Native hepatitis B virions and capsids visualized by electron cryomicroscopy

Hepatitis B virus (HBV) infects more than 350 million people, of which one million will die every year. The infectious virion is an enveloped capsid containing the viral polymerase and double-stranded DNA genome. The structure of the capsid assembled in vitro from expressed core protein has been studied intensively. However, little is known about the structure and assembly of native capsids present in infected cells, and even less is known about the structure of mature virions. We used electron cryomicroscopy (cryo-EM) and image analysis to examine HBV virions (Dane particles) isolated from patient serum and capsids positive and negative for HBV DNA isolated from the livers of transgenic mice. Both types of capsids assembled as icosahedral particles indistinguishable from previous image reconstructions of capsids. Likewise, the virions contained capsids with either T = 3 or T = 4 icosahedral symmetry. Projections extending from the lipid envelope were attributed to surface glycoproteins. Their packing was unexpectedly nonicosahedral but conformed to an ordered lattice. These structural features distinguish HBV from other enveloped viruses.     

Structure and assembly of a T=1 virus-like particle in BK polyomavirus

In polyomaviruses the pentameric capsomers are interlinked by the long C-terminal arm of the structural protein VP1. The T=7 icosahedral structure of these viruses is possible due to an intriguing adaptability of this linker arm to the different local environments in the capsid. To explore the assembly process, we have compared the structure of two virus-like particles (VLPs) formed, as we found, in a calcium-dependent manner by the VP1 protein of human polyomavirus BK. The structures were determined using electron cryomicroscopy (cryo-EM), and the three-dimensional reconstructions were interpreted by atomic modeling. In the small VP1 particle, 26.4 nm in diameter, the pentameric capsomers form an icosahedral T=1 surface lattice with meeting densities at the threefold axes that interlinked three capsomers. In the larger particle, 50.6 nm in diameter, the capsomers form a T=7 icosahedral shell with three unique contacts. A folding model of the BKV VP1 protein was obtained by alignment with the VP1 protein of simian virus 40 (SV40). The model fitted well into the cryo-EM density of the T=7 particle. However, residues 297 to 362 of the C-terminal arm had to be remodeled to accommodate the higher curvature of the T=1 particle. The loops, before and after the C-terminal short helix, were shown to provide the hinges that allowed curvature variation in the particle shell. The meeting densities seen at the threefold axes in the T=1 particle were consistent with the triple-helix interlinking contact at the local threefold axes in the T=7 structure.     

Three-dimensional organization of Rift Valley fever virus revealed by cryoelectron tomography

Rift Valley fever virus (RVFV) is a member of the Bunyaviridae virus family (genus Phlebovirus) and is considered to be one of the most important pathogens in Africa, causing viral zoonoses in livestock and humans. Here, we report the characterization of the three-dimensional structural organization of RVFV vaccine strain MP-12 by cryoelectron tomography. Vitrified-hydrated virions were found to be spherical, with an average diameter of 100 nm. The virus glycoproteins formed cylindrical hollow spikes that clustered into distinct capsomeres. In contrast to previous assertions that RVFV is pleomorphic, the structure of RVFV MP-12 was found to be highly ordered. The three-dimensional map was resolved to a resolution of 6.1 nm, and capsomeres were observed to be arranged on the virus surface in an icosahedral lattice with clear T=12 quasisymmetry. All icosahedral symmetry axes were visible in self-rotation functions calculated using the Fourier transform of the RVFV MP-12 tomogram. To the best of our knowledge, a triangulation number of 12 had previously been reported only for Uukuniemi virus, a bunyavirus also within the Phlebovirus genus. The results presented in this study demonstrate that RVFV MP-12 possesses T=12 icosahedral symmetry and suggest that other members of the Phlebovirus genus, as well as of the Bunyaviridae family, may adopt icosahedral symmetry. Knowledge of the virus architecture may provide a structural template to develop vaccines and diagnostics, since no effective anti-RVFV treatments are available for human use.     

Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody

Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection.     

