Josué Gómez-Blanco

Asymmetric cryo-EM reconstruction of phage MS2 reveals genome structure in situ

In single-stranded ribonucleic acid (RNA) viruses, virus capsid assembly and genome packaging are intertwined processes. Using cryo-electron microscopy and single particle analysis we determined the asymmetric virion structure of bacteriophage MS2, which includes 178 copies of the coat protein, a single copy of the A-protein and the RNA genome. This reveals that in situ, the viral RNA genome can adopt a defined conformation. The RNA forms a branched network of stem-loops that almost all allocate near the capsid inner surface, while predominantly binding to coat protein dimers that are located in one-half of the capsid. This suggests that genomic RNA is highly involved in genome packaging and virion assembly.

Epitope Insertion at the N-Terminal Molecular Switch of the Rabbit Hemorrhagic Disease Virus T=3 Capsid Protein Leads to Larger T=4 Capsids.

ABSTRACT Viruses need only one or a few structural capsid proteins to build an infectious particle. This is possible through the extensive use of symmetry and the conformational polymorphism of the structural proteins. Using virus-like particles (VLP) from rabbit hemorrhagic disease virus (RHDV) as a model, we addressed the basis of calicivirus capsid assembly and their application in vaccine design. The RHDV capsid is based on a T=3 lattice containing 180 identical subunits (VP1). We determined the structure of RHDV VLP to 8.0-Å resolution by three-dimensional cryoelectron microscopy; in addition, we used San Miguel sea lion virus (SMSV) and feline calicivirus (FCV) capsid subunit structures to establish the backbone structure of VP1 by homology modeling and flexible docking analysis. Based on the three-domain VP1 model, several insertion mutants were designed to validate the VP1 pseudoatomic model, and foreign epitopes were placed at the N- or C-terminal end, as well as in an exposed loop on the capsid surface. We selected a set of T and B cell epitopes of various lengths derived from viral and eukaryotic origins. Structural analysis of these chimeric capsids further validates the VP1 model to design new chimeras. Whereas most insertions are well tolerated, VP1 with an FCV capsid protein-neutralizing epitope at the N terminus assembled into mixtures of T=3 and larger T=4 capsids. The calicivirus capsid protein, and perhaps that of many other viruses, thus can encode polymorphism modulators that are not anticipated from the plane sequence, with important implications for understanding virus assembly and evolution.

Cryo-EM near-atomic structure of a dsRNA fungal virus shows ancient structural motifs preserved in the dsRNA viral lineage

Significance Viruses that are seemingly unrelated in genomic studies, and which infect hosts in different domains of life, show similarities in virion structure that indicate deep evolutionary relationships. We report the cryo-EM structure, at near-atomic resolution, of the fungal dsRNA Penicillium chrysogenum virus. Its capsid protein is a duplication of a single primordial α-helical domain. This domain has a fold that is conserved among dsRNA viruses; it has increased its complexity through an early gene duplication event, followed by insertion of distinct segments in preferential “hotspots.” We show evidence that this preserved hallmark indicates an ancestral fold, and we suggest a relationship among current viral lineages. Viruses evolve so rapidly that sequence-based comparison is not suitable for detecting relatedness among distant viruses. Structure-based comparisons suggest that evolution led to a small number of viral classes or lineages that can be grouped by capsid protein (CP) folds. Here, we report that the CP structure of the fungal dsRNA Penicillium chrysogenum virus (PcV) shows the progenitor fold of the dsRNA virus lineage and suggests a relationship between lineages. Cryo-EM structure at near-atomic resolution showed that the 982-aa PcV CP is formed by a repeated α-helical core, indicative of gene duplication despite lack of sequence similarity between the two halves. Superimposition of secondary structure elements identified a single “hotspot” at which variation is introduced by insertion of peptide segments. Structural comparison of PcV and other distantly related dsRNA viruses detected preferential insertion sites at which the complexity of the conserved α-helical core, made up of ancestral structural motifs that have acted as a skeleton, might have increased, leading to evolution of the highly varied current structures. Analyses of structural motifs only apparent after systematic structural comparisons indicated that the hallmark fold preserved in the dsRNA virus lineage shares a long (spinal) α-helix tangential to the capsid surface with the head-tailed phage and herpesvirus viral lineage.

Cryphonectria nitschkei virus 1 structure shows that the capsid protein of chrysoviruses is a duplicated helix-rich fold conserved in fungal double-stranded RNA viruses

ABSTRACT Cryoelectron microscopy reconstruction of Cryphonectria nitschkei virus 1, a double-stranded RNA (dsRNA) virus, shows that the capsid protein (60 copies/particle) is formed by a repeated helical core, indicative of gene duplication. This unusual organization is common to chrysoviruses. The arrangement of many of these putative α-helices is conserved in the totivirus L-A capsid protein, suggesting a shared motif. Our results indicate that a 120-subunit T=1 capsid is a conserved architecture that optimizes dsRNA replication and organization.

