Ribosome-Associated Vesicles promote activity-dependent local translation.

Local protein synthesis in axons and dendrites underpins synaptic plasticity. However, the composition of the protein synthesis machinery in distal neuronal processes and the mechanisms for its activity-driven deployment to local translation sites remain unclear. Here, we employed cryo-electron tomography, volume electron microscopy, and live-cell imaging to identify Ribosome-Associated Vesicles (RAVs) as a dynamic platform for moving ribosomes to distal processes. Stimulation via chemically-induced long-term potentiation causes RAV accumulation in distal sites to drive local translation. We also demonstrate activity-driven changes in RAV generation and dynamics in vivo, identifying tubular ER shaping proteins in RAV biogenesis. Together, our work identifies a mechanism for ribosomal delivery to distal sites in neurons to promote activity-dependent local translation.

Characterization of the three-dimensional synaptic and mitochondrial nanoarchitecture within glutamatergic synaptic complexes in postmortem human brain via focused ion beam-scanning electron microscopy.

Glutamatergic synapses are the primary site of excitatory synaptic signaling and neural communication in the cerebral cortex. Electron microscopy (EM) studies in non-human model organisms have demonstrated that glutamate synaptic activity and functioning are directly reflected in quantifiable ultrastructural features. Thus, quantitative EM analysis of glutamate synapses in ex vivo preserved human brain tissue has the potential to provide novel insight into in vivo synaptic functioning. However, factors associated with the acquisition and preservation of human brain tissue have resulted in persistent concerns regarding the potential confounding effects of antemortem and postmortem biological processes on synaptic and sub-synaptic ultrastructural features. Thus, we sought to determine how well glutamate synaptic relationships and nanoarchitecture are preserved in postmortem human dorsolateral prefrontal cortex (DLPFC), a region that substantially differs in size and architecture from model systems. Focused ion beam-scanning electron microscopy (FIB-SEM), a powerful volume EM (VEM) approach, was employed to generate high-fidelity, fine-resolution, three-dimensional (3D) micrographic datasets appropriate for quantitative analyses. Using postmortem human DLPFC with a 6-hour postmortem interval, we optimized a tissue preservation and staining workflow that generated samples of excellent ultrastructural preservation and the high-contrast staining intensity required for FIB-SEM imaging. Quantitative analysis of sub-cellular, sub-synaptic and organelle components within glutamate axo-spinous synapses revealed that ultrastructural features of synaptic function and activity were well-preserved within and across individual synapses in postmortem human brain tissue. The synaptic, sub-synaptic and organelle measures were highly consistent with findings from experimental models that are free from antemortem or postmortem effects. Further, dense reconstruction of neuropil revealed a unique, ultrastructurally-complex, spiny dendritic shaft that exhibited features characteristic of neuronal processes with heightened synaptic communication, integration and plasticity. Altogether, our findings provide a critical proof-of-concept that ex vivo VEM analysis provides a valuable and informative means to infer in vivo functioning of human brain.

Structure of native HIV-1 cores and their interactions with IP6 and CypA.

The viral capsid plays essential roles in HIV replication and is a major platform engaging host factors. To overcome challenges in study native capsid structure, we used the perfringolysin O to perforate the membrane of HIV-1 particles, thus allowing host proteins and small molecules to access the native capsid while improving cryo­electron microscopy image quality. Using cryo­electron tomography and subtomogram averaging, we determined the structures of native capsomers in the presence and absence of inositol hexakisphosphate (IP6) and cyclophilin A and constructed an all-atom model of a complete HIV-1 capsid. Our structures reveal two IP6 binding sites and modes of cyclophilin A interactions. Free energy calculations substantiate the two binding sites at R18 and K25 and further show a prohibitive energy barrier for IP6 to pass through the pentamer. Our results demonstrate that perfringolysin O perforation is a valuable tool for structural analyses of enveloped virus capsids and interactions with host cell factors.

CryoET structures of immature HIV Gag reveal six-helix bundle.

Gag is the HIV structural precursor protein which is cleaved by viral protease to produce mature infectious viruses. Gag is a polyprotein composed of MA (matrix), CA (capsid), SP1, NC (nucleocapsid), SP2 and p6 domains. SP1, together with the last eight residues of CA, have been hypothesized to form a six-helix bundle responsible for the higher-order multimerization of Gag necessary for HIV particle assembly. However, the structure of the complete six-helix bundle has been elusive. Here, we determined the structures of both Gag in vitro assemblies and Gag viral-like particles (VLPs) to 4.2 Å and 4.5 Å resolutions using cryo-electron tomography and subtomogram averaging by emClarity. A single amino acid mutation (T8I) in SP1 stabilizes the six-helix bundle, allowing to discern the entire CA-SP1 helix connecting to the NC domain. These structures provide a blueprint for future development of small molecule inhibitors that can lock SP1 in a stable helical conformation, interfere with virus maturation, and thus block HIV-1 infection.