Cryo-EM of the injectisome and type III secretion systems

Double-membrane-spanning protein complexes, such as the T3SS, had long presented an intractable challenge for structural biology. As a consequence, until a few years ago, our molecular understanding of this fascinating complex was limited to composite models, consisting of structures of isolated domains, positioned within the overall complex. Most of the membrane-embedded components remained completely uncharacterized.In recent years, the emergence of cryo-electron microscopy (cryo-EM) as a method for determining protein structures to high resolution, has be transformative to our capacity to understand the architecture of this complex, and its mechanism of substrate transport.In this review, we summarize the recent structures of the various T3SS components, determined by cryo-EM, and highlight the regions of the complex that remain to be characterized. We also discuss the recent structural insights into the mechanism of effector transport through the T3SS. Finally, we highlight some of the challenges that remain to be tackled.     

Cationic lipid/DNA complex-adjuvanted influenza A virus vaccination induces robust cross-protective immunity

Influenza A virus is a negative-strand segmented RNA virus in which antigenically distinct viral subtypes are defined by the hemagglutinin (HA) and neuraminidase (NA) major viral surface proteins. An ideal inactivated vaccine for influenza A virus would induce not only highly robust strain-specific humoral and T-cell immune responses but also cross-protective immunity in which an immune response to antigens from a particular viral subtype (e.g., H3N2) would protect against other viral subtypes (e.g., H1N1). Cross-protective immunity would help limit outbreaks from newly emerging antigenically novel strains. Here, we show in mice that the addition of cationic lipid/noncoding DNA complexes (CLDC) as adjuvant to whole inactivated influenza A virus vaccine induces significantly more robust adaptive immune responses both in quantity and quality than aluminum hydroxide (alum), which is currently the most widely used adjuvant in clinical human vaccination. CLDC-adjuvanted vaccine induced higher total influenza virus-specific IgG, particularly for the IgG2a/c subclass. Higher levels of multicytokine-producing influenza virus-specific CD4 and CD8 T cells were induced by CLDC-adjuvanted vaccine than with alum-adjuvanted vaccine. Importantly, CLDC-adjuvanted vaccine provided significant cross-protection from either a sublethal or lethal influenza A viral challenge with a different subtype than that used for vaccination. This superior cross-protection afforded by the CLDC adjuvant required CD8 T-cell recognition of viral peptides presented by classical major histocompatibility complex class I proteins. Together, these results suggest that CLDC has particular promise for vaccine strategies in which T cells play an important role and may offer new opportunities for more effective control of human influenza epidemics and pandemics by inactivated influenza virus vaccine.     

Gene therapy meets vaccine development

Therapeutic vaccines such as those used to combat cancer or persistent viral infection are required to reprogramme a downregulated immune system. This presents a difficult challenge for vaccine design and merits the development of novel immunization protocols. Currently, we know that mobilization of dendritic cells (DCs) to present antigens to T lymphocytes is crucial for effective immunization. Our increasing understanding of DC biology, coupled with the growing sophistication of viral vectors developed for gene therapy, makes more rational vaccine design an exciting possibility. Here we propose that engineering viral vectors to express antigens in activated DCs will provide the most effective vaccines for priming an immune response.     

The 2.3-angstrom structure of porcine circovirus 2

Porcine circovirus 2 (PCV2) is a T=1 nonenveloped icosahedral virus that has had severe impact on the swine industry. Here we report the crystal structure of an N-terminally truncated PCV2 virus-like particle at 2.3-Å resolution, and the cryo-electron microscopy (cryo-EM) image reconstruction of a full-length PCV2 virus-like particle at 9.6-Å resolution. This is the first atomic structure of a circovirus. The crystal structure revealed that the capsid protein fold is a canonical viral jelly roll. The loops connecting the strands of the jelly roll define the limited features of the surface. Sulfate ions interacting with the surface and electrostatic potential calculations strongly suggest a heparan sulfate binding site that allows PCV2 to gain entry into the cell. The crystal structure also allowed previously determined epitopes of the capsid to be visualized. The cryo-EM image reconstruction showed that the location of the N terminus, absent in the crystal structure, is inside the capsid. As the N terminus was previously shown to be antigenic, it may externalize through viral “breathing.”     

