Conventional Electron Microscopy, Cryogenic Electron Microscopy, and Cryogenic Electron Tomography of Viruses.

Electron microscopy (EM) techniques have been crucial for understanding the structure of biological specimens such as cells, tissues and macromolecular assemblies. Viruses and related viral assemblies are ideal targets for structural studies that help to define essential biological functions. Whereas conventional EM methods use chemical fixation, dehydration, and staining of the specimens, cryogenic electron microscopy (cryo-EM) preserves the native hydrated state.

The Basic Architecture of Viruses.

Viruses are elegant macromolecular assemblies and constitute a paradigm of the economy of genomic resources; they must use simple general principles to complete their life cycles successfully. Viruses need only one or a few different capsid structural subunits to build an infectious particle, which is possible for two reasons: extensive use of symmetry and built-in conformational flexibility. Although viruses come in many shapes and sizes, two major symmetric assemblies are found: icosahedral and helical.

A RNA Dodecahedral Cage Inside a Human Virus Plays a Dual Biological Role in Virion Assembly and Genome Release Control.

Human rhinoviruses (RV) are among the most frequent human pathogens. As major causative agents of common colds they originate serious socioeconomic problems and huge expenditure every year, and they also exacerbate severe respiratory diseases. No anti-rhinoviral drugs or vaccines are available so far.

Structure of the aminoterminal domain of the birnaviral multifunctional VP3 protein and its unexplored critical role.

To overcome their limited genetic capacity, numerous viruses encode multifunctional proteins. The birnavirus VP3 protein plays key roles during infection, including scaffolding of the viral capsid during morphogenesis, recruitment, and regulation of the viral RNA polymerase, shielding of the double-stranded RNA genome and targeting of host endosomes for genome replication, and immune evasion. The dimeric form of VP3 is critical for these functions.

Mechanical disassembly of human picobirnavirus like particles indicates that cargo retention is tuned by the RNA-coat protein interaction.

Here we investigate the cargo retention of individual human picobirnavirus (hPBV) virus-like particles (VLPs) which differ in the N-terminal of their capsid protein (CP): (i) hPBV CP contains the full-length CP sequence; (ii) hPBV Δ45-CP lacks the first 45 N-terminal residues; and (iii) hPBV Ht-CP is the full-length CP with a N-terminal 36-residue tag that includes a 6-His segment. Consequently, each VLP variant holds a different interaction with the ssRNA cargo. We used atomic force microscopy (AFM) to induce and monitor the mechanical disassembly of individual hPBV particles.

Equilibrium Dynamics of a Biomolecular Complex Analyzed at Single-amino Acid Resolution by Cryo-electron Microscopy.

The biological function of macromolecular complexes depends not only on large-scale transitions between conformations, but also on small-scale conformational fluctuations at equilibrium. Information on the equilibrium dynamics of biomolecular complexes could, in principle, be obtained from local resolution (LR) data in cryo-electron microscopy (cryo-EM) maps. However, this possibility had not been validated by comparing, for a same biomolecular complex, LR data with quantitative information on equilibrium dynamics obtained by an established solution technique.

Cryo-EM structures show the mechanistic basis of pan-peptidase inhibition by human α-macroglobulin.

Human α2-macroglobulin (hα2M) is a multidomain protein with a plethora of essential functions, including transport of signaling molecules and endopeptidase inhibition in innate immunity. Here, we dissected the molecular mechanism of the inhibitory function of the ∼720-kDa hα2M tetramer through eight cryo–electron microscopy (cryo-EM) structures of complexes from human plasma. In the native complex, the hα2M subunits are organized in two flexible modules in expanded conformation, which enclose a highly porous cavity in which the proteolytic activity of circulating plasma proteins is tested.

Immunogenicity of Multi-Target Chimeric RHDV Virus-Like Particles Delivering Foreign B-Cell Epitopes.

The rabbit hemorrhagic disease virus (RHDV) vaccine platform is a nanoparticle composed of 180 copies of the viral capsid protein, VP60, self-assembled into virus-like particles (VLPs). RHDV VLPs are able to accept the simultaneous incorporation of target epitopes at different insertion sites. The resulting chimeric RHDV VLPs displaying immunogenic foreign antigens have been shown to induce specific protective immune responses against inserted heterologous T-cytotoxic and B-cell epitopes in the mouse and pig models.

Nanotechnological Applications Based on Bacterial Encapsulins.

Encapsulins are proteinaceous nanocontainers, constructed by a single species of shell protein that self-assemble into 20-40 nm icosahedral particles. Encapsulins are structurally similar to the capsids of viruses of the HK97-like lineage, to which they are evolutionarily related. Nearly all these nanocontainers encase a single oligomeric protein that defines the physiological role of the complex, although a few encapsulate several activities within a single particle.

Chimeric RHDV Virus-Like Particles Displaying Foot-and-Mouth Disease Virus Epitopes Elicit Neutralizing Antibodies and Confer Partial Protection in Pigs.

Currently there is a clear trend towards the establishment of virus-like particles (VLPs) as a powerful tool for vaccine development. VLPs are tunable nanoparticles that can be engineered to be used as platforms for multimeric display of foreign antigens. We have previously reported that VLPs derived from rabbit hemorrhagic disease virus (RHDV) constitute an excellent vaccine vector, capable of inducing specific protective immune responses against inserted heterologous T-cytotoxic and B-cell epitopes.

Cryo-EM structure of a thermophilic encapsulin offers clues to its functions.

Wiryaman & Toor [ (2021). , 342-350] report the cryo-EM structure of a encapsulin, a nanocompartment that encapsulates a ferritin-like protein cargo. The 2 Å resolution structure offers insights into the active role of this thermostable encapsulin in regulating iron homeostasis to reduce oxidative stress.