Structural and functional insights into Escherichia coli α2-macroglobulin endopeptidase snap-trap inhibition

Significance Proteolytic enzymes are inhibited in vivo by protein inhibitors. Such inhibitors are used by symbiotic bacteria in our gut to protect themselves from digestive peptidases. This is the case for Escherichia coli, which has acquired a large, multidomain inhibitor of broad inhibitory spectrum [Escherichia coli α2-macroglobulin (ECAM)]. We studied ECAM and found it is cleaved by host peptidases, which triggers large conformational rearrangement of the inhibitor—shown by protein crystallography and electron microscopy reconstructions—as well as covalent binding of the peptidase. The latter is blocked similarly to a mouse by a snap trap, which prevents damage to the bacterial envelope. Prey peptidases, however, can still be active in the digestion of intake proteins. The survival of commensal bacteria requires them to evade host peptidases. Gram-negative bacteria from the human gut microbiome encode a relative of the human endopeptidase inhibitor, α2-macroglobulin (α2M). Escherichia coli α2M (ECAM) is a ∼180-kDa multidomain membrane-anchored pan-peptidase inhibitor, which is cleaved by host endopeptidases in an accessible bait region. Structural studies by electron microscopy and crystallography reveal that this cleavage causes major structural rearrangement of more than half the 13-domain structure from a native to a compact induced form. It also exposes a reactive thioester bond, which covalently traps the peptidase. Subsequently, peptidase-laden ECAM is shed from the membrane and may dimerize. Trapped peptidases are still active except against very large substrates, so inhibition potentially prevents damage of large cell envelope components, but not host digestion. Mechanistically, these results document a novel monomeric “snap trap.”

Chrysovirus structure: repeated helical core as evidence of gene duplication.

Chrysoviruses are double-stranded RNA viruses with a multipartite genome. Structure of two fungal chrysoviruses, Penicillium chrysogenum virus and Cryphonectria nitschkei chrysovirus 1, has been determined by three-dimensional cryo-electron microscopy analysis and in hydrodynamic studies. The capsids of both viruses are based on a T=1 lattice containing 60 subunits, remain structurally undisturbed throughout the viral cycle, and participate in genome metabolism. The capsid protein is formed by a repeated α-helical core, indicative of gene duplication. Whereas the chrysovirus capsid protein has two motifs with the same fold, most dsRNA virus capsid subunits consist of dimers of a single protein with similar folds. The arrangement of the chrysovirus α-helical core is conserved in the totivirus L-A capsid protein, suggesting a shared basic fold. The encapsidated genome is organized in concentric shells; whereas inner dsRNA shells are diffuse, the outermost layer is organized into a dodecahedral cage beneath the protein capsid. This genome ordering could constitute a framework for dsRNA transcription in the capsid interior and/or have a structural role for capsid stability.

Heterodimers as the structural unit of the T=1 capsid of the fungal double-stranded RNA Rosellinia necatrix quadrivirus 1

ABSTRACT Most double-stranded RNA (dsRNA) viruses are transcribed and replicated in a specialized icosahedral capsid with a T=1 lattice consisting of 60 asymmetric capsid protein (CP) dimers. These capsids help to organize the viral genome and replicative complex(es). They also act as molecular sieves that isolate the virus genome from host defense mechanisms and allow the passage of nucleotides and viral transcripts. Rosellinia necatrix quadrivirus 1 (RnQV1), the type species of the family Quadriviridae, is a dsRNA fungal virus with a multipartite genome consisting of four monocistronic segments (segments 1 to 4). dsRNA-2 and dsRNA-4 encode two CPs (P2 and P4, respectively), which coassemble into ∼450-Å-diameter capsids. We used three-dimensional cryo-electron microscopy combined with complementary biophysical techniques to determine the structures of RnQV1 virion strains W1075 and W1118. RnQV1 has a quadripartite genome, and the capsid is based on a single-shelled T=1 lattice built of P2-P4 dimers. Whereas the RnQV1-W1118 capsid is built of full-length CP, P2 and P4 of RnQV1-W1075 are cleaved into several polypeptides, maintaining the capsid structural organization. RnQV1 heterodimers have a quaternary organization similar to that of homodimers of reoviruses and other dsRNA mycoviruses. The RnQV1 capsid is the first T=1 capsid with a heterodimer as an asymmetric unit reported to date and follows the architectural principle for dsRNA viruses that a 120-subunit capsid is a conserved assembly that supports dsRNA replication and organization. IMPORTANCE Given their importance to health, members of the family Reoviridae are the basis of most structural and functional studies and provide much of our knowledge of dsRNA viruses. Analysis of bacterial, protozoal, and fungal dsRNA viruses has improved our understanding of their structure, function, and evolution, as well. Here, we studied a dsRNA virus that infects the fungus Rosellinia necatrix, an ascomycete that is pathogenic to a wide range of plants. Using three-dimensional cryo-electron microscopy and analytical ultracentrifugation analysis, we determined the structure and stoichiometry of Rosellinia necatrix quadrivirus 1 (RnQV1). The RnQV1 capsid is a T=1 capsid with 60 heterodimers as the asymmetric units. The large amount of genetic information used by RnQV1 to construct a simple T=1 capsid is probably related to the numerous virus-host and virus-virus interactions that it must face in its life cycle, which lacks an extracellular phase.

