Delineating organizational principles of the endogenous L-A virus by cryo-EM and computational analysis of native cell extracts.

The high abundance of most viruses in infected host cells benefits their structural characterization. However, endogenous viruses are present in low copy numbers and are therefore challenging to investigate. Here, we retrieve cell extracts enriched with an endogenous virus, the yeast L-A virus. The determined cryo-EM structure discloses capsid-stabilizing cation-π stacking, widespread across viruses and within the Totiviridae, and an interplay of non-covalent interactions from ten distinct capsomere interfaces. The capsid-embedded mRNA decapping active site trench is supported by a constricting movement of two flexible opposite-facing loops. tRNA-loaded polysomes and other biomacromolecules, presumably mRNA, are found in virus proximity within the cell extract. Mature viruses participate in larger viral communities resembling their rare in-cell equivalents in terms of size, composition, and inter-virus distances. Our results collectively describe a 3D-architecture of a viral milieu, opening the door to cell-extract-based high-resolution structural virology.

Point mutation in a virus-like capsid drives symmetry reduction to form tetrahedral cages.

Protein capsids are a widespread form of compartmentalization in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry maximizes the internal volume that can be enclosed within. Despite the strong preference for icosahedral symmetry, we show that simple point mutations in a virus-like capsid can drive the assembly of unique symmetry-reduced structures. Starting with the encapsulin from Myxococcus xanthus, a 180-mer bacterial capsid that adopts the well-studied viral HK97 fold, we use mass photometry and native charge detection mass spectrometry to identify a triple histidine point mutant that forms smaller dimorphic assemblies. Using cryoelectron microscopy, we determine the structures of a precedented 60-mer icosahedral assembly and an unexpected 36-mer tetrahedron that features significant geometric rearrangements around a new interaction surface between capsid protomers. We subsequently find that the tetrahedral assembly can be generated by triple-point mutation to various amino acids and that even a single histidine point mutation is sufficient to form tetrahedra. These findings represent a unique example of tetrahedral geometry when surveying all characterized encapsulins, HK97-like capsids, or indeed any virus-derived capsids reported in the Protein Data Bank, revealing the surprising plasticity of capsid self-assembly that can be accessed through minimal changes in the protein sequence.

Structural morphing in the viral portal vertex of bacteriophage lambda.

The portal protein of tailed bacteriophage plays essential roles in various aspects of capsid assembly, motor assembly, genome packaging, connector formation, and infection processes. After DNA packaging is complete, additional proteins are assembled onto the portal to form the connector complex, which is crucial as it bridges the mature head and tail. In this study, we report high-resolution cryo-electron microscopy (cryo-EM) structures of the portal vertex from bacteriophage lambda in both its prohead and mature virion states. Comparison of these structures shows that during head maturation, in addition to capsid expansion, the portal protein undergoes conformational changes to establish interactions with the connector proteins. Additionally, the independently assembled tail undergoes morphological alterations at its proximal end, facilitating its connection to the head-tail joining protein and resulting in the formation of a stable portal-connector-tail complex. The B-DNA molecule spirally glides through the tube, interacting with the nozzle blade region of the middle-ring connector protein. These insights elucidate a mechanism for portal maturation and DNA translocation within the phage lambda system. IMPORTANCE: The tailed bacteriophages possess a distinct portal vertex that consists of a ring of 12 portal proteins associated with a 5-fold capsid shell. This portal protein is crucial in multiple stages of virus assembly and infection. Our research focused on examining the structures of the portal vertex in both its preliminary prohead state and the fully mature virion state of bacteriophage lambda. By analyzing these structures, we were able to understand how the portal protein undergoes conformational changes during maturation, the mechanism by which it prevents DNA from escaping, and the process of DNA spirally gliding.

High-resolution comparative atomic structures of two Giardiavirus prototypes infecting G. duodenalis parasite.