Cytoplasmic CPSF6 Regulates HIV-1 Capsid Trafficking and Infection in a Cyclophilin A-Dependent Manner.

Human immunodeficiency virus type 1 (HIV-1) capsid binds host proteins during infection, including cleavage and polyadenylation specificity factor 6 (CPSF6) and cyclophilin A (CypA). We observe that HIV-1 infection induces higher-order CPSF6 formation, and capsid-CPSF6 complexes cotraffic on microtubules. CPSF6-capsid complex trafficking is impacted by capsid alterations that reduce CPSF6 binding or by excess cytoplasmic CPSF6 expression, both of which are associated with decreased HIV-1 infection. Higher-order CPSF6 complexes bind and disrupt HIV-1 capsid assemblies in vitro Disruption of HIV-1 capsid binding to CypA leads to increased CPSF6 binding and altered capsid trafficking, resulting in reduced infectivity. Our data reveal an interplay between CPSF6 and CypA that is important for cytoplasmic capsid trafficking and HIV-1 infection. We propose that CypA prevents HIV-1 capsid from prematurely engaging cytoplasmic CPSF6 and that differences in CypA cellular localization and innate immunity may explain variations in HIV-1 capsid trafficking and uncoating in CD4+ T cells and macrophages.IMPORTANCE HIV is the causative agent of AIDS, which has no cure. The protein shell that encases the viral genome, the capsid, is critical for HIV replication in cells at multiple steps. HIV capsid has been shown to interact with multiple cell proteins during movement to the cell nucleus in a poorly understood process that may differ during infection of different cell types. In this study, we show that premature or too much binding of one human protein, cleavage and polyadenylation specificity factor 6 (CPSF6), disrupts the ability of the capsid to deliver the viral genome to the cell nucleus. Another human protein, cyclophilin A (CypA), can shield HIV capsid from premature binding to CPSF6, which can differ in CD4+ T cells and macrophages. Better understanding of how HIV infects cells will allow better drugs to prevent or inhibit infection and pathogenesis.

Serial cryoFIB/SEM Reveals Cytoarchitectural Disruptions in Leigh Syndrome Patient Cells.

The advancement of serial cryoFIB/SEM offers an opportunity to study large volumes of near-native, fully hydrated frozen cells and tissues at voxel sizes of 10 nm and below. We explored this capability for pathologic characterization of vitrified human patient cells by developing and optimizing a serial cryoFIB/SEM volume imaging workflow. We demonstrate profound disruption of subcellular architecture in primary fibroblasts from a Leigh syndrome patient harboring a disease-causing mutation in USMG5 protein responsible for impaired mitochondrial energy production.

Intrinsic curvature of the HIV-1 CA hexamer underlies capsid topology and interaction with cyclophilin A.

The mature retrovirus capsid consists of a variably curved lattice of capsid protein (CA) hexamers and pentamers. High-resolution structures of the curved assembly, or in complex with host factors, have not been available. By devising cryo-EM methodologies for exceedingly flexible and pleomorphic assemblies, we have determined cryo-EM structures of apo-CA hexamers and in complex with cyclophilin A (CypA) at near-atomic resolutions. The CA hexamers are intrinsically curved, flexible and asymmetric, revealing the capsomere and not the previously touted dimer or trimer interfaces as the key contributor to capsid curvature. CypA recognizes specific geometries of the curved lattice, simultaneously interacting with three CA protomers from adjacent hexamers via two noncanonical interfaces, thus stabilizing the capsid. By determining multiple structures from various helical symmetries, we further revealed the essential plasticity of the CA molecule, which allows formation of continuously curved conical capsids and the mechanism of capsid pattern sensing by CypA.

AutoCLEM: An Automated Workflow for Correlative Live-Cell Fluorescence Microscopy and Cryo-Electron Tomography.

