Targeted mutagenesis of the herpesvirus fusogen central helix captures transition states.

Herpesviruses remain a burden for animal and human health, including the medically important varicella-zoster virus (VZV). Membrane fusion mediated by conserved core glycoproteins, the fusogen gB and the heterodimer gH-gL, enables herpesvirus cell entry. The ectodomain of gB orthologs has five domains and is proposed to transition from a prefusion to postfusion conformation but the functional relevance of the domains for this transition remains poorly defined. Here we describe structure-function studies of the VZV gB DIII central helix targeting residues 526EHV528. Critically, a H527P mutation captures gB in a prefusion conformation as determined by cryo-EM, a loss of membrane fusion in a virus free assay, and failure of recombinant VZV to spread in cell monolayers. Importantly, two predominant cryo-EM structures of gB[H527P] are identified by 3D classification and focused refinement, suggesting they represented gB conformations in transition. These studies reveal gB DIII as a critical element for herpesvirus gB fusion function.

The Cryo-EM structures of two amphibian antimicrobial cross-ß amyloid fibrils.

The amyloid-antimicrobial link hypothesis is based on antimicrobial properties found in human amyloids involved in neurodegenerative and systemic diseases, along with amyloidal structural properties found in antimicrobial peptides (AMPs). Supporting this hypothesis, we here determined the fibril structure of two AMPs from amphibians, uperin 3.5 and aurein 3.3, by cryogenic electron microscopy (cryo-EM), revealing amyloid cross-ß fibrils of mated ß-sheets at atomic resolution. Uperin 3.5 formed a 3-blade symmetrical propeller of nine peptides per fibril layer including tight ß-sheet interfaces. This cross-ß cryo-EM structure complements the cross-α fibril conformation previously determined by crystallography, substantiating a secondary structure switch mechanism of uperin 3.5. The aurein 3.3 arrangement consisted of six peptides per fibril layer, all showing kinked ß-sheets allowing a rounded compactness of the fibril. The kinked ß-sheets are similar to LARKS (Low-complexity, Amyloid-like, Reversible, Kinked Segments) found in human functional amyloids.

The giant mimivirus 1.2 Mb genome is elegantly organized into a 30-nm diameter helical protein shield.

Mimivirus is the prototype of the Mimiviridae family of giant dsDNA viruses. Little is known about the organization of the 1.2 Mb genome inside the membrane-limited nucleoid filling the ~0.5 µm icosahedral capsids. Cryo-electron microscopy, cryo-electron tomography, and proteomics revealed that it is encased into a ~30-nm diameter helical protein shell surprisingly composed of two GMC-type oxidoreductases, which also form the glycosylated fibrils decorating the capsid. The genome is arranged in 5- or 6-start left-handed super-helices, with each DNA-strand lining the central channel. This luminal channel of the nucleoprotein fiber is wide enough to accommodate oxidative stress proteins and RNA polymerase subunits identified by proteomics. Such elegant supramolecular organization would represent a remarkable evolutionary strategy for packaging and protecting the genome, in a state ready for immediate transcription upon unwinding in the host cytoplasm. The parsimonious use of the same protein in two unrelated substructures of the virion is unexpected for a giant virus with thousand genes at its disposal.

Human Adenovirus Type 5 Infection Leads to Nuclear Envelope Destabilization and Membrane Permeability Independently of Adenovirus Death Protein.

The human adenovirus type 5 (HAdV5) infects epithelial cells of the upper and lower respiratory tract. The virus causes lysis of infected cells and thus enables spread of progeny virions to neighboring cells for the next round of infection. The mechanism of adenovirus virion egress across the nuclear barrier is not known. The human adenovirus death protein (ADP) facilitates the release of virions from infected cells and has been hypothesized to cause membrane damage. Here, we set out to answer whether ADP does indeed increase nuclear membrane damage. We analyzed the nuclear envelope morphology using a combination of fluorescence and state-of-the-art electron microscopy techniques, including serial block-face scanning electron microscopy and electron cryo-tomography of focused ion beam-milled cells. We report multiple destabilization phenotypes of the nuclear envelope in HAdV5 infection. These include reduction of lamin A/C at the nuclear envelope, large-scale membrane invaginations, alterations in double membrane separation distance and small-scale membrane protrusions. Additionally, we measured increased nuclear membrane permeability and detected nuclear envelope lesions under cryoconditions. Unexpectedly, and in contrast to previous hypotheses, ADP did not have an effect on lamin A/C reduction or nuclear permeability.