Structural Basis for the Development of Avian Virus Capsids That Display Influenza Virus Proteins and Induce Protective Immunity

ABSTRACT Bioengineering of viruses and virus-like particles (VLPs) is a well-established approach in the development of new and improved vaccines against viral and bacterial pathogens. We report here that the capsid of a major avian pathogen, infectious bursal disease virus (IBDV), can accommodate heterologous proteins to induce protective immunity. The structural units of the ∼70-nm-diameter T=13 IBDV capsid are trimers of VP2, which is made as a precursor (pVP2). The pVP2 C-terminal domain has an amphipathic α helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, 466-residue pVP2 intermediates bearing this α helix assemble into genuine VLPs only when expressed with an N-terminal His6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for protein insertion, as they are large enough (cargo space, ∼78,000 nm3) and are assembled from a single protein. We explored HT-VP2-466-based chimeric capsids initially using enhanced green fluorescent protein (EGFP). The VLP assembly yield was efficient when we coexpressed EGFP-HT-VP2-466 and HT-VP2-466 from two recombinant baculoviruses. The native EGFP structure (∼240 copies/virion) was successfully inserted in a functional form, as VLPs were fluorescent, and three-dimensional cryo-electron microscopy showed that the EGFP molecules incorporated at the inner capsid surface. Immunization of mice with purified EGFP-VLPs elicited anti-EGFP antibodies. We also inserted hemagglutinin (HA) and matrix (M2) protein epitopes derived from the mouse-adapted A/PR/8/34 influenza virus and engineered several HA- and M2-derived chimeric capsids. Mice immunized with VLPs containing the HA stalk, an M2 fragment, or both antigens developed full protection against viral challenge. IMPORTANCE Virus-like particles (VLPs) are multimeric protein cages that mimic the infectious virus capsid and are potential candidates as nonliving vaccines that induce long-lasting protection. Chimeric VLPs can display or include foreign antigens, which could be a conserved epitope to elicit broadly neutralizing antibodies or several variable epitopes effective against a large number of viral strains. We report the biochemical, structural, and immunological characterization of chimeric VLPs derived from infectious bursal disease virus (IBDV), an important poultry pathogen. To test the potential of IBDV VLPs as a vaccine vehicle, we used the enhanced green fluorescent protein and two fragments derived from the hemagglutinin and the M2 matrix protein of the human murine-adapted influenza virus. The IBDV capsid protein fused to influenza virus peptides formed assemblies able to protect mice against viral challenge. Our studies establish the basis for a new generation of multivalent IBDV-based vaccines.

Acquisition of functions on the outer capsid surface during evolution of double-stranded RNA fungal viruses

Unlike their counterparts in bacterial and higher eukaryotic hosts, most fungal viruses are transmitted intracellularly and lack an extracellular phase. Here we determined the cryo-EM structure at 3.7 Å resolution of Rosellinia necatrix quadrivirus 1 (RnQV1), a fungal double-stranded (ds)RNA virus. RnQV1, the type species of the family Quadriviridae, has a multipartite genome consisting of four monocistronic segments. Whereas most dsRNA virus capsids are based on dimers of a single protein, the ~450-Å-diameter, T = 1 RnQV1 capsid is built of P2 and P4 protein heterodimers, each with more than 1000 residues. Despite a lack of sequence similarity between the two proteins, they have a similar α-helical domain, the structural signature shared with the lineage of the dsRNA bluetongue virus-like viruses. Domain insertions in P2 and P4 preferential sites provide additional functions at the capsid outer surface, probably related to enzyme activity. The P2 insertion has a fold similar to that of gelsolin and profilin, two actin-binding proteins with a function in cytoskeleton metabolism, whereas the P4 insertion suggests protease activity involved in cleavage of the P2 383-residue C-terminal region, absent in the mature viral particle. Our results indicate that the intimate virus-fungus partnership has altered the capsid genome-protective and/or receptor-binding functions. Fungal virus evolution has tended to allocate enzyme activities to the virus capsid outer surface.

Denoising of high-resolution single-particle electron-microscopy density maps by their approximation using three-dimensional Gaussian functions.

Cryo-electron microscopy (cryo-EM) of frozen-hydrated preparations of isolated macromolecular complexes is the method of choice to obtain the structure of complexes that cannot be easily studied by other experimental methods due to their flexibility or large size. An increasing number of macromolecular structures are currently being obtained at subnanometer resolution but the interpretation of structural details in such EM-derived maps is often difficult because of noise at these high-frequency signal components that reduces their contrast. In this paper, we show that the method for EM density-map approximation using Gaussian functions can be used for denoising of single-particle EM maps of high (typically subnanometer) resolution. We show its denoising performance using simulated and experimental EM density maps of several complexes.