The Giardia lamblia virus (GLV) is a non-enveloped icosahedral dsRNA and endosymbiont virus that infects the zoonotic protozoan parasite Giardia duodenalis (syn. G. lamblia, G. intestinalis), which is a pathogen of mammals, including humans. Elucidating the transmission mechanism of GLV is crucial for gaining an in-depth understanding of the virulence of the virus in G. duodenalis. GLV belongs to the family Totiviridae, which infects yeast and protozoa intracellularly; however, it also transmits extracellularly, similar to the phylogenetically, distantly related toti-like viruses that infect multicellular hosts. The GLV capsid structure is extensively involved in the longstanding discussion concerning extracellular transmission in Totiviridae and toti-like viruses. Hence, this study constructed the first high-resolution comparative atomic models of two GLV strains, namely GLV-HP and GLV-CAT, which showed different intracellular localization and virulence phenotypes, using cryogenic electron microscopy single-particle analysis. The atomic models of the GLV capsids presented swapped C-terminal extensions, extra surface loops, and a lack of cap-snatching pockets, similar to those of toti-like viruses. However, their open pores and absence of the extra crown protein resemble those of other yeast and protozoan Totiviridae viruses, demonstrating the essential structures for extracellular cell-to-cell transmission. The structural comparison between GLV-HP and GLV-CAT indicates the first evidence of critical structural motifs for the transmission and virulence of GLV in G. duodenalis.

RNA genome packaging and capsid assembly of bluetongue virus visualized in host cells.

Unlike those of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and ssRNA viruses, the mechanism of genome packaging of dsRNA viruses is poorly understood. Here, we combined the techniques of high-resolution cryoelectron microscopy (cryo-EM), cellular cryoelectron tomography (cryo-ET), and structure-guided mutagenesis to investigate genome packaging and capsid assembly of bluetongue virus (BTV), a member of the Reoviridae family of dsRNA viruses. A total of eleven assembly states of BTV capsid were captured, with resolutions up to 2.8 Å, with most visualized in the host cytoplasm. ATPase VP6 was found underneath the vertices of capsid shell protein VP3 as an RNA-harboring pentamer, facilitating RNA packaging. RNA packaging expands the VP3 shell, which then engages middle- and outer-layer proteins to generate infectious virions. These revealed "duality" characteristics of the BTV assembly mechanism reconcile previous contradictory co-assembly and core-filling models and provide insights into the mysterious RNA packaging and capsid assembly of Reoviridae members and beyond.

The reversible activation of norovirus by metal ions.

Murine norovirus (MNV) undergoes extremely large conformational changes in response to the environment. The T = 3 icosahedral capsid is composed of 180 copies of ~58-kDa VP1 comprised of N-terminus (N), shell (S), and C-terminal protruding (P) domains. At neutral pH, the P domains are loosely tethered to the shell and float ~15 Å above the surface. At low pH or in the presence of bile salts, the P domain drops onto the shell and this movement is accompanied by conformational changes within the P domain that enhance receptor interactions while blocking antibody binding. While previous crystallographic studies identified metal binding sites in the isolated P domain, the ~2.7-Å cryo-electron microscopy structures of MNV in the presence of Mg2+ or Ca2+ presented here show that metal ions can recapitulate the contraction observed at low pH or in the presence of bile. Further, we show that these conformational changes are reversed by dialysis against EDTA. As observed in the P domain crystal structures, metal ions bind to and contract the G'H' loop. This movement is correlated with the lifting of the C'D' loop and rotation of the P domain dimers about each other, exposing the bile salt binding pocket. Isothermal titration calorimetry experiments presented here demonstrate that the activation signals (bile salts, low pH, and metal ions) act in a synergistic manner that, individually, all result in the same activated structure. We present a model whereby these reversible conformational changes represent a uniquely dynamic and tissue-specific structural adaptation to the in vivo environment.IMPORTANCEThe highly mobile protruding domains on the calicivirus capsids are recognized by cell receptor(s) and antibodies. At neutral pH, they float ~15 Å above the shell but at low pH or in the presence of bile salts, they contract onto the surface. Concomitantly, changes within the P domain block antibody binding while enhancing receptor binding. While we previously demonstrated that metals also block antibody binding, it was unknown whether they might also cause similar conformational changes in the virion. Here, we present the near atomic cryo-electron microscopy structures of infectious murine norovirus (MNV) in the presence of calcium or magnesium ions. The metal ions reversibly induce the same P domain contraction as low pH and bile salts and act in a synergistic manner with the other stimuli. We propose that, unlike most other viruses, MNV facilely changes conformations as a unique means to escape immune surveillance as it moves through various tissues.

