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.

RetroCHMP3 blocks budding of enveloped viruses without blocking cytokinesis.

Many enveloped viruses require the endosomal sorting complexes required for transport (ESCRT) pathway to exit infected cells. This highly conserved pathway mediates essential cellular membrane fission events, which restricts the acquisition of adaptive mutations to counteract viral co-option. Here, we describe duplicated and truncated copies of the ESCRT-III factor CHMP3 that block ESCRT-dependent virus budding and arose independently in New World monkeys and mice. When expressed in human cells, these retroCHMP3 proteins potently inhibit release of retroviruses, paramyxoviruses, and filoviruses. Remarkably, retroCHMP3 proteins have evolved to reduce interactions with other ESCRT-III factors and have little effect on cellular ESCRT processes, revealing routes for decoupling cellular ESCRT functions from viral exploitation. The repurposing of duplicated ESCRT-III proteins thus provides a mechanism to generate broad-spectrum viral budding inhibitors without blocking highly conserved essential cellular ESCRT functions.

Structural and Proteomic Characterization of the Initiation of Giant Virus Infection.

Since their discovery, giant viruses have expanded our understanding of the principles of virology. Due to their gargantuan size and complexity, little is known about the life cycles of these viruses. To answer outstanding questions regarding giant virus infection mechanisms, we set out to determine biomolecular conditions that promote giant virus genome release. We generated four infection intermediates in Samba virus (Mimivirus genus, lineage A) as visualized by cryoelectron microscopy (cryo-EM), cryoelectron tomography (cryo-ET), and scanning electron microscopy (SEM). Each of these four intermediates reflects similar morphology to a stage that occurs in vivo. We show that these genome release stages are conserved in other mimiviruses. Finally, we identified proteins that are released from Samba and newly discovered Tupanvirus through differential mass spectrometry. Our work revealed the molecular forces that trigger infection are conserved among disparate giant viruses. This study is also the first to identify specific proteins released during the initial stages of giant virus infection.

Molecular Architecture of the SARS-CoV-2 Virus.

SARS-CoV-2 is an enveloped virus responsible for the COVID-19 pandemic. Despite recent advances in the structural elucidation of SARS-CoV-2 proteins, the detailed architecture of the intact virus remains to be unveiled. Here we report the molecular assembly of the authentic SARS-CoV-2 virus using cryoelectron tomography (cryo-ET) and subtomogram averaging (STA). Native structures of the S proteins in pre- and postfusion conformations were determined to average resolutions of 8.7-11 Å. Compositions of the N-linked glycans from the native spikes were analyzed by mass spectrometry, which revealed overall processing states of the native glycans highly similar to that of the recombinant glycoprotein glycans. The native conformation of the ribonucleoproteins (RNPs) and their higher-order assemblies were revealed. Overall, these characterizations revealed the architecture of the SARS-CoV-2 virus in exceptional detail and shed light on how the virus packs its ∼30-kb-long single-segmented RNA in the ∼80-nm-diameter lumen.

Low-Fidelity Assembly of Influenza A Virus Promotes Escape from Host Cells.

Influenza viruses inhabit a wide range of host environments using a limited repertoire of protein components. Unlike viruses with stereotyped shapes, influenza produces virions with significant morphological variability even within clonal populations. Whether this tendency to form pleiomorphic virions is coupled to compositional heterogeneity and whether it affects replicative fitness remains unclear. Here, we address these questions by developing a strain of influenza A virus amenable to rapid compositional characterization through quantitative, site-specific labeling of viral proteins. Using this strain, we find that influenza A produces virions with broad variations in size and composition from even single infected cells. This phenotypic variability contributes to virus survival during environmental challenges, including exposure to antivirals. Complementing genetic adaptations that act over larger populations and longer times, this "low-fidelity" assembly of influenza A virus allows small populations to survive environments that fluctuate over individual replication cycles.

DNA-Packing Portal and Capsid-Associated Tegument Complexes in the Tumor Herpesvirus KSHV.

Assembly of Kaposi's sarcoma-associated herpesvirus (KSHV) begins at a bacteriophage-like portal complex that nucleates formation of an icosahedral capsid with capsid-associated tegument complexes (CATCs) and facilitates translocation of an ∼150-kb dsDNA genome, followed by acquisition of a pleomorphic tegument and envelope. Because of deviation from icosahedral symmetry, KSHV portal and tegument structures have largely been obscured in previous studies. Using symmetry-relaxed cryo-EM, we determined the in situ structure of the KSHV portal and its interactions with surrounding capsid proteins, CATCs, and the terminal end of KSHV's dsDNA genome. Our atomic models of the portal and capsid/CATC, together with visualization of CATCs' variable occupancy and alternate orientation of CATC-interacting vertex triplexes, suggest a mechanism whereby the portal orchestrates procapsid formation and asymmetric long-range determination of CATC attachment during DNA packaging prior to pleomorphic tegumentation/envelopment. Structure-based mutageneses confirm that a triplex deep binding groove for CATCs is a hotspot that holds promise for antiviral development.

Electron Cryo-microscopy Structure of Ebola Virus Nucleoprotein Reveals a Mechanism for Nucleocapsid-like Assembly.

