As we respond to viral epidemics and accelerate the discovery of new viruses, sifting through vast volumes of structural virology data could rapidly become an impossible task. virusMED is a curated ...atlas of metal/drug-binding and immunological hotspots in viral protein structures that provides a navigation guide for structure-function analysis and the development of antiviral strategies.
Many bacterial pathogens have long, slender pili through which they adhere to host cells. The crystal structure of the major pilin subunit from the Gram-positive human pathogen Streptococcus pyogenes ...at 2.2 angstroms resolution reveals an extended structure comprising two all-β domains. The molecules associate in columns through the crystal, with each carboxyl terminus adjacent to a conserved lysine of the next molecule. This lysine forms the isopeptide bonds that link the subunits in native pili, validating the relevance of the crystal assembly. Each subunit contains two lysine-asparagine isopeptide bonds generated by an intramolecular reaction, and we find evidence for similar isopeptide bonds in other cell surface proteins of Gram-positive bacteria. The present structure explains the strength and stability of such Gram-positive pili and could facilitate vaccine development.
Flagellotropic bacteriophages engage flagella to reach the bacterial surface as an effective means to increase the capture radius for predation. Structural details of these viruses are of great ...interest given the substantial drag forces and torques they face when moving down the spinning flagellum. We show that the main capsid and auxiliary proteins form two nested chainmails that ensure the integrity of the bacteriophage head. Core stabilising structures are conserved in herpesviruses suggesting their ancestral origin. The structure of the tail also reveals a robust yet pliable assembly. Hexameric rings of the tail-tube protein are braced by the N-terminus and a β-hairpin loop, and interconnected along the tail by the splayed β-hairpins. By contrast, we show that the β-hairpin has an inhibitory role in the tail-tube precursor, preventing uncontrolled self-assembly. Dyads of acidic residues inside the tail-tube present regularly-spaced motifs well suited to DNA translocation into bacteria through the tail.
Abstract
The epidemic emergence of relatively rare and geographically isolated flaviviruses adds to the ongoing disease burden of viruses such as dengue. Structural analysis is key to understand and ...combat these pathogens. Here, we present a chimeric platform based on an insect-specific flavivirus for the safe and rapid structural analysis of pathogenic viruses. We use this approach to resolve the architecture of two neurotropic viruses and a structure of dengue virus at 2.5 Å, the highest resolution for an enveloped virion. These reconstructions allow improved modelling of the stem region of the envelope protein, revealing two lipid-like ligands within highly conserved pockets. We show that these sites are essential for viral growth and important for viral maturation. These findings define a hallmark of flavivirus virions and a potential target for broad-spectrum antivirals and vaccine design. We anticipate the chimeric platform to be widely applicable for investigating flavivirus biology.
Poxviruses are large DNA viruses that cause disease in animals and humans. They differ from classical enveloped viruses, because their membrane is acquired from cytoplasmic membrane precursors ...assembled onto a viral protein scaffold formed by the D13 protein rather than budding through cellular compartments. It was found three decades ago that the antibiotic rifampicin blocks this process and prevents scaffold formation. To elucidate the mechanism of action of rifampicin, we have determined the crystal structures of six D13–rifamycin complexes. These structures reveal that rifamycin compounds bind to a phenylalanine-rich region, or F-ring, at the membrane-proximal opening of the central channel of the D13 trimer. We show by NMR, surface plasmon resonance (SPR), and site-directed mutagenesis that A17, a membrane-associated viral protein, mediates the recruitment of the D13 scaffold by also binding to the F-ring. This interaction is the target of rifampicin, which prevents A17 binding, explaining the inhibition of viral morphogenesis. The F-ring of D13 is both conserved in sequence in mammalian poxviruses and essential for interaction with A17, defining a target for the development of assembly inhibitors. The model of the A17–D13 interaction describes a two-component system for remodeling nascent membranes that may be conserved in other large and giant DNA viruses.
Abstract
The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30 kb), ...infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled together with a linker fragment containing CMV promoter into a circular full-length viral cDNA in a single reaction. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with ~20 kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical sample. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions.
