

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude. Heavily glycosylated S trimers bind the ACE2 receptor and mediate entry of virions into target cells. S exhibits extensive conformational flexibility: it modulates the exposure of its receptor binding site and later undergoes complete structural rearrangement to drive fusion of viral and cellular membranes. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy. The structure and distribution of S on the virion surface, however, has not been characterised. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions, determining the high-resolution structure, conformational flexibility and distributions of S trimers in situ on the virion surface. These results provide a basis for understanding the conformations of S present on the virion, and for studying their interactions with neutralizing antibodies.

microbiology↗