HIV-1 Env trimers asymmetrically engage CD4 receptors in membranes.

Human immunodeficiency virus 1 (HIV-1) infection is initiated by binding of the viral envelope glycoprotein (Env) to the cell-surface receptor CD41-4. Although high-resolution structures of Env in a complex with the soluble domains of CD4 have been determined, the binding process is less understood in native membranes5-13. Here we used cryo-electron tomography to monitor Env-CD4 interactions at the membrane-membrane interfaces formed between HIV-1 and CD4-presenting virus-like particles. Env-CD4 complexes organized into clusters and rings, bringing the opposing membranes closer together. Env-CD4 clustering was dependent on capsid maturation. Subtomogram averaging and classification revealed that Env bound to one, two and finally three CD4 molecules, after which Env adopted an open state. Our data indicate that asymmetric HIV-1 Env trimers bound to one and two CD4 molecules are detectable intermediates during virus binding to host cell membranes, which probably has consequences for antibody-mediated immune responses and vaccine immunogen design.

HSV-1 exploits host heterochromatin for nuclear egress.

Herpes simplex virus (HSV-1) progeny form in the nucleus and exit to successfully infect other cells. Newly formed capsids navigate complex chromatin architecture to reach the inner nuclear membrane (INM) and egress. Here, we demonstrate by transmission electron microscopy (TEM) that HSV-1 capsids traverse heterochromatin associated with trimethylation on histone H3 lysine 27 (H3K27me3) and the histone variant macroH2A1. Through chromatin profiling during infection, we revealed global redistribution of these marks whereby massive host genomic regions bound by macroH2A1 and H3K27me3 correlate with decreased host transcription in active compartments. We found that the loss of these markers resulted in significantly lower viral titers but did not impact viral genome or protein accumulation. Strikingly, we discovered that loss of macroH2A1 or H3K27me3 resulted in nuclear trapping of capsids. Finally, by live-capsid tracking, we quantified this decreased capsid movement. Thus, our work demonstrates that HSV-1 takes advantage of the dynamic nature of host heterochromatin formation during infection for efficient nuclear egress.

The Structures and Functions of Parvovirus Capsids and Missing Pieces: the Viral DNA and Its Packaging, Asymmetrical Features, Nonprotein Components, and Receptor or Antibody Binding and Interactions.

Parvoviruses are among the smallest and superficially simplest animal viruses, infecting a broad range of hosts, including humans, and causing some deadly infections. In 1990, the first atomic structure of the canine parvovirus (CPV) capsid revealed a 26-nm-diameter T=1 particle made up of two or three versions of a single protein, and packaging about 5,100 nucleotides of single-stranded DNA. Our structural and functional understanding of parvovirus capsids and their ligands has increased as imaging and molecular techniques have advanced, and capsid structures for most groups within the Parvoviridae family have now been determined. Despite those advances, significant questions remain unanswered about the functioning of those viral capsids and their roles in release, transmission, or cellular infection. In addition, the interactions of capsids with host receptors, antibodies, or other biological components are also still incompletely understood. The parvovirus capsid's apparent simplicity likely conceals important functions carried out by small, transient, or asymmetric structures. Here, we highlight some remaining open questions that may need to be answered to provide a more thorough understanding of how these viruses carry out their various functions. The many different members of the family Parvoviridae share a capsid architecture, and while many functions are likely similar, others may differ in detail. Many of those parvoviruses have not been experimentally examined in detail (or at all in some cases), so we, therefore, focus this minireview on the widely studied protoparvoviruses, as well as the most thoroughly investigated examples of adeno-associated viruses.

Structural Insights into the Assembly of the African Swine Fever Virus Inner Capsid.