In Vitro Viral Evolution Identifies a Critical Residue in the Alphaherpesvirus Fusion Glycoprotein B Ectodomain That Controls gH/gL-Independent Entry.

Herpesvirus entry and spread requires fusion of viral and host cell membranes, which is mediated by the conserved surface glycoprotein B (gB). Upon activation, gB undergoes a major conformational change and transits from a metastable prefusion to a stable postfusion conformation. Although gB is a structural homolog of low-pH-triggered class III fusogens, its fusion activity depends strictly on the presence of the conserved regulatory gH/gL complex and nonconserved receptor binding proteins, which ensure that fusion occurs at the right time and space. How gB maintains its prefusion conformation and how gB fusogenicity is controlled remain poorly understood. Here, we report the isolation and characterization of a naturally selected pseudorabies virus (PrV) gB able to mediate efficient gH/gL-independent virus-cell and cell-cell fusion. We found that the control exerted on gB by the accompanying viral proteins is mediated via its cytosolic domain (CTD). Whereas gB variants lacking the CTD are inactive, a single mutation of a conserved asparagine residue in an alpha-helical motif of the ectodomain recently shown to be at the core of the gB prefusion trimer compensated for CTD absence and uncoupled gB from regulatory viral proteins, resulting in a hyperfusion phenotype. This phenotype was transferred to gB homologs from different alphaherpesvirus genera. Overall, our data propose a model in which the central helix acts as a molecular switch for the gB pre-to-postfusion transition by conveying the structural status of the endo- to the ectodomain, thereby governing their cross talk for fusion activation, providing a new paradigm for herpesvirus fusion regulation.IMPORTANCE The class III fusion protein glycoprotein B (gB) drives membrane fusion during entry and spread of herpesviruses. To mediate fusion, gB requires activation by the conserved gH/gL complex by a poorly defined mechanism. A detailed molecular-level understanding of herpesvirus membrane fusion is of fundamental virological interest and has considerable potential for the development of new therapeutics blocking herpesvirus cell invasion and spread. Using in vitro evolution and targeted mutagenesis of three different animal alphaherpesviruses, we identified a single conserved amino acid in a regulatory helix in the center of the gB ectodomain that enables efficient gH/gL-independent entry and plays a crucial role in the pre-to-postfusion transition of gB. Our results propose that the central helix is a key regulatory element involved in the intrastructural signal transduction between the endo- and ectodomain for fusion activation. This study expands our understanding of herpesvirus membrane fusion and uncovers potential targets for therapeutic interventions.

Fluorescent protein tagging of adenoviral proteins pV and pIX reveals 'late virion accumulation compartment'.

The human adenovirus type 5 (HAdV5) causes disease of the upper and lower respiratory tract. The early steps of HAdV5 entry up to genome replication in the host nucleus have been extensively studied. However, late stages of infection remain poorly understood. Here, we set out to elucidate the spatiotemporal orchestration of late adenovirus nuclear remodeling in living cells. We generated virus mutants expressing fluorescently tagged protein IX (pIX) and protein V (pV), a capsid and viral genome associated protein, respectively. We found that during progeny virion production both proteins localize to a membrane-less, nuclear compartment, which is highly impermeable such that in immunofluorescence microscopy antibodies can hardly penetrate it. We termed this compartment 'late virion accumulation compartment' (LVAC). Correlation between light- and electron microscopy revealed that the LVAC contains paracrystalline arrays of viral capsids that arrange tightly packed within a honeycomb-like organization of viral DNA. Live-cell microscopy as well as FRAP measurements showed that the LVAC is rigid and restricts diffusion of larger molecules, indicating that capsids are trapped inside.

Herpesvirus membrane fusion - a team effort.

One of the essential steps in every viral 'life' cycle is entry into the host cell. Membrane-enveloped viruses carry dedicated proteins to catalyse the fusion of the viral and cellular membrane. Herpesviruses feature a set of essential, structurally diverse glycoproteins on the viral surface that form a multicomponent fusion machinery, necessary for the entry mechanism. For Herpes simplex virus 1, these essential glycoproteins are gD, gH, gL and gB. In this review we describe the functions of the individual components, the potential interactions between them as well as the influence of post-translational modifications on the fusion mechanism.

Protein interactions and consensus clustering analysis uncover insights into herpesvirus virion structure and function relationships.