Viral infection causes comprehensive rearrangements of the cell that reflect as much host defense mechanisms as virus-induced structures assembled to facilitate infection. Regardless of their pro- or ...antiviral role, large intracellular structures are readily detectable by microscopy and often provide a signature characteristic of a specific viral infection. The structural features and localization of these assemblies have thus been commonly used for the diagnostic and classification of viruses since the early days of virology. More recently, characterization of viral superstructures using molecular and structural approaches have revealed very diverse organizations and roles, ranging from dynamic viral factories behaving like liquid organelles to ultra-stable crystals embedding and protecting virions. This chapter reviews the structures, functions and biotechnological applications of virus-induced superstructures with a focus on assemblies that have a regular organization, for which detailed structural descriptions are available. Examples span viruses infecting all domains of life including the assembly of virions into crystalline arrays in eukaryotic and bacterial viruses, nucleus-like compartments involved in the replication of large bacteriophages, and pyramid-like structures mediating the egress of archaeal viruses. Among these superstructures, high-resolution structures are available for crystalline objects produced by insect viruses: viral polyhedra which function as the infectious form of occluded viruses, and spindles which are potent virulence factors of entomopoxviruses. In turn, some of these highly symmetrical objects have been used to develop and validate advanced structural approaches, pushing the boundary of structural biology.
Dengue virus (DENV) causes the major arboviral disease of the tropics, characterized in its severe forms by signs of hemorrhage and plasma leakage. DENV encodes a nonstructural glycoprotein, NS1, ...that associates with intracellular membranes and the cell surface. NS1 is eventually secreted as a soluble hexamer from DENV-infected cells and circulates in the bloodstream of infected patients. Extracellular NS1 has been shown to modulate the complement system and to enhance DENV infection, yet its structure and function remain essentially unknown. By combining cryoelectron microscopy analysis with a characterization of NS1 amphipathic properties, we show that the secreted NS1 hexamer forms a lipoprotein particle with an open-barrel protein shell and a prominent central channel rich in lipids. Biochemical and NMR analyses of the NS1 lipid cargo reveal the presence of triglycerides, bound at an equimolar ratio to the NS1 protomer, as well as cholesteryl esters and phospholipids, a composition evocative of the plasma lipoproteins involved in vascular homeostasis. This study suggests that DENV NS1, by mimicking or hijacking lipid metabolic pathways, contributes to endothelium dysfunction, a key feature of severe dengue disease.
Natural killer cells and cytotoxic T lymphocytes accomplish the critically important function of killing virus-infected and neoplastic cells. They do this by releasing the pore-forming protein ...perforin and granzyme proteases from cytoplasmic granules into the cleft formed between the abutting killer and target cell membranes. Perforin, a 67-kilodalton multidomain protein, oligomerizes to form pores that deliver the pro-apoptopic granzymes into the cytosol of the target cell. The importance of perforin is highlighted by the fatal consequences of congenital perforin deficiency, with more than 50 different perforin mutations linked to familial haemophagocytic lymphohistiocytosis (type 2 FHL). Here we elucidate the mechanism of perforin pore formation by determining the X-ray crystal structure of monomeric murine perforin, together with a cryo-electron microscopy reconstruction of the entire perforin pore. Perforin is a thin 'key-shaped' molecule, comprising an amino-terminal membrane attack complex perforin-like (MACPF)/cholesterol dependent cytolysin (CDC) domain followed by an epidermal growth factor (EGF) domain that, together with the extreme carboxy-terminal sequence, forms a central shelf-like structure. A C-terminal C2 domain mediates initial, Ca2+-dependent membrane binding. Most unexpectedly, however, electron microscopy reveals that the orientation of the perforin MACPF domain in the pore is inside-out relative to the subunit arrangement in CDCs. These data reveal remarkable flexibility in the mechanism of action of the conserved MACPF/CDC fold and provide new insights into how related immune defence molecules such as complement proteins assemble into pores.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Spindles are intracellular crystals of the fusolin protein that enhances the oral virulence of insect poxviruses by disruption of the larval chitinous peritrophic matrix. The enigmatic fusolin ...protein is classified as a lytic polysaccharide monooxygenase (LPMO) by sequence and structure. Although circumstantial evidence points towards a role for fusolin in chitin degradation, no biochemical data exist to verify this claim. In the present study, we demonstrate that fusolin released from over 40‐year‐old spindles, stored for 10 years at 4 °C, are chitin‐degrading LPMOs. Not only was fusolin active after long‐term storage, but it also withstood high temperature and oxidative stress in its crystalline form, highlighting extreme stability that is beneficial to viral persistence and desirable for potential biotechnology applications.
Most insect poxviruses produce spindles, ultrastable crystals of the fusolin protein, that increase their oral virulence. Fusolin is known to disrupt the chitinous peritrophic matrix lining the larval digestive tract. In keeping with a known structural homology, we show that fusolin is a lytic polysaccharide monooxygenase (LPMO) that can depolymerize chitin by oxidation.
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