African swine fever virus (ASFV), the cause of a highly contagious hemorrhagic and fatal disease of domestic pigs, has a complex multilayer structure. The inner capsid of ASFV located underneath the inner membrane enwraps the genome-containing nucleoid and is likely the assembly of proteolytic products from the virally encoded polyproteins pp220 and pp62. Here, we report the crystal structure of ASFV p150△NC, a major middle fragment of the pp220 proteolytic product p150. The structure of ASFV p150△NC contains mainly helices and has a triangular plate-like shape. The triangular plate is approximately 38 Šin thickness, and the edge of the triangular plate is approximately 90 Šlong. The structure of ASFV p150△NC is not homologous to any of the known viral capsid proteins. Further analysis of the cryo-electron microscopy maps of the ASFV and the homologous faustovirus inner capsids revealed that p150 or the p150-like protein of faustovirus assembles to form screwed propeller-shaped hexametric and pentametric capsomeres of the icosahedral inner capsids. Complexes of the C terminus of p150 and other proteolytic products of pp220 likely mediate interactions between the capsomeres. Together, these findings provide new insights into the assembling of ASFV inner capsid and provide a reference for understanding the assembly of the inner capsids of nucleocytoplasmic large DNA viruses (NCLDV). IMPORTANCE African swine fever virus has caused catastrophic destruction to the pork industry worldwide since it was first discovered in Kenya in 1921. The architecture of ASFV is complicated, with two protein shells and two membrane envelopes. Currently, mechanisms involved in the assembly of the ASFV inner core shell are less understood. The structural studies of the ASFV inner capsid protein p150 performed in this research enable the building of a partial model of the icosahedral ASFV inner capsid, which provides a structural basis for understanding the structure and assembly of this complex virion. Furthermore, the structure of ASFV p150△NC represents a new type of fold for viral capsid assembly, which could be a common fold for the inner capsid assembly of nucleocytoplasmic large DNA viruses (NCLDV) and would facilitate the development of vaccine and antivirus drugs against these complex viruses.

Structural atlas of a human gut crassvirus.

CrAssphage and related viruses of the order Crassvirales (hereafter referred to as crassviruses) were originally discovered by cross-assembly of metagenomic sequences. They are the most abundant viruses in the human gut, are found in the majority of individual gut viromes, and account for up to 95% of the viral sequences in some individuals1-4. Crassviruses are likely to have major roles in shaping the composition and functionality of the human microbiome, but the structures and roles of most of the virally encoded proteins are unknown, with only generic predictions resulting from bioinformatic analyses4,5. Here we present a cryo-electron microscopy reconstruction of Bacteroides intestinalis virus ΦcrAss0016, providing the structural basis for the functional assignment of most of its virion proteins. The muzzle protein forms an assembly about 1 MDa in size at the end of the tail and exhibits a previously unknown fold that we designate the 'crass fold', that is likely to serve as a gatekeeper that controls the ejection of cargos. In addition to packing the approximately 103 kb of virus DNA, the ΦcrAss001 virion has extensive storage space for virally encoded cargo proteins in the capsid and, unusually, within the tail. One of the cargo proteins is present in both the capsid and the tail, suggesting a general mechanism for protein ejection, which involves partial unfolding of proteins during their extrusion through the tail. These findings provide a structural basis for understanding the mechanisms of assembly and infection of these highly abundant crassviruses.

Structural Study of the Cobetia marina Bacteriophage 1 (Carin-1) by Cryo-EM.