Correlative light and electron microscopy (CLEM) combines the strengths of both light and electron imaging modalities and enables linking of biological spatiotemporal information from live-cell fluorescence light microscopy (fLM) to high-resolution cellular ultra-structures from cryo-electron microscopy and tomography (cryoEM/ET). This has been previously achieved by using fLM signals to localize the regions of interest under cryogenic conditions. The correlation process, however, is often tedious and time-consuming with low throughput and limited accuracy, because multiple correlation steps at different length scales are largely carried out manually. Here, we present an experimental workflow, AutoCLEM, which overcomes the existing limitations and improves the performance and throughput of CLEM methods, and associated software. The AutoCLEM system encompasses a high-speed confocal live-cell imaging module to acquire an automated fLM grid atlas that is linked to the cryoEM grid atlas, followed by cryofLM imaging after freezing. The fLM coordinates of the targeted areas are automatically converted to cryoEM/ET and refined using fluorescent fiducial beads. This AutoCLEM workflow significantly accelerates the correlation efficiency between live-cell fluorescence imaging and cryoEM/ET structural analysis, as demonstrated by visualizing human immunodeficiency virus type 1 (HIV-1) interacting with host cells.

Truncated CPSF6 Forms Higher-Order Complexes That Bind and Disrupt HIV-1 Capsid.

Cleavage and polyadenylation specificity factor 6 (CPSF6) is a human protein that binds HIV-1 capsid and mediates nuclear transport and integration targeting of HIV-1 preintegration complexes. Truncation of the protein at its C-terminal nuclear-targeting arginine/serine-rich (RS) domain produces a protein, CPSF6-358, that potently inhibits HIV-1 infection by targeting the capsid and inhibiting nuclear entry. To understand the molecular mechanism behind this restriction, the interaction between CPSF6-358 and HIV-1 capsid was characterized using in vitro and in vivo assays. Purified CPSF6-358 protein formed oligomers and bound in vitro-assembled wild-type (WT) capsid protein (CA) tubes, but not CA tubes containing a mutation in the putative binding site of CPSF6. Intriguingly, binding of CPSF6-358 oligomers to WT CA tubes physically disrupted the tubular assemblies into small fragments. Furthermore, fixed- and live-cell imaging showed that stably expressed CPSF6-358 forms cytoplasmic puncta upon WT HIV-1 infection and leads to capsid permeabilization. These events did not occur when the HIV-1 capsid contained a mutation known to prevent CPSF6 binding, nor did they occur in the presence of a small-molecule inhibitor of capsid binding to CPSF6-358. Together, our in vitro biochemical and transmission electron microscopy data and in vivo intracellular imaging results provide the first direct evidence for an oligomeric nature of CPSF6-358 and suggest a plausible mechanism for restriction of HIV-1 infection by CPSF6-358.IMPORTANCE After entry into cells, the HIV-1 capsid, which contains the viral genome, interacts with numerous host cell factors to facilitate crucial events required for replication, including uncoating. One such host cell factor, called CPSF6, is predominantly located in the cell nucleus and interacts with HIV-1 capsid. The interaction between CA and CPSF6 is critical during HIV-1 replication in vivo Truncation of CPSF6 leads to its localization to the cell cytoplasm and inhibition of HIV-1 infection. Here, we determined that truncated CPSF6 protein forms large higher-order complexes that bind directly to HIV-1 capsid, leading to its disruption. Truncated CPSF6 expression in cells leads to premature capsid uncoating that is detrimental to HIV-1 infection. Our study provides the first direct evidence for an oligomeric nature of truncated CPSF6 and insights into the highly regulated process of HIV-1 capsid uncoating.

CryoEM Structure Refinement by Integrating NMR Chemical Shifts with Molecular Dynamics Simulations.

Single particle cryoEM has emerged as a powerful method for structure determination of proteins and complexes, complementing X-ray crystallography and NMR spectroscopy. Yet, for many systems, the resolution of cryoEM density map has been limited to 4-6 Å, which only allows for resolving bulky amino acids side chains, thus hindering accurate model building from the density map. On the other hand, experimental chemical shifts (CS) from solution and solid state MAS NMR spectra provide atomic level data for each amino acid within a molecule or a complex; however, structure determination of large complexes and assemblies based on NMR data alone remains challenging. Here, we present a novel integrated strategy to combine the highly complementary experimental data from cryoEM and NMR computationally by molecular dynamics simulations to derive an atomistic model, which is not attainable by either approach alone. We use the HIV-1 capsid protein (CA) C-terminal domain as well as the large capsid assembly to demonstrate the feasibility of this approach, termed NMR CS-biased cryoEM structure refinement.

Preparation and characterization of zein thermo-modified starch films.