Structure Determination of Feline Calicivirus Virus-Like Particles in the Context of a Pseudo-Octahedral Arrangement

The vesivirus feline calicivirus (FCV) is a positive strand RNA virus encapsidated by an icosahedral T=3 shell formed by the viral VP1 protein. Upon its expression in the insect cell - baculovirus system in the context of vaccine development, two types of virus-like particles (VLPs) were formed, a majority built of 60 subunits (T=1) and a minority probably built of 180 subunits (T=3). The structure of the small particles was determined by x-ray crystallography at 0.8 nm resolution helped by cryo-electron microscopy in order to understand their formation. Cubic crystals belonged to space group P2₁3. Their self-rotation function showed the presence of an octahedral pseudo-symmetry similar to the one described previously by Agerbandje and co-workers for human parvovirus VLPs. The crystal structure could be solved starting from the published VP1 structure in the context of the T=3 viral capsid. In contrast to viral capsids, where the capsomers are interlocked by the exchange of the N-terminal arm (NTA) domain, this domain is disordered in the T=1 capsid of the VLPs. Furthermore it is prone to proteolytic cleavage. The relative orientation of P (protrusion) and S (shell) domains is alerted so as to fit VP1 to the smaller T=1 particle whereas the intermolecular contacts around 2-fold, 3-fold and 5-fold axes are conserved. By consequence the surface of the VLP is very similar compared to the viral capsid and suggests a similar antigenicity. The knowledge of the structure of the VLPs will help to improve their stability, in respect to a use for vaccination.     

High-resolution 3D structures reveal the biological functions of reoviruses

Viruses in the family Reoviridae are non-enveloped particles comprising a segmented double-stranded RNA genome surrounded by a two-layered or multi-layered icosahedral protein capsid. These viruses are classified into two sub-families based on their particle structural organization. Recent studies have focused on high-resolution three-dimensional structures of reovirus particles by using cryo-electron microscopy （cryo-EM） to approach the resolutions seen in X-ray crystallographic structures. The results of cryo-EM image reconstructions allow tracing of most of the protein side chains, and thus permit integration of structural and functional information into a coherent mechanism for reovirus assembly and entry.     

Identification of Class I HLA T Cell Control Epitopes for West Nile Virus

The recent West Nile virus (WNV) outbreak in the United States underscores the importance of understanding human immune responses to this pathogen. Via the presentation of viral peptide ligands at the cell surface, class I HLA mediate the T cell recognition and killing of WNV infected cells. At this time, there are two key unknowns in regards to understanding protective T cell immunity: 1) the number of viral ligands presented by the HLA of infected cells, and 2) the distribution of T cell responses to these available HLA/viral complexes. Here, comparative mass spectroscopy was applied to determine the number of WNV peptides presented by the HLA-A*11:01 of infected cells after which T cell responses to these HLA/WNV complexes were assessed. Six viral peptides derived from capsid, NS3, NS4b, and NS5 were presented. When T cells from infected individuals were tested for reactivity to these six viral ligands, polyfunctional T cells were focused on the GTL9 WNV capsid peptide, ligands from NS3, NS4b, and NS5 were less immunogenic, and two ligands were largely inert, demonstrating that class I HLA reduce the WNV polyprotein to a handful of immune targets and that polyfunctional T cells recognize infections by zeroing in on particular HLA/WNV epitopes. Such dominant HLA/peptide epitopes are poised to drive the development of WNV vaccines that elicit protective T cells as well as providing key antigens for immunoassays that establish correlates of viral immunity.     

Template recognition and formation of initiation complexes by the replicase of a segmented double-stranded RNA virus

Replication of the segmented double-stranded (ds) RNA genome of viruses belonging to the Reoviridae family requires the RNA-dependent RNA polymerase (RdRP) to use 10-12 different mRNAs as templates for (-) strand synthesis. Rotavirus serves as a model system for study of this process, since its RdRP (VP1) is catalytically active and can specifically recognize template mRNAs in vitro. Here, we have analyzed the requirements for template recognition by the rotavirus RdRP and compared those to the requirements for formation of (-) strand initiation complexes. The results show that multiple functionally independent recognition signals are present at the 3'-end of viral mRNAs, some positioned in nonconserved regions upstream of the highly conserved 3'-terminal consensus sequence. We also found that RdRP recognition signals are distinct from cis-acting signals that promote (-) strand synthesis, because deletions of portions of the 3'-consensus sequence that caused viral mRNAs to be poorly replicated in vitro did not necessarily prevent efficient recognition of the RNA by the RdRP. Although the RdRP alone can specifically bind to viral mRNAs, our analysis reveals that this interaction is not sufficient to generate initiation complexes, even in the presence of nucleotides and divalent cations. Rather, the formation of initiation complexes also requires the core lattice protein (VP2), a virion component that forms a T = 1 icosahedral shell that encapsidates the segmented dsRNA genome. The essential role that the core lattice protein has in (-) strand initiation provides a mechanism for the coordination of genome replication and virion assembly.     