Most of studied bacteriophages (phages) are terrestrial viruses. However, marine phages are shown to be highly involved in all levels of oceanic regulation. They are, however, still largely overlooked by the scientific community. By inducing cell lysis on half of the bacterial population daily, their role and influence on the bacterial biomass and evolution, as well as their impact in the global biogeochemical cycles, is undeniable. Cobetia marina virus 1 (Carin-1) is a member of the Podoviridae family infecting the γ-protoabacteria C. marina. Here, we present the almost complete, nearly-atomic resolution structure of Carin-1 comprising capsid, portal, and tail machineries at 3.5 Å, 3.8 Å and 3.9 Å, respectively, determined by cryo-electron microscopy (cryo-EM). Our experimental results, combined with AlphaFold2 (AF), allowed us to obtain the nearly-atomic structure of Carin-1 by fitting and refining the AF atomic models in the high resolution cryo-EM map, skipping the bottleneck of de-novo manual building and speeding up the structure determination process. Our structural results highlighted the T7-like nature of Carin1, as well as several novel structural features like the presence of short spikes on the capsid, reminiscent those described for Rhodobacter capsulatus gene transfer agent (RcGTA). This is, to our knowledge, the first time such assembly is described for a bacteriophage, shedding light into the common evolution and shared mechanisms between gene transfer agents and phages. This first full structure determined for a marine podophage allowed to propose an infection mechanism different than the one proposed for the archetypal podophage T7. IMPORTANCE Oceans play a central role in the carbon cycle on Earth and on the climate regulation (half of the planet's CO2 is absorbed by phytoplankton photosynthesis in the oceans and just as much O2 is liberated). The understanding of the biochemical equilibriums of marine biology represents a major goal for our future. By lysing half of the bacterial population every day, marine bacteriophages are key actors of these equilibriums. Despite their importance, these marine phages have, so far, only been studied a little and, in particular, structural insights are currently lacking, even though they are fundamental for the understanding of the molecular mechanisms of their mode of infection. The structures described in our manuscript allow us to propose an infection mechanism that differs from the one proposed for the terrestrial T7 virus, and might also allow us to, in the future, better understand the way bacteriophages shape the global ecosystem.

Structure of Human Enterovirus 70 and Its Inhibition by Capsid-Binding Compounds.

Enterovirus 70 (EV70) is a human pathogen belonging to the family Picornaviridae. EV70 is transmitted by eye secretions and causes acute hemorrhagic conjunctivitis, a serious eye disease. Despite the severity of the disease caused by EV70, its structure is unknown. Here, we present the structures of the EV70 virion, altered particle, and empty capsid determined by cryo-electron microscopy. The capsid of EV70 is composed of the subunits VP1, VP2, VP3, and VP4. The partially collapsed hydrophobic pocket located in VP1 of the EV70 virion is not occupied by a pocket factor, which is commonly present in other enteroviruses. Nevertheless, we show that the pocket can be targeted by the antiviral compounds WIN51711 and pleconaril, which block virus infection. The inhibitors prevent genome release by stabilizing EV70 particles. Knowledge of the structures of complexes of EV70 with inhibitors will enable the development of capsid-binding therapeutics against this virus. IMPORTANCE Globally distributed enterovirus 70 (EV70) causes local outbreaks of acute hemorrhagic conjunctivitis. The discharge from infected eyes enables the high-efficiency transmission of EV70 in overcrowded areas with low hygienic standards. Currently, only symptomatic treatments are available. We determined the structures of EV70 in its native form, the genome release intermediate, and the empty capsid resulting from genome release. Furthermore, we elucidated the structures of EV70 in complex with two inhibitors that block virus infection, and we describe the mechanism of their binding to the virus capsid. These results enable the development of therapeutics against EV70.

Subcellular organization of viral particles during maturation of nucleus-forming jumbo phage.

Many eukaryotic viruses assemble mature particles within distinct subcellular compartments, but bacteriophages are generally assumed to assemble randomly throughout the host cell cytoplasm. Here, we show that viral particles of Pseudomonas nucleus-forming jumbo phage PhiPA3 assemble into a unique structure inside cells we term phage bouquets. We show that after capsids complete DNA packaging at the surface of the phage nucleus, tails assemble and attach to capsids, and these particles accumulate over time in a spherical pattern, with tails oriented inward and the heads outward to form bouquets at specific subcellular locations. Bouquets localize at the same fixed distance from the phage nucleus even when it is mispositioned, suggesting an active mechanism for positioning. These results mark the discovery of a pathway for organizing mature viral particles inside bacteria and demonstrate that nucleus-forming jumbo phages, like most eukaryotic viruses, are highly spatially organized during all stages of their lytic cycle.

Atomic Structure of the Human Sapovirus Capsid Reveals a Unique Capsid Protein Conformation in Caliciviruses.