Infections with human herpesviruses are ubiquitous and a public health concern worldwide. Current treatments reduce the severity of some symptoms associated to herpetic infections but neither remove the viral reservoir from the infected host nor protect from the recurrent symptom outbreaks that characterise herpetic infections. The difficulty in therapeutically tackling these viral systems stems in part from their remarkably large proteomes and the complex networks of physical and functional associations that they tailor. This study presents our efforts to unravel the complexity of the interactome of herpes simplex virus type 1 (HSV1), the prototypical herpesvirus species. Inspired by our previous work, we present an improved and more integrative computational pipeline for the protein-protein interaction (PPI) network reconstruction in HSV1, together with a newly developed consensus clustering framework, which allowed us to extend the analysis beyond binary physical interactions and revealed a system-level layout of higher-order functional associations in the virion proteome. Additionally, the analysis provided new functional annotation for the currently undercharacterised protein pUS10. In-depth bioinformatics sequence analysis unravelled structural features in pUS10 reminiscent of those observed in some capsid-associated proteins in tailed bacteriophages, with which herpesviruses are believed to share a common ancestry. Using immunoaffinity purification (IP)-mass spectrometry (MS), we obtained additional support for our bioinformatically predicted interaction between pUS10 and the inner tegument protein pUL37, which binds cytosolic capsids, contributing to initial tegumentation and eventually virion maturation. In summary, this study unveils new, to our knowledge, insights at both the system and molecular levels that can help us better understand the complexity behind herpesvirus infections.

Protein assemblies ejected directly from native membranes yield complexes for mass spectrometry.

Membrane proteins reside in lipid bilayers and are typically extracted from this environment for study, which often compromises their integrity. In this work, we ejected intact assemblies from membranes, without chemical disruption, and used mass spectrometry to define their composition. From Escherichia coli outer membranes, we identified a chaperone-porin association and lipid interactions in the ß-barrel assembly machinery. We observed efflux pumps bridging inner and outer membranes, and from inner membranes we identified a pentameric pore of TonB, as well as the protein-conducting channel SecYEG in association with F1FO adenosine triphosphate (ATP) synthase. Intact mitochondrial membranes from Bos taurus yielded respiratory complexes and fatty acid-bound dimers of the ADP (adenosine diphosphate)/ATP translocase (ANT-1). These results highlight the importance of native membrane environments for retaining small-molecule binding, subunit interactions, and associated chaperones of the membrane proteome.

Multi-layered control of Galectin-8 mediated autophagy during adenovirus cell entry through a conserved PPxY motif in the viral capsid.

Cells employ active measures to restrict infection by pathogens, even prior to responses from the innate and humoral immune defenses. In this context selective autophagy is activated upon pathogen induced membrane rupture to sequester and deliver membrane fragments and their pathogen contents for lysosomal degradation. Adenoviruses, which breach the endosome upon entry, escape this fate by penetrating into the cytosol prior to autophagosome sequestration of the ruptured endosome. We show that virus induced membrane damage is recognized through Galectin-8 and sequesters the autophagy receptors NDP52 and p62. We further show that a conserved PPxY motif in the viral membrane lytic protein VI is critical for efficient viral evasion of autophagic sequestration after endosomal lysis. Comparing the wildtype with a PPxY-mutant virus we show that depletion of Galectin-8 or suppression of autophagy in ATG5-/- MEFs rescues infectivity of the PPxY-mutant virus while depletion of the autophagy receptors NDP52, p62 has only minor effects. Furthermore we show that wildtype viruses exploit the autophagic machinery for efficient nuclear genome delivery and control autophagosome formation via the cellular ubiquitin ligase Nedd4.2 resulting in reduced antigenic presentation. Our data thus demonstrate that a short PPxY-peptide motif in the adenoviral capsid permits multi-layered viral control of autophagic processes during entry.

Cellular electron cryo tomography and in situ sub-volume averaging reveal the context of microtubule-based processes.

Electron cryo-tomography (cryoET) is currently the only technique that allows the direct observation of proteins in their native cellular environment. Sub-volume averaging of electron tomograms offers a route to increase the signal-to-noise of repetitive biological structures, such improving the information content and interpretability of tomograms. We discuss the potential for sub-volume averaging in highlighting and investigating specific processes in situ, focusing on microtubule structure and viral infection. We show that (i) in situ sub-volume averaging from single tomograms can guide and complement segmentation of biological features, (ii) the in situ determination of the structure of individual viruses is possible as they infect a cell, and (iii) novel, transient processes can be imaged with high levels of detail.

Native structure of a retroviral envelope protein and its conformational change upon interaction with the target cell.

