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Cryo–electron microscopy (cryo-EM) continues its remarkable growth as a method for visualizing biological objects, which has been driven by advances across the entire pipeline. Developments in both single-particle analysis and in situ tomography have enabled more structures to be imaged and determined to better resolutions, at faster speeds, and with more scientists having improved access. This review highlights recent advances at each stageof the cryo-EM pipeline and provides examples of how these techniques have been used to investigate real-world problems, including antibody development against the SARS-CoV-2 spike during the recent COVID-19 pandemic.

Flaviviruses such as Dengue (DENV) or Zika virus (ZIKV) assemble into an immature form within the endoplasmatic reticulum (ER), and are then processed by furin protease in the trans-Golgi. To better grasp maturation, we carry out cryo-EM reconstructions of immature Spondweni virus (SPOV), a human flavivirus of the same serogroup as ZIKV. By employing asymmetric localised reconstruction we push the resolution to 3.8 Å, enabling us to refine an atomic model which includes the crucial furin protease recognition site and a conserved Histidine pH-sensor. For direct comparison, we also solve structures of the mature forms of SPONV and DENV to 2.6 Å and 3.1 Å, respectively. We identify an ordered lipid that is present in only the mature forms of ZIKV, SPOV, and DENV and can bind as a consequence of rearranging amphipathic stem-helices of E during maturation. We propose a structural role for the pocket and suggest it stabilizes mature E., Here, the authors provide cryo-EM structures of mature and immature Spondweni virus, defining the furin recognition site at high resolution, and identifying a lipid that binds E upon capsid maturation and is also present in Zika and Dengue virions.

Mosquito-transmitted flaviviruses, such as Dengue virus (DENV) or Zika virus (ZIKV), are responsible for significant economic damage and human misery. In infected cells, flaviviruses first assemble into an immature form within the endoplasmatic reticulum (ER), and then undergo further processing by furin protease in the trans-Golgi. Despite substantial efforts, previous cryogenic electron microscopy (cryo-EM) studies of immature flaviviruses were restricted to low to medium resolutions, limiting our understanding of maturation. To better grasp the process of maturation, we have carried out cryo-EM reconstructions of immature Spondweni virus (SPOV), an emerging human flavivirus belonging to the same serogroup as ZIKV (~75% amino acid identity). By combining localized reconstruction and focused refinement, we were able to improve the resolution to 3.8 Å, yielding unprecedented insight into the immature form. The structure elucidates how, at neutral pH, polar interactions conceal the furin recognition site within trimeric envelope (E) protein spikes. Furthermore, we identify how a strictly conserved pH sensor anchors the precursor membrane (prM) protein to immature E. We reconstructed mature forms of SPONV and DENV to 2.6Å and 3.1Å, respectively. Comparison with immature virus shows a conserved binding pocket for a lipid headgroup, which forms as a consequence of the rearrangement of amphipathic stem-helices of E. We propose a structural role for the pocket and suggest it stabilizes mature E. Taken together, our data suggest a compelling rationale for low-pH triggered conformational rearrangement in the Golgi, which occurs during flavivirus maturation.

Technological advances in transmission electron microscopes and detectors have turned cryogenic electron microscopy (cryo-EM) into an essential tool for structural biology. A commonly used cryo-EM data analysis method, single particle analysis, averages hundreds of thousands of low-dose images of individual macromolecular complexes to determine a density map of the complex. The presence of symmetry in the complex is beneficial since each projection image can be assigned to multiple views of the complex. However, data processing that applies symmetry can average out asymmetric features and consequently data analysis methods are required to resolve asymmetric structural features. Scipion is a cryo-EM image processing framework that integrates functionalities from different image processing packages as plugins. To extend its functionality for handling symmetry mismatches, we present here a Scipion plugin termed LocalRec implementing the localized reconstruction method. When tested on an adenovirus data set, the plugin enables resolving the symmetry-mismatched trimeric fibre bound to the five-fold vertices of the capsid. Furthermore, it improves the structure determination of the icosahedral capsid by dealing with the defocus gradient across the particle. LocalRec is expected to be widely applicable in a range of cryo-EM investigations of flexible and symmetry mismatched complexes.