Sapovirus (SaV) is a member of the Caliciviridae family, which causes acute gastroenteritis in humans and animals. Human sapoviruses (HuSaVs) are genetically and antigenically diverse, but the lack of a viral replication system and structural information has hampered the development of vaccines and therapeutics. Here, we successfully produced a self-assembled virus-like particle (VLP) from the HuSaV GI.6 VP1 protein, and the first atomic structure was determined using single-particle cryo-electron microscopy (cryo-EM) at a 2.9-Å resolution. The atomic model of the VP1 protein revealed a unique capsid protein conformation in caliciviruses. All N-terminal arms in the A, B, and C subunits interacted with adjacent shell domains after extending through their subunits. The roof of the arched VP1 dimer was formed between the P2 subdomains by the interconnected ß strands and loops, and its buried surface was minimized compared to those of other caliciviruses. Four hypervariable regions that are potentially involved in the antigenic diversity of SaV formed extensive clusters on top of the P domain. Potential receptor binding regions implied by tissue culture mutants of porcine SaV were also located near these hypervariable clusters. Conserved sequence motifs of the VP1 protein, "PPG" and "GWS," may stabilize the inner capsid shell and the outer protruding domain, respectively. These findings will provide the structural basis for the medical treatment of HuSaV infections and facilitate the development of vaccines, antivirals, and diagnostic systems. IMPORTANCE SaV and norovirus, belonging to the Caliciviridae family, are common causes of acute gastroenteritis in humans and animals. SaV and norovirus infections are public health problems in all age groups, which occur explosively and sporadically worldwide. HuSaV is genetically and antigenically diverse and is currently classified into 4 genogroups consisting of 18 genotypes based on the sequence similarity of the VP1 proteins. Despite these detailed genetic analyses, the lack of structural information on viral capsids has become a problem for the development of vaccines or antiviral drugs. The 2.9-Å atomic model of the HuSaV GI.6 VLP presented here not only revealed the location of the amino acid residues involved in immune responses and potential receptor binding sites but also provided essential information for the design of stable constructs needed for the development of vaccines and antivirals.

Particle Morphology of Medusavirus Inside and Outside the Cells Reveals a New Maturation Process of Giant Viruses.

Medusavirus, a giant virus, is phylogenetically closer to eukaryotes than the other giant viruses and has been recently classified as an independent species. However, details of its morphology and maturation process in host cells remain unclear. Here, we investigated the particle morphology of medusavirus inside and outside infected cells using conventional transmission electron microscopy (C-TEM) and cryo-electron microscopy (cryo-EM). The C-TEM of amoebae infected with the medusavirus showed four types of particles, i.e., pseudo-DNA-empty (p-Empty), DNA-empty (Empty), semi-DNA-full (s-Full), and DNA-full (Full). Time-dependent changes in the four types of particles and their intracellular localization suggested a new maturation process for the medusavirus. Viral capsids and viral DNAs are produced independently in the cytoplasm and nucleus, respectively, and only the empty particles located near the host nucleus can incorporate the viral DNA into the capsid. Therefore, all four types of particles were found outside the cells. The cryo-EM of these particles showed that the intact virus structure, covered with three different types of spikes, was preserved among all particle types, although with minor size-related differences. The internal membrane exhibited a structural array similar to that of the capsid, interacted closely with the capsid, and displayed open membrane structures in the Empty and p-Empty particles. The results suggest that these open structures in the internal membrane are used for an exchange of scaffold proteins and viral DNA during the maturation process. This new model of the maturation process of medusavirus provides insight into the structural and behavioral diversity of giant viruses. IMPORTANCE Giant viruses exhibit diverse morphologies and maturation processes. In this study, medusavirus showed four types of particle morphologies, both inside and outside the infected cells, when propagated in amoeba culture. Time-course analysis and intracellular localization of the medusavirus in the infected cells suggested a new maturation process via the four types of particles. Like the previously reported pandoravirus, the viral DNA of medusavirus is replicated in the host's nucleus. However, viral capsids are produced independently in the host cytoplasm, and only empty capsids near the nucleus can take up viral DNA. As a result, many immature particles were released from the host cell along with the mature particles. The capsid structure is well conserved among the four types of particles, except for the open membrane structures in the empty particles, suggesting that they are used to exchange scaffold proteins for viral DNAs. These findings indicate that medusavirus has a unique maturation process.

Structural Assembly of Qß Virion and Its Diverse Forms of Virus-like Particles.

