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African swine fever virus (ASFV) is a giant and complex DNA virus that causes a highly contagious and often lethal swine disease for which no vaccine is available. Using an optimized image reconstruction strategy, we solved the ASFV capsid structure up to 4.1 angstroms, which is built from 17,280 proteins, including one major (p72) and four minor (M1249L, p17, p49, and H240R) capsid proteins organized into pentasymmetrons and trisymmetrons. The atomic structure of the p72 protein informs putative conformational epitopes, distinguishing ASFV from other nucleocytoplasmic large DNA viruses. The minor capsid proteins form a complicated network below the outer capsid shell, stabilizing the capsid by holding adjacent capsomers together. Acting as core organizers, 100-nanometer-long M1249L proteins run along each edge of the trisymmetrons that bridge two neighboring pentasymmetrons and form extensive intermolecular networks with other capsid proteins, driving the formation of the capsid framework. These structural details unveil the basis of capsid stability and assembly, opening up new avenues for African swine fever vaccine development.

HIV-1 contains a cone-shaped capsid encasing the viral genome. This capsid is thought to follow fullerene geometry—a curved hexameric lattice of the capsid protein, CA, closed by incorporating 12 CA pentamers. Current models for core structure are based on crystallography of hexameric and cross-linked pentameric CA, electron microscopy of tubular CA arrays, and simulations. Here, we report subnanometer-resolution cryo-electron tomography structures of hexameric and pentameric CA within intact HIV-1 particles. Whereas the hexamer structure is compatible with crystallography studies, the pentamer forms using different interfaces. Determining multiple structures revealed how CA flexes to form the variably curved core shell. We show that HIV-1 CA assembles both aberrant and perfect fullerene cones, supporting models in which conical cores assemble de novo after maturation.

Studies on viruses infecting archaea living in the most extreme environments continue to show a remarkable diversity of structures, suggesting that the sampling continues to be very sparse.We have used electron cryo-microscopy to study at 3.7-Å resolution the structure of the Sulfolobus polyhedral virus 1 (SPV1), which was originally isolated from a hot, acidic spring in Beppu, Japan. The 2 capsid proteins with variant single jelly-roll folds form pentamers and hexamers which assemble into a T = 43 icosahedral shell. In contrast to tailed icosahedral double-stranded DNA (dsDNA) viruses infecting bacteria and archaea, and herpesviruses infecting animals and humans, where naked DNA is packed under very high pressure due to the repulsion between adjacent layers of DNA, the circular dsDNA in SPV1 is fully covered with a viral protein forming a nucleoprotein filament with attractive interactions between layers. Most strikingly, we have been able to show that the DNA is in an A-form, as it is in the filamentous viruses infecting hyperthermophilic acidophiles. Previous studies have suggested that DNA is in the B-form in bacteriophages, and our study is a direct visualization of the structure of DNA in an icosahedral virus.

A high-resolution structure of the bacteriophage MS2 sheds light on the structure of the genome and how the genome is delivered into a bacterium. Atomic structure of an ssRNA genome Hong Zhou and colleagues provide the first description of genome–capsid interactions in a spherical single-stranded RNA (ssRNA) virus, using the bacteriophage MS2 as a model. Unlike double-stranded DNA viruses that pump their genome into a preformed capsid, ssRNA viruses co-assemble their capsid with their genome. Here the authors determine the MS2 structure at 3.6 Å resolution and are able to trace around 80% of the backbone of the viral genome, identifying regions that react with the maturation protein and providing insights into the ssRNA capsid co-assembly process. Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid 1 , 2 , 3 , single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome 4 , 5 , 6 , 7 ; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host ‘sex pilus’ (F-pilus) 8 ; it was the first fully sequenced organism 9 and is a model system for studies of translational gene regulation 10 , 11 , RNA–protein interactions 12 , 13 , 14 , and RNA virus assembly 15 , 16 , 17 . Its positive-sense ssRNA genome of 3,569 bases is enclosed in a capsid with one maturation protein monomer and 89 coat protein dimers arranged in a T  = 3 icosahedral lattice 18 , 19 . The maturation protein is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection 8 , but how the genome is organized and delivered is not known. Here we describe the MS2 structure at 3.6 Å resolution, determined by electron-counting cryo-electron microscopy (cryoEM) and asymmetric reconstruction. We traced approximately 80% of the backbone of the viral genome, built atomic models for 16 RNA stem–loops, and identified three conserved motifs of RNA–coat protein interactions among 15 of these stem–loops with diverse sequences. The stem–loop at the 3′ end of the genome interacts extensively with the maturation protein, which, with just a six-helix bundle and a six-stranded β-sheet, forms a genome-delivery apparatus and joins 89 coat protein dimers to form a capsid. This atomic description of genome–capsid interactions in a spherical ssRNA virus provides insight into genome delivery via the host sex pilus and mechanisms underlying ssRNA–capsid co-assembly, and inspires speculation about the links between nucleoprotein complexes and the origins of viruses.