Cryo-electron Microscopy Structure, Assembly, and Mechanics Show Morphogenesis and Evolution of Human Picobirnavirus.

Despite their diversity, most double-stranded-RNA (dsRNA) viruses share a specialized T=1 capsid built from dimers of a single protein that provides a platform for genome transcription and replication. This ubiquitous capsid remains structurally undisturbed throughout the viral cycle, isolating the genome to avoid triggering host defense mechanisms. Human picobirnavirus (hPBV) is a dsRNA virus frequently associated with gastroenteritis, although its pathogenicity is yet undefined.

Precise location of linear epitopes on the capsid surface of feline calicivirus recognized by neutralizing and non-neutralizing monoclonal antibodies.

We report the generation, characterization and epitope mapping of a panel of 26 monoclonal antibodies (MAbs) against the VP1 capsid protein of feline calicivirus (FCV). Two close but distinct linear epitopes were identified at the capsid outermost surface (P2 subdomain) of VP1, within the E5'HVR antigenic hypervariable region: one spanning amino acids 431-435 (PAGDY), highly conserved and recognized by non-neutralizing MAbs; and a second epitope spanning amino acids 445-451 (ITTANQY), highly variable and recognized by neutralizing MAbs. These antibodies might be valuable for diagnostic applications, as well as for further research in different aspects of the biology of FCV.

Cryo-electron microscopy for the study of virus assembly.

Although viruses are extremely diverse in shape and size, evolution has led to a limited number of viral classes or lineages, which is probably linked to the assembly constraints of a viable capsid. Viral assembly mechanisms are restricted to two general pathways, (i) co-assembly of capsid proteins and single-stranded nucleic acids and (ii) a sequential mechanism in which scaffolding-mediated capsid precursor assembly is followed by genome packaging. Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), which are revolutionizing structural biology, are central to determining the high-resolution structures of many viral assemblies as well as those of assembly intermediates.

ICTV Virus Taxonomy Profile: .

Members of the family are isometric, non-enveloped viruses with segmented, linear, dsRNA genomes. There are 3-7 genomic segments, each of which is individually encapsidated. Chrysoviruses infect fungi, plants and possibly insects, and may cause hypovirulence in their fungal hosts.

Structure and assembly of double-stranded RNA mycoviruses.

Mycoviruses are a diverse group that includes ssRNA, dsRNA, and ssDNA viruses, with or without a protein capsid, as well as with a complex envelope. Most mycoviruses are transmitted by cytoplasmic interchange and are thought to lack an extracellular phase in their infection cycle. Structural analysis has focused on dsRNA mycoviruses, which usually package their genome in a 120-subunit T=1 icosahedral capsid, with a capsid protein (CP) dimer as the asymmetric unit.

Programmed Recognition between Complementary Dinucleolipids To Control the Self-Assembly of Lipidic Amphiphiles.

One of the major goals in systems chemistry is to create molecular assemblies with emergent properties that are characteristic of life. An interesting approach toward this goal is based on merging different biological building blocks into synthetic systems with properties arising from the combination of their molecular components. The covalent linkage of nucleic acids (or their constituents: nucleotides, nucleosides and nucleobases) with lipids in the same hybrid molecule leads, for example, to the so-called nucleolipids.

Structural nanotechnology: three-dimensional cryo-EM and its use in the development of nanoplatforms for in vitro catalysis.

The organization of enzymes into different subcellular compartments is essential for correct cell function. Protein-based cages are a relatively recently discovered subclass of structurally dynamic cellular compartments that can be mimicked in the laboratory to encapsulate enzymes. These synthetic structures can then be used to improve our understanding of natural protein-based cages, or as nanoreactors in industrial catalysis, metabolic engineering, and medicine.

ICTV Virus Taxonomy Profile: Quadriviridae.

The Quadriviridae is a monogeneric family of non-enveloped spherical viruses with quadripartite dsRNA genomes, each segment of 3.5-5.0 kbp, comprising 16.

Mechanics of Virus-like Particles Labeled with Green Fluorescent Protein.

Nanoindentation with an atomic force microscope was used to investigate the mechanical properties of virus-like particles (VLPs) derived from the avian pathogen infectious bursal disease virus, in which the major capsid protein was modified by fusion with enhanced green fluorescent protein (EGFP). These VLPs assemble as ∼70-nm-diameter T = 13 icosahedral capsids with large cargo space. The effect of the insertion of heterologous proteins in the capsid was characterized in the elastic regime, revealing that EGFP-labeled chimeric VLPs are more rigid than unmodified VLPs.

Capsid Structure of dsRNA Fungal Viruses.

Most fungal, double-stranded (ds) RNA viruses lack an extracellular life cycle stage and are transmitted by cytoplasmic interchange. dsRNA mycovirus capsids are based on a 120-subunit T = 1 capsid, with a dimer as the asymmetric unit. These capsids, which remain structurally undisturbed throughout the viral cycle, nevertheless, are dynamic particles involved in the organization of the viral genome and the viral polymerase necessary for RNA synthesis.

Biophysical properties of single rotavirus particles account for the functions of protein shells in a multilayered virus.

The functions performed by the concentric shells of multilayered dsRNA viruses require specific protein interactions that can be directly explored through their mechanical properties. We studied the stiffness, breaking force, critical strain and mechanical fatigue of individual Triple, Double and Single layered rotavirus (RV) particles. Our results, in combination with Finite Element simulations, demonstrate that the mechanics of the external layer provides the resistance needed to counteract the stringent conditions of extracellular media.