Characterizing the genome of mature virions is pivotal to understanding the highly dynamic processes of virus assembly and infection. Owing to the different cellular fates of DNA and RNA, the life cycles of double-stranded (ds) DNA and dsRNA viruses are dissimilar. In terms of nucleic acid packing, dsDNA viruses, which lack genome segmentation and intra-capsid transcriptional machinery, predominantly display single-spooled genome organizations(1-8). Because the release of dsRNA into the cytoplasm triggers host defence mechanisms(9), dsRNA viruses retain their genomes within a core particle that contains the enzymes required for RNA replication and transcription(10-12). The genomes of dsRNA viruses vary greatly in the degree of segmentation. In members of the Reoviridae family, genomes consist of 10-12 segments and exhibit a non-spooled arrangement mediated by RNA-dependent RNA polymerases(11-14). However, whether this arrangement is a general feature of dsRNA viruses remains unknown. Here, using cryo-electron microscopy to resolve the dsRNA genome structure of the tri-segmented bacteriophage Phi 6 of the Cystoviridae family, we show that dsRNA viruses can adopt a dsDNA-like single-spooled genome organization. We find that in this group of viruses, RNA-dependent RNA polymerases do not direct genome ordering, and the dsRNA can adopt multiple conformations. We build a model that encompasses 90% of the genome, and use this to quantify variation in the packing density and to characterize the different liquid crystalline geometries that are exhibited by the tightly compacted nucleic acid. Our results demonstrate that the canonical model for the packing of dsDNA can be extended to dsRNA viruses.

Double-stranded RNA viruses infect a wide spectrum of hosts, including animals, plants, fungi, and bacteria. Yet genome replication mechanisms of these viruses are conserved. During the infection cycle, a proteinaceous capsid, the polymerase complex, is formed. An essential component of this capsid is the viral RNA polymerase that replicates and transcribes the enclosed viral genome. The polymerase complex structure is well characterized for many double-stranded RNA viruses. However, much less is known about the hierarchical molecular interactions that take place in building up such complexes. Using the bacteriophage Φ6 self-assembly system, we obtained novel insights into the processes that mediate polymerase subunit incorporation into the polymerase complex for generation of functional structures. The results presented pave the way for the exploitation and engineering of viral self-assembly processes for biomedical and synthetic biology applications. An understanding of viral assembly processes at the molecular level may also facilitate the development of antivirals that target viral capsid assembly., Double-stranded RNA (dsRNA) viruses package several RNA-dependent RNA polymerases (RdRp) together with their dsRNA genome into an icosahedral protein capsid known as the polymerase complex. This structure is highly conserved among dsRNA viruses but is not found in any other virus group. RdRp subunits typically interact directly with the main capsid proteins, close to the 5-fold symmetric axes, and perform viral genome replication and transcription within the icosahedral protein shell. In this study, we utilized Pseudomonas phage Φ6, a well-established virus self-assembly model, to probe the potential roles of the RdRp in dsRNA virus assembly. We demonstrated that Φ6 RdRp accelerates the polymerase complex self-assembly process and contributes to its conformational stability and integrity. We highlight the role of specific amino acid residues on the surface of the RdRp in its incorporation during the self-assembly reaction. Substitutions of these residues reduce RdRp incorporation into the polymerase complex during the self-assembly reaction. Furthermore, we determined that the overall transcription efficiency of the Φ6 polymerase complex increased when the number of RdRp subunits exceeded the number of genome segments. These results suggest a mechanism for RdRp recruitment in the polymerase complex and highlight its novel role in virion assembly, in addition to the canonical RNA transcription and replication functions.