The capsids of double-stranded DNA viruses protect the viral genome from the harsh extracellular environment, while maintaining stability against the high internal pressure of packaged DNA. To elucidate how capsids maintain stability in an extreme environment, we use cryoelectron microscopy to determine the capsid structure of thermostable phage P74-26 to 2.8-Å resolution. We find P74-26 capsids exhibit an overall architecture very similar to those of other tailed bacteriophages, allowing us to directly compare structures to derive the structural basis for enhanced stability. Our structure reveals lasso-like interactions that appear to function like catch bonds. This architecture allows the capsid to expand during genome packaging, yet maintain structural stability. The P74-26 capsid has T = 7 geometry despite being twice as large as mesophilic homologs. Capsid capacity is increased with a larger, flatter major capsid protein. Given these results, we predict decreased icosahedral complexity (i.e. T ≤ 7) leads to a more stable capsid assembly. Viral capsids need to protect the genome against harsh environmental conditions and cope with high internal pressure from the packaged genome. Here, the authors determine the structure of the thermostable phage P74-26 capsid at 2.8-Å resolution and identify features underlying enhanced capsid capacity and structural stability.

Retroviruses assemble and bud from infected cells in an immature form and require proteolytic maturation for infectivity. The CA (capsid) domains of the Gag polyproteins assemble a protein lattice as a truncated sphere in the immature virion. Proteolytic cleavage of Gag induces dramatic structural rearrangements; a subset of cleaved CA subsequently assembles into the mature core, whose architecture varies among retroviruses. Murine leukemia virus (MLV) is the prototypical γ-retrovirus and serves as the basis of retroviral vectors, but the structure of the MLV CA layer is unknown. Here we have combined X-ray crystallography with cryoelectron tomography to determine the structures of immature and mature MLV CA layers within authentic viral particles. This reveals the structural changes associated with maturation, and, by comparison with HIV-1, uncovers conserved and variable features. In contrast to HIV-1, most MLV CA is used for assembly of the mature core, which adopts variable, multilayered morphologies and does not form a closed structure. Unlike in HIV-1, there is similarity between protein–protein interfaces in the immature MLV CA layer and those in the mature CA layer, and structural maturation of MLV could be achieved through domain rotations that largely maintain hexameric interactions. Nevertheless, the dramatic architectural change on maturation indicates that extensive disassembly and reassembly are required for mature core growth. The core morphology suggests that wrapping of the genome in CA sheets may be sufficient to protect the MLV ribonucleoprotein during cell entry.

The structure of the HIV-1 capsid is analysed by cryo-electron microscopy and cryo-electron tomography, allowing presentation of an all-atom molecular dynamics model of the entire capsid. Atomic structure of the HIV-1 capsid Human immunodeficiency virus-1 (HIV-1), the predominant AIDS virus, contains a spheroidal capsid enclosing the viral RNA genome. As the retrovirus matures, the capsid forms through spontaneous oligomerization of the capsid protein CA. Using cryo-electron microscopy and cryo-electron tomography, combined with all-atom large-scale molecular dynamics simulations, Gongpu Zhao et al . have determined a complete atomic structure of the HIV-1 capsid. The resulting structural models reveal elements that are essential for capsid formation, stability and viral infectivity. Of special interest are the hydrophobic interactions evident in a novel three-fold interface between the carboxy-terminal domains of CA protein, a feature that appears to be unique to the mature capsid and which has previously been suggested as a potentially attractive therapeutic target. Retroviral capsid proteins are conserved structurally but assemble into different morphologies 1 . The mature human immunodeficiency virus-1 (HIV-1) capsid is best described by a ‘fullerene cone’ model 2 , 3 , in which hexamers of the capsid protein are linked to form a hexagonal surface lattice that is closed by incorporating 12 capsid-protein pentamers. HIV-1 capsid protein contains an amino-terminal domain (NTD) comprising seven α-helices and a β-hairpin 4 , 5 , a carboxy-terminal domain (CTD) comprising four α-helices 6 , 7 , and a flexible linker with a 3 10 -helix connecting the two structural domains 8 . Structures of the capsid-protein assembly units have been determined by X-ray crystallography 9 , 10 ; however, structural information regarding the assembled capsid and the contacts between the assembly units is incomplete. Here we report the cryo-electron microscopy structure of a tubular HIV-1 capsid-protein assembly at 8 Å resolution and the three-dimensional structure of a native HIV-1 core by cryo-electron tomography. The structure of the tubular assembly shows, at the three-fold interface 11 , a three-helix bundle with critical hydrophobic interactions. Mutagenesis studies confirm that hydrophobic residues in the centre of the three-helix bundle are crucial for capsid assembly and stability, and for viral infectivity. The cryo-electron-microscopy structures enable modelling by large-scale molecular dynamics simulation, resulting in all-atom models for the hexamer-of-hexamer and pentamer-of-hexamer elements as well as for the entire capsid. Incorporation of pentamers results in closer trimer contacts and induces acute surface curvature. The complete atomic HIV-1 capsid model provides a platform for further studies of capsid function and for targeted pharmacological intervention.