Enveloped viruses enter their host cells by membrane fusion. The process of attachment and fusion in retroviruses is mediated by a single viral envelope glycoprotein (Env). Conformational changes of Env in the course of fusion are a focus of intense studies. Here we provide further insight into the changes occurring in retroviral Env during its initial interaction with the cell, employing murine leukemia virus (MLV) as model system. We first determined the structure of both natively membrane anchored MLV Env and MLV Env tagged with YFP in the proline rich region (PRR) by electron cryo tomography (cET) and sub-volume averaging. At a resolution of ∼20Å, native MLV Env presents as a hollow trimer (height ∼85Å, diameter ∼120Å) composed of step-shaped protomers. The major difference to the YFP-tagged protein was in regions outside of the central trimer. Next, we focused on elucidating the changes in MLV Env upon interaction with a host cell. Virus interaction with the plasma membrane occurred over a large surface and Env clustering on the binding site was observed. Sub-volume averaging did yield a low-resolution structure of Env interacting with the cell, which had lost its threefold symmetry and was elongated by ∼35Å in comparison to the unbound protein. This indicates a major rearrangement of Env upon host cell binding. At the site of virus interaction, the otherwise clearly defined bilayer structure of the host cell plasma membrane was much less evident, indicative of integral membrane protein accumulation and/or a change in membrane lipid composition.

Two distinct trimeric conformations of natively membrane-anchored full-length herpes simplex virus 1 glycoprotein B.

Many viruses are enveloped by a lipid bilayer acquired during assembly, which is typically studded with one or two types of glycoproteins. These viral surface proteins act as the primary interface between the virus and the host. Entry of enveloped viruses relies on specialized fusogen proteins to help merge the virus membrane with the host membrane. In the multicomponent herpesvirus fusion machinery, glycoprotein B (gB) acts as this fusogen. Although the structure of the gB ectodomain postfusion conformation has been determined, any other conformations (e.g., prefusion, intermediate conformations) have so far remained elusive, thus restricting efforts to develop antiviral treatments and prophylactic vaccines. Here, we have characterized the full-length herpes simplex virus 1 gB in a native membrane by displaying it on cell-derived vesicles and using electron cryotomography. Alongside the known postfusion conformation, a novel one was identified. Its structure, in the context of the membrane, was determined by subvolume averaging and found to be trimeric like the postfusion conformation, but appeared more condensed. Hierarchical constrained density-fitting of domains unexpectedly revealed the fusion loops in this conformation to be apart and pointing away from the anchoring membrane. This vital observation is a substantial step forward in understanding the complex herpesvirus fusion mechanism, and opens up new opportunities for more targeted intervention of herpesvirus entry.

Extracellular vesicles: a platform for the structure determination of membrane proteins by Cryo-EM.

Membrane protein-enriched extracellular vesicles (MPEEVs) provide a platform for studying intact membrane proteins natively anchored with the correct topology in genuine biological membranes. This approach circumvents the need to conduct tedious detergent screens for solubilization, purification, and reconstitution required in classical membrane protein studies. We have applied this method to three integral type I membrane proteins, namely the Caenorhabditis elegans cell-cell fusion proteins AFF-1 and EFF-1 and the glycoprotein B (gB) from Herpes simplex virus type 1 (HSV1). Electron cryotomography followed by subvolume averaging allowed the 3D reconstruction of EFF-1 and HSV1 gB in the membrane as well as an analysis of the spatial distribution and interprotein interactions on the membrane. MPEEVs have many applications beyond structural/functional investigations, such as facilitating the raising of antibodies, for protein-protein interaction assays or for diagnostics use, as biomarkers, and possibly therapeutics.

High-precision correlative fluorescence and electron cryo microscopy using two independent alignment markers.

Correlative light and electron microscopy (CLEM) is an emerging technique which combines functional information provided by fluorescence microscopy (FM) with the high-resolution structural information of electron microscopy (EM). So far, correlative cryo microscopy of frozen-hydrated samples has not reached better than micrometre range accuracy. Here, a method is presented that enables the correlation between fluorescently tagged proteins and electron cryo tomography (cryoET) data with nanometre range precision. Specifically, thin areas of vitrified whole cells are examined by correlative fluorescence cryo microscopy (cryoFM) and cryoET. Novel aspects of the presented cryoCLEM workflow not only include the implementation of two independent electron dense fluorescent markers to improve the precision of the alignment, but also the ability of obtaining an estimate of the correlation accuracy for each individual object of interest. The correlative workflow from plunge-freezing to cryoET is detailed step-by-step for the example of locating fluorescence-labelled adenovirus particles trafficking inside a cell.