The coat proteins (CPs) of single-stranded RNA bacteriophages (ssRNA phages) directly assemble around the genomic RNA (gRNA) to form a near-icosahedral capsid with a single maturation protein (Mat) that binds the gRNA and interacts with the retractile pilus during infection of the host. Understanding the assembly of ssRNA phages is essential for their use in biotechnology, such as RNA protection and delivery. Here, we present the complete gRNA model of the ssRNA phage Qß, revealing that the 3' untranslated region binds to the Mat and the 4127 nucleotides fold domain-by-domain, and is connected through long-range RNA-RNA interactions, such as kissing loops. Thirty-three operator-like RNA stem-loops are located and primarily interact with the asymmetric A/B CP-dimers, suggesting a pathway for the assembly of the virions. Additionally, we have discovered various forms of the virus-like particles (VLPs), including the canonical T = 3 icosahedral, larger T = 4 icosahedral, prolate, oblate forms, and a small prolate form elongated along the 3-fold axis. These particles are all produced during a normal infection, as well as when overexpressing the CPs. When overexpressing the shorter RNA fragments encoding only the CPs, we observed an increased percentage of the smaller VLPs, which may be sufficient to encapsidate a shorter RNA.

Cryo-EM Structure of a Possum Enterovirus.

Enteroviruses (EVs) represent a substantial concern to global health. Here, we present the cryo-EM structure of a non-human enterovirus, EV-F4, isolated from the Australian brushtail possum to assess the structural diversity of these picornaviruses. The capsid structure, determined to ~3 Å resolution by single particle analysis, exhibits a largely smooth surface, similar to EV-F3 (formerly BEV-2). Although the cellular receptor is not known, the absence of charged residues on the outer surface of the canyon suggest a different receptor type than for EV-F3. Density for the pocket factor is clear, with the entrance to the pocket being smaller than for other enteroviruses.

Refined Capsid Structure of Human Adenovirus D26 at 3.4 Å Resolution.

Various adenoviruses are being used as viral vectors for the generation of vaccines against chronic and emerging diseases (e.g., AIDS, COVID-19). Here, we report the improved capsid structure for one of these vectors, human adenovirus D26 (HAdV-D26), at 3.4 Å resolution, by reprocessing the previous cryo-electron microscopy dataset and obtaining a refined model. In addition to overall improvements in the model, the highlights of the structure include (1) locating a segment of the processed peptide of VIII that was previously believed to be released from the mature virions, (2) reorientation of the helical appendage domain (APD) of IIIa situated underneath the vertex region relative to its counterpart observed in the cleavage defective (ts1) mutant of HAdV-C5 that resulted in the loss of interactions between the APD and hexon bases, and (3) the revised conformation of the cleaved N-terminal segments of pre-protein VI (pVIn), located in the hexon cavities, is highly conserved, with notable stacking interactions between the conserved His13 and Phe18 residues. Taken together, the improved model of HAdV-D26 capsid provides a better understanding of protein-protein interactions in HAdV capsids and facilitates the efforts to modify and/or design adenoviral vectors with altered properties. Last but not least, we provide some insights into clotting factors (e.g., FX and PF4) binding to AdV vectors.

Structural characterization of an envelope-associated adeno-associated virus type 2 capsid.

Adeno-associated virus (AAV) are classified as non-enveloped ssDNA viruses. However, AAV capsids embedded within exosomes have been observed, and it has been suggested that the AAV membrane associated accessory protein (MAAP) may play a role in envelope-associated AAV (EA-AAV) capsid formation. Here, we observed and selected sufficient homogeneous EA-AAV capsids of AAV2, produced using the Sf9 baculoviral expression system, to determine the cryo-electron microscopy (cryo-EM) structure at 3.14 Å resolution. The reconstructed map confirmed that the EA-AAV capsid, showed no significant structural variation compared to the non-envelope capsid. In addition, the Sf9 expression system used implies the notion that MAAP may enhance exosome AAV encapsulation. Furthermore, we speculate that these EA-AAV capsids may have therapeutic benefits over the currently used non-envelope AAV capsids, with advantages in immune evasion and/or improved infectivity.