A new method of preparing films from zein thermo-modified starch was presented. According to data of micro-visco-amylography and rheometer, dry heating with zein significantly decreased the pasting properties of waxy corn starch and distarch phosphate, the values of G' and G″ of starches dry heated with zein decreased, while the loss tangent increased as a function of heating time. Scanning electron microscopy images showed that the surfaces of starch granules became rough after dry heating with zein, suggesting interactions between zein and the granule surface. In films made from mixtures, the starch/zein compatibility was improved through dry heating. Compared with films cast from mixtures of separately heat-treated starch and zein, films made from the starch/zein mixtures heated together possessed higher water contact angle and tensile strength. Simple dry heating with zein could thus be used as a promising modification method for improving the functionality of edible films from starches.

In vitro protease cleavage and computer simulations reveal the HIV-1 capsid maturation pathway.

HIV-1 virions assemble as immature particles containing Gag polyproteins that are processed by the viral protease into individual components, resulting in the formation of mature infectious particles. There are two competing models for the process of forming the mature HIV-1 core: the disassembly and de novo reassembly model and the non-diffusional displacive model. To study the maturation pathway, we simulate HIV-1 maturation in vitro by digesting immature particles and assembled virus-like particles with recombinant HIV-1 protease and monitor the process with biochemical assays and cryoEM structural analysis in parallel. Processing of Gag in vitro is accurate and efficient and results in both soluble capsid protein and conical or tubular capsid assemblies, seemingly converted from immature Gag particles. Computer simulations further reveal probable assembly pathways of HIV-1 capsid formation. Combining the experimental data and computer simulations, our results suggest a sequential combination of both displacive and disassembly/reassembly processes for HIV-1 maturation.

Cyclophilin A stabilizes the HIV-1 capsid through a novel non-canonical binding site.

The host cell factor cyclophilin A (CypA) interacts directly with the HIV-1 capsid and regulates viral infectivity. Although the crystal structure of CypA in complex with the N-terminal domain of the HIV-1 capsid protein (CA) has been known for nearly two decades, how CypA interacts with the viral capsid and modulates HIV-1 infectivity remains unclear. We determined the cryoEM structure of CypA in complex with the assembled HIV-1 capsid at 8-Å resolution. The structure exhibits a distinct CypA-binding pattern in which CypA selectively bridges the two CA hexamers along the direction of highest curvature. EM-guided all-atom molecular dynamics simulations and solid-state NMR further reveal that the CypA-binding pattern is achieved by single-CypA molecules simultaneously interacting with two CA subunits, in different hexamers, through a previously uncharacterized non-canonical interface. These results provide new insights into how CypA stabilizes the HIV-1 capsid and is recruited to facilitate HIV-1 infection.

Controlled bacterial lysis for electron tomography of native cell membranes.

Cryo-electron tomography (cryoET) has become a powerful tool for direct visualization of 3D structures of native biological specimens at molecular resolution, but its application is limited to thin specimens (<300 nm). Recently, vitreous sectioning and cryoFIB milling technologies were developed to physically reduce the specimen thickness; however, cryoET analysis of membrane protein complexes within native cell membranes remains a great challenge. Here, we use phage ΦX174 lysis gene E to rapidly produce native, intact, bacterial cell membranes for high resolution cryoET. We characterized E gene-induced cell lysis using FIB/SEM and cryoEM and showed that the bacteria cytoplasm was largely depleted through spot lesion, producing ghosts with the cell membranes intact. We further demonstrated the utility of E-gene-induced lysis for cryoET using the bacterial chemotaxis receptor signaling complex array. The described method should have a broad application for structural and functional studies of native, intact cell membranes and membrane protein complexes.

Correlative microscopy for 3D structural analysis of dynamic interactions.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state(1). However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging(2), due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-1(3-7), the resolution afforded by live-cell microscopy is limited (~200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target. In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

Mature HIV-1 capsid structure by cryo-electron microscopy and all-atom molecular dynamics.

Retroviral capsid proteins are conserved structurally but assemble into different morphologies. The mature human immunodeficiency virus-1 (HIV-1) capsid is best described by a 'fullerene cone' model, in which hexamers of the capsid protein are linked to form a hexagonal surface lattice that is closed by incorporating 12 capsid-protein pentamers. HIV-1 capsid protein contains an amino-terminal domain (NTD) comprising seven α-helices and a ß-hairpin, a carboxy-terminal domain (CTD) comprising four α-helices, and a flexible linker with a 310-helix connecting the two structural domains. Structures of the capsid-protein assembly units have been determined by X-ray crystallography; however, structural information regarding the assembled capsid and the contacts between the assembly units is incomplete. Here we report the cryo-electron microscopy structure of a tubular HIV-1 capsid-protein assembly at 8 Å resolution and the three-dimensional structure of a native HIV-1 core by cryo-electron tomography. The structure of the tubular assembly shows, at the three-fold interface, a three-helix bundle with critical hydrophobic interactions. Mutagenesis studies confirm that hydrophobic residues in the centre of the three-helix bundle are crucial for capsid assembly and stability, and for viral infectivity. The cryo-electron-microscopy structures enable modelling by large-scale molecular dynamics simulation, resulting in all-atom models for the hexamer-of-hexamer and pentamer-of-hexamer elements as well as for the entire capsid. Incorporation of pentamers results in closer trimer contacts and induces acute surface curvature. The complete atomic HIV-1 capsid model provides a platform for further studies of capsid function and for targeted pharmacological intervention.