The structure of herpesvirus fusion glycoprotein B-bilayer complex reveals the protein-membrane and lateral protein-protein interaction.

Glycoprotein B (gB) is a key component of the complex herpesvirus fusion machinery. We studied membrane interaction of two gB ectodomain forms and present an electron cryotomography structure of the gB-bilayer complex. The two forms differed in presence or absence of the membrane proximal region (MPR) but showed an overall similar trimeric shape. The presence of the MPR impeded interaction with liposomes. In contrast, the MPR-lacking form interacted efficiently with liposomes. Lateral interaction resulted in coat formation on the membranes. The structure revealed that interaction of gB with membranes was mediated by the fusion loops and limited to the outer membrane leaflet. The observed intrinsic propensity of gB to cluster on membranes indicates an additional role of gB in driving the fusion process forward beyond the transient fusion pore opening and subsequently leading to fusion pore expansion.

Conserved eukaryotic fusogens can fuse viral envelopes to cells.

Caenorhabditis elegans proteins AFF-1 and EFF-1 [C. elegans fusion family (CeFF) proteins] are essential for developmental cell-to-cell fusion and can merge insect cells. To study the structure and function of AFF-1, we constructed vesicular stomatitis virus (VSV) displaying AFF-1 on the viral envelope, substituting the native fusogen VSV glycoprotein. Electron microscopy and tomography revealed that AFF-1 formed distinct supercomplexes resembling pentameric and hexameric "flowers" on pseudoviruses. Viruses carrying AFF-1 infected mammalian cells only when CeFFs were on the target cell surface. Furthermore, we identified fusion family (FF) proteins within and beyond nematodes, and divergent members from the human parasitic nematode Trichinella spiralis and the chordate Branchiostoma floridae could also fuse mammalian cells. Thus, FF proteins are part of an ancient family of cellular fusogens that can promote fusion when expressed on a viral particle.

Cryo electron tomography of native HIV-1 budding sites.

The structure of immature and mature HIV-1 particles has been analyzed in detail by cryo electron microscopy, while no such studies have been reported for cellular HIV-1 budding sites. Here, we established a system for studying HIV-1 virus-like particle assembly and release by cryo electron tomography of intact human cells. The lattice of the structural Gag protein in budding sites was indistinguishable from that of the released immature virion, suggesting that its organization is determined at the assembly site without major subsequent rearrangements. Besides the immature lattice, a previously not described Gag lattice was detected in some budding sites and released particles; this lattice was found at high frequencies in a subset of infected T-cells. It displays the same hexagonal symmetry and spacing in the MA-CA layer as the immature lattice, but lacks density corresponding to NC-RNA-p6. Buds and released particles carrying this lattice consistently lacked the viral ribonucleoprotein complex, suggesting that they correspond to aberrant products due to premature proteolytic activation. We hypothesize that cellular and/or viral factors normally control the onset of proteolytic maturation during assembly and release, and that this control has been lost in a subset of infected T-cells leading to formation of aberrant particles.

The three-dimensional organization of polyribosomes in intact human cells.

Structural studies have provided detailed insights into different functional states of the ribosome and its interaction with factors involved in nascent peptide folding, processing, and targeting. However, how the translational machinery is organized spatially in native cellular environments is not yet well understood. Here we have mapped individual ribosomes in electron tomograms of intact human cells by template matching and determined the average structure of the ribosome in situ. Characteristic features of active ribosomes in the cellular environment were assigned to the tRNA channel, elongation factors, and additional densities near the peptide tunnel. Importantly, the relative spatial configuration of neighboring ribosomes in the cell is clearly nonrandom. The preferred configurations are specific for active polysomes and were largely abrogated in puromycin-treated control cells. The distinct neighbor orientations found in situ resemble configurations of bacterial polysomes in vitro, indicating a conserved supramolecular organization with implications for nascent polypeptide folding.

Viral fusion: how Flu induces dimples on liposomes.

Membrane fusion--a basic cellular process enabling crucial cellular functions such as membrane trafficking--is mechanistically only partially understood. Most of the existing knowledge has come from studying host-cell entry of viruses with lipid bilayer envelopes, which proceeds through fusion of viral and host-cell membranes.Crystal structures for a number of viral fusion proteins facilitating this process have contributed substantially to our understanding. One of the next challenges is to merge these high-resolution structures of soluble parts of fusion effectors with native structural information on the proteins in the course of their functional interactions with the target membranes. In this issue of The EMBO Journal,Lee (2010) presents an elegant example of such a study,probing the early interaction of the influenza virus with liposomes by cryo-electron tomography (cryoET).