Cryo-electron microscopy reconstruction of a poliovirus-receptor-membrane complex

To study non-enveloped virus cell entry, a versatile in vitro model system was developed in which liposomes containing nickel-chelating lipids were decorated with His-tagged poliovirus receptors and bound to virus. This system provides an exciting opportunity for structural characterization of the early steps in cell entry in the context of a membrane. Here we report the three-dimensional structure of a poliovirus-receptor-membrane complex solved by cryo-electron microscopy (cryo-EM) at a resolution of 32 A. Methods were developed to establish the symmetry of the complex objectively. This reconstruction demonstrates that receptor binding brings a viral five-fold axis close to the membrane. Density is clearly defined for the icosahedral virus, for receptors (including known glycosylation sites) and for the membrane bilayer. Apparent perturbations of the bilayer close to the viral five-fold axis may function in subsequent steps of cell entry.     

Visualizing transiently associated catalytic domains in assembly-line biosynthesis using cryo-electron microscopy

While cryo-electron microscopy (cryo-EM) has revolutionized the structure determination of supramolecular protein complexes that are refractory to structure determination by X-ray crystallography, structure determination by cryo-EM can nonetheless be complicated by excessive conformational flexibility or structural heterogeneity resulting from weak or transient protein–protein association. Since such transient complexes are often critical for function, specialized approaches must be employed for the determination of meaningful structure–function relationships. Here, we outline examples in which transient protein–protein interactions have been visualized successfully by cryo-EM in the biosynthesis of fatty acids, polyketides, and terpenes. These studies demonstrate the utility of chemical crosslinking to stabilize transient protein–protein complexes for cryo-EM structural analysis, as well as the use of partial signal subtraction and localized reconstruction to extract useful structural information out of cryo-EM data collected from inherently dynamic systems. While these approaches do not always yield atomic resolution insights on protein–protein interactions, they nonetheless enable direct experimental observation of complexes in assembly-line biosynthesis that would otherwise be too fleeting for structural analysis.     

Protruding Features of Viral Capsids Are Clustered on Icosahedral Great Circles

Spherical viruses are remarkably well characterized by the Triangulation (T) number developed by Casper and Klug. The T-number specifies how many viral capsid proteins are required to cover the virus, as well as how they are further subdivided into pentamer and hexamer subunits. The T-number however does not constrain the orientations of these proteins within the subunits or dictate where the proteins should place their protruding features. These protrusions often take the form of loops, spires and helices, and are significant because they aid in stability of the capsid as well as recognition by the host organism. Until now there has be no overall understanding of the placement of protrusions for spherical viruses, other than they have icosahedral symmetry. We constructed a set of gauge points based upon the work affine extensions of Keef and Twarock, which have fixed relative angular locations with which to measure the locations of these features. This work adds a new element to our understanding of the geometric arrangement of spherical viral capsid proteins; chiefly that the locations of protruding features are not found stochastically distributed in an icosahedral manner across the viral surface, but instead these features are found only in specific locations along the 15 icosahedral great circles. We have found that this result holds true as the T number and viral capsids size increases, suggesting an underlying geometric constraint on their locations. This is in spite of the fact that the constraints on the pentamers and hexamer orientations change as a function of T-number, as you need to accommodate more hexamers in the same solid angle between pentamers. The existence of this angular constraint of viral capsids suggests that there is a fitness or energetic benefit to the virus placing its protrusions in this manner. This discovery may have profound impacts on identifying and eliminating viral pathogens, understanding evolutionary constraints as well as bioengineering for capsid drug delivery systems. This result also suggests that in addition to biochemical attachment restrictions, there are additional geometric constraints that should be adhered to when modifying protein capsids.     

New insights into GPCR coupling and dimerisation from cryo-EM structures

Over the past three years (2020–2022) more structures of GPCRs have been determined than in the previous twenty years (2000–2019), primarily of GPCR complexes that are large enough for structure determination by single-particle cryo-EM. This review will present some structural highlights that have advanced our molecular understanding of promiscuous G protein coupling, how a G protein receptor kinase and β-arrestins couple to GPCRs, and GPCR dimerisation. We will also discuss advances in the use of gene fusions, nanobodies, and F_ab fragments to facilitate the structure determination of GPCRs in the inactive state that, on their own, are too small for structure determination by single-particle cryo-EM.