Structural insight into HIV-1 capsid recognition by rhesus TRIM5α.

Tripartite motif protein isoform 5 alpha (TRIM5α) is a potent antiviral protein that restricts infection by HIV-1 and other retroviruses. TRIM5α recognizes the lattice of the retrovirus capsid through its B30.2 (PRY/SPRY) domain in a species-specific manner. Upon binding, TRIM5α induces premature disassembly of the viral capsid and activates the downstream innate immune response. We have determined the crystal structure of the rhesus TRIM5α PRY/SPRY domain that reveals essential features for capsid binding. Combined cryo-electron microscopy and biochemical data show that the monomeric rhesus TRIM5α PRY/SPRY, but not the human TRIM5α PRY/SPRY, can bind to HIV-1 capsid protein assemblies without causing disruption of the capsid. This suggests that the PRY/SPRY domain alone constitutes an important pattern-sensing component of TRIM5α that is capable of interacting with viral capsids of different curvatures. Our results provide molecular insights into the mechanisms of TRIM5α-mediated retroviral restriction.

Protease cleavage leads to formation of mature trimer interface in HIV-1 capsid.

During retrovirus particle maturation, the assembled Gag polyprotein is cleaved by the viral protease into matrix (MA), capsid (CA), and nucleocapsid (NC) proteins. To form the mature viral capsid, CA rearranges, resulting in a lattice composed of hexameric and pentameric CA units. Recent structural studies of assembled HIV-1 CA revealed several inter-subunit interfaces in the capsid lattice, including a three-fold interhexamer interface that is critical for proper capsid stability. Although a general architecture of immature particles has been provided by cryo-electron tomographic studies, the structural details of the immature particle and the maturation pathway remain unknown. Here, we used cryo-electron microscopy (cryoEM) to determine the structure of tubular assemblies of the HIV-1 CA-SP1-NC protein. Relative to the mature assembled CA structure, we observed a marked conformational difference in the position of the CA-CTD relative to the NTD in the CA-SP1-NC assembly, involving the flexible hinge connecting the two domains. This difference was verified via engineered disulfide crosslinking, revealing that inter-hexamer contacts, in particular those at the pseudo three-fold axis, are altered in the CA-SP1-NC assemblies compared to the CA assemblies. Results from crosslinking analyses of mature and immature HIV-1 particles containing the same Cys substitutions in the Gag protein are consistent with these findings. We further show that cleavage of preassembled CA-SP1-NC by HIV-1 protease in vitro leads to release of SP1 and NC without disassembly of the lattice. Collectively, our results indicate that the proteolytic cleavage of Gag leads to a structural reorganization of the polypeptide and creates the three-fold interhexamer interface, important for the formation of infectious HIV-1 particles.

Functional assembly of bacterial communities with activity for the biodegradation of an organophosphorus pesticide in the rape phyllosphere.

Although most pesticides sprayed on terrestrial plants remain on their leaf surfaces, the relationship between leaf-associated microbial populations and pesticide degradation remains unclear. Here we examined changes in the bacterial community composition in the rape phyllosphere after treatment with dichlorvos, an organophosphorus pesticide. Results indicate that the bacterial community showed marked changes after treatment. We evaluated the rate of dichlorvos degradation by a natural microbial community on rape leaves and found that more dichlorvos was degraded on microbial-population-inhabited leaves than on surface-sterilized leaves. Six dichlorvos-degrading bacteria with 16S rRNA gene sequences that are most similar to those of members of the genera Pseudomonas, Xanthomonas, Sphingomonas, Acidovorax, Agrobacterium and Chryseobacterium were isolated from the natural community. We report for the first time that three of these epiphytic bacterial species, from the genera Sphingomonas, Acidovorax and Chryseobacterium, can degrade organophosphorus compounds. Collectively, these results provide direct evidence that bacteria on leaves can degrade organophosphate pesticides, and demonstrate that phyllosphere bacteria have great potential for the bioremediation of pesticides in situ, where the environment is hostile to nonepiphytic bacteria.