The mature retrovirus capsid consists of a variably curved lattice of capsid protein (CA) hexamers and pentamers. High-resolution structures of the curved assembly, or in complex with host factors, have not been available. By devising cryo-EM methodologies for exceedingly flexible and pleomorphic assemblies, we have determined cryo-EM structures of apo-CA hexamers and in complex with cyclophilin A (CypA) at near-atomic resolutions. The CA hexamers are intrinsically curved, flexible and asymmetric, revealing the capsomere and not the previously touted dimer or trimer interfaces as the key contributor to capsid curvature. CypA recognizes specific geometries of the curved lattice, simultaneously interacting with three CA protomers from adjacent hexamers via two noncanonical interfaces, thus stabilizing the capsid. By determining multiple structures from various helical symmetries, we further revealed the essential plasticity of the CA molecule, which allows formation of continuously curved conical capsids and the mechanism of capsid pattern sensing by CypA.Cryo-EM structures of HIV-1 capsid in tubular assemblies feature intrinsically curved and asymmetric hexamers and provide insight into cyclophilin A binding.

Cryo-electron tomography and subtomogram averaging methods reveal the structure of the capsid lattice within intact heterogeneous immature HIV-1 particles. Capsid structure in a mixed immature HIV-1 population Our understanding of immature HIV — the form of the virus leaves the cell — is based on purified components assembled in vitro in a manner thought to mimic the true state. Here John Briggs and colleagues resolve the structure of the protein shell within intact heterogeneous immature HIV-1 particles. Using cryo-electron tomography and sub-tomogram averaging (averaging thousands of aligned sub-volumes containing the same structural unit), the authors determine the structure of the capsid lattice at at 8.8 Å. The structure shows that the arrangement of capsid in immature HIV-1 is very different from that seen in the mature HIV-1 capsid core, and reveals the tertiary and quaternary interactions that mediate HIV-1 assembly. Human immunodeficiency virus type 1 (HIV-1) assembly proceeds in two stages. First, the 55 kilodalton viral Gag polyprotein assembles into a hexameric protein lattice at the plasma membrane of the infected cell, inducing budding and release of an immature particle. Second, Gag is cleaved by the viral protease, leading to internal rearrangement of the virus into the mature, infectious form 1 . Immature and mature HIV-1 particles are heterogeneous in size and morphology, preventing high-resolution analysis of their protein arrangement in situ by conventional structural biology methods. Here we apply cryo-electron tomography and sub-tomogram averaging methods to resolve the structure of the capsid lattice within intact immature HIV-1 particles at subnanometre resolution, allowing unambiguous positioning of all α-helices. The resulting model reveals tertiary and quaternary structural interactions that mediate HIV-1 assembly. Strikingly, these interactions differ from those predicted by the current model based on in vitro -assembled arrays of Gag-derived proteins from Mason–Pfizer monkey virus 2 . To validate this difference, we solve the structure of the capsid lattice within intact immature Mason–Pfizer monkey virus particles. Comparison with the immature HIV-1 structure reveals that retroviral capsid proteins, while having conserved tertiary structures, adopt different quaternary arrangements during virus assembly. The approach demonstrated here should be applicable to determine structures of other proteins at subnanometre resolution within heterogeneous environments.

Although the nucleocytoplasmic large DNA viruses (NCLDVs) are one of the largest group of viruses that infect many eukaryotic hosts, the near-atomic resolution structures of these viruses have remained unknown. Here we describe a 3.5 Å resolution icosahedrally averaged capsid structure of Paramecium bursaria chlorella virus 1 (PBCV-1). This structure consists of 5040 copies of the major capsid protein, 60 copies of the penton protein and 1800 minor capsid proteins of which there are 13 different types. The minor capsid proteins form a hexagonal network below the outer capsid shell, stabilizing the capsid by binding neighboring capsomers together. The size of the viral capsid is determined by a tape-measure, minor capsid protein of which there are 60 copies in the virion. Homologs of the tape-measure protein and some of the other minor capsid proteins exist in other NCLDVs. Thus, a similar capsid assembly pathway might be used by other NCLDVs. Nucleocytoplasmic large DNA viruses are a group of viruses that infect many eukaryotic hosts. Here, the authors provide a 3.5 Å resolution icosahedrally-averaged capsid structure of Paramecium bursaria chlorella virus 1 and show how 1800 minor capsid proteins form a hexagonal network below the outer capsid shell.