Cryogenic transmission electron microscopy (cryo-EM) is widely used to determine high-resolution structures of symmetric virus capsids. The method holds promise for extending studies beyond purified capsids and their symmetric protein shells, The non-symmetric genome component has been addressed in dsRNA cypoviruses and ssRNA bacteriophages Q beta and MS2. The structure of human herpes simplex virus type 1 capsids has been determined within intact virions to resolve capsid-tegument interactions. Electron tomography under cryogenic conditions (cryo-ET), has allowed resolving an early membrane fusion intermediate of Rift Valley fever virus. Antibody-affinity based sample grids allow capturing of virions directly from cell cultures or even clinical samples. These and other emerging methods will support studies to address viral entry, assembly and neutralization processes at increasingly high resolutions and native conditions.

Foot-and-mouth disease virus (FMDV) is highly contagious and infects cloven-hoofed domestic livestock leading to foot-and-mouth disease (FMD). FMD outbreaks have severe economic impact due to production losses and associated control measures. FMDV is found as seven distinct serotypes, but there are numerous subtypes within each serotype, and effective vaccines must match the subtypes circulating in the field. In addition, the O and Southern African Territories (SAT) serotypes, are relatively more thermolabile and their viral capsids readily dissociate into non-immunogenic pentameric subunits, which can compromise the effectiveness of FMD vaccines. Here we report the construction of a chimeric clone between the SAT2 and O serotypes, designed to have SAT2 antigenicity. Characterisation of the chimeric virus showed growth kinetics equal to that of the wild type SAT2 virus with better thermostability, attributable to changes in the VP4 structural protein. Sequence and structural analyses confirmed that no changes from SAT2 were present elsewhere in the capsid as a consequence of the VP4 changes. Following exposure to an elevated temperature the thermostable SAT2-O1K chimera induced higher neutralizing-antibody titres in comparison to wild type SAT2 virus.

Correct outer protein shell assembly is a prerequisite for virion infectivity in many multi-shelled dsRNA viruses. In the prototypic dsRNA bacteriophage φ6, the assembly reaction is promoted by calcium ions but its biomechanics remain poorly understood. Here, we describe the near-atomic resolution structure of the φ6 double-shelled particle. The outer T=13 shell protein P8 consists of two alpha-helical domains joined by a linker, which allows the trimer to adopt either a closed or an open conformation. The trimers in an open conformation swap domains with each other. Our observations allow us to propose a mechanistic model for calcium concentration regulated outer shell assembly. Furthermore, the structure provides a prime exemplar of bona fide domain-swapping. This leads us to extend the theory of domain-swapping from the level of monomeric subunits and multimers to closed spherical shells, and to hypothesize a mechanism by which closed protein shells may arise in evolution., Double-shelled bacteriophage φ6 is a well-studied model system used to understand assembly of dsRNA viruses. Here the authors report a near-atomic resolution cryo-EM structure of φ6 and propose a model for the structural transitions occurring in the outer shell during genome packaging.

Electron cryomicroscopy can yield near-atomic resolution structures of highly ordered macromolecular complexes. Often however some subunits bind in a flexible manner, have different symmetry from the rest of the complex, or are present in sub-stoichiometric amounts, limiting the attainable resolution. Here we report a general method for the localized three-dimensional reconstruction of such subunits. After determining the particle orientations, local areas corresponding to the subunits can be extracted and treated as single particles. We demonstrate the method using three examples including a flexible assembly and complexes harbouring subunits with either partial occupancy or mismatched symmetry. Most notably, the method allows accurate fitting of the monomeric RNA-dependent RNA polymerase bound at the threefold axis of symmetry inside a viral capsid, revealing for the first time its exact orientation and interactions with the capsid proteins. Localized reconstruction is expected to provide novel biological insights in a range of challenging biological systems., Electron cryomicroscopy can allow the elucidation of macromolecular structures; however, mismatches in symmetry between different components limit the attainable resolution. Here, the authors set out a computational method for extracting and retaining information from such components.