Ebola virus nucleoprotein (eNP) assembles into higher-ordered structures that form the viral nucleocapsid (NC) and serve as the scaffold for viral RNA synthesis. However, molecular insights into the NC assembly process are lacking. Using a hybrid approach, we characterized the NC-like assembly of eNP, identified novel regulatory elements, and described how these elements impact function. We generated a three-dimensional structure of the eNP NC-like assembly at 5.8 Å using electron cryo-microscopy and identified a new regulatory role for eNP helices α22-α23. Biochemical, biophysical, and mutational analyses revealed that inter-eNP contacts within α22-α23 are critical for viral NC assembly and regulate viral RNA synthesis. These observations suggest that the N terminus and α22-α23 of eNP function as context-dependent regulatory modules (CDRMs). Our current study provides a framework for a structural mechanism for NC-like assembly and a new therapeutic target.

The structure and physical properties of a packaged bacteriophage particle.

A string of nucleotides confined within a protein capsid contains all the instructions necessary to make a functional virus particle, a virion. Although the structure of the protein capsid is known for many virus species1,2, the three-dimensional organization of viral genomes has mostly eluded experimental probes3,4. Here we report all-atom structural models of an HK97 virion5, including its entire 39,732 base pair genome, obtained through multiresolution simulations. Mimicking the action of a packaging motor6, the genome was gradually loaded into the capsid. The structure of the packaged capsid was then refined through simulations of increasing resolution, which produced a 26 million atom model of the complete virion, including water and ions confined within the capsid. DNA packaging occurs through a loop extrusion mechanism7 that produces globally different configurations of the packaged genome and gives each viral particle individual traits. Multiple microsecond-long all-atom simulations characterized the effect of the packaged genome on capsid structure, internal pressure, electrostatics and diffusion of water, ions and DNA, and revealed the structural imprints of the capsid onto the genome. Our approach can be generalized to obtain complete all-atom structural models of other virus species, thereby potentially revealing new drug targets at the genome-capsid interface.

A virally encoded high-resolution screen of cytomegalovirus dependencies.

Genetic screens have transformed our ability to interrogate cellular factor requirements for viral infections1,2, but most current approaches are limited in their sensitivity, biased towards early stages of infection and provide only simplistic phenotypic information that is often based on survival of infected cells2-4. Here, by engineering human cytomegalovirus to express single guide RNA libraries directly from the viral genome, we developed virus-encoded CRISPR-based direct readout screening (VECOS), a sensitive, versatile, viral-centric approach that enables profiling of different stages of viral infection in a pooled format. Using this approach, we identified hundreds of host dependency and restriction factors and quantified their direct effects on viral genome replication, viral particle secretion and infectiousness of secreted particles, providing a multi-dimensional perspective on virus-host interactions. These high-resolution measurements reveal that perturbations altering late stages in the life cycle of human cytomegalovirus (HCMV) mostly regulate viral particle quality rather than quantity, establishing correct virion assembly as a critical stage that is heavily reliant on virus-host interactions. Overall, VECOS facilitates systematic high-resolution dissection of the role of human proteins during the infection cycle, providing a roadmap for in-depth study of host-herpesvirus interactions.

Global changes in the RNA binding specificity of HIV-1 gag regulate virion genesis.

The HIV-1 Gag protein orchestrates all steps of virion genesis, including membrane targeting and RNA recruitment into virions. Using crosslinking-immunoprecipitation (CLIP) sequencing, we uncover several dramatic changes in the RNA-binding properties of Gag that occur during virion genesis, coincident with membrane binding, multimerization, and proteolytic maturation. Prior to assembly, and after virion assembly and maturation, the nucleocapsid domain of Gag preferentially binds to psi and Rev Response elements in the viral genome, and GU-rich mRNA sequences. However, during virion genesis, this specificity transiently changes in a manner that facilitates genome packaging; nucleocapsid binds to many sites on the HIV-1 genome and to mRNA sequences with a HIV-1-like, A-rich nucleotide composition. Additionally, we find that the matrix domain of Gag binds almost exclusively to specific tRNAs in the cytosol, and this association regulates Gag binding to cellular membranes.

A viral packaging motor varies its DNA rotation and step size to preserve subunit coordination as the capsid fills.

Multimeric, ring-shaped molecular motors rely on the coordinated action of their subunits to perform crucial biological functions. During these tasks, motors often change their operation in response to regulatory signals. Here, we investigate a viral packaging machine as it fills the capsid with DNA and encounters increasing internal pressure. We find that the motor rotates the DNA during packaging and that the rotation per base pair increases with filling. This change accompanies a reduction in the motor's step size. We propose that these adjustments preserve motor coordination by allowing one subunit to make periodic, specific, and regulatory contacts with the DNA. At high filling, we also observe the downregulation of the ATP-binding rate and the emergence of long-lived pauses, suggesting a throttling-down mechanism employed by the motor near the completion of packaging. This study illustrates how a biological motor adjusts its operation in response to changing conditions, while remaining highly coordinated.

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 rearrangement of ebola virus VP40 begets multiple functions in the virus life cycle.

Proteins, particularly viral proteins, can be multifunctional, but the mechanisms behind multifunctionality are not fully understood. Here, we illustrate through multiple crystal structures, biochemistry, and cellular microscopy that VP40 rearranges into different structures, each with a distinct function required for the ebolavirus life cycle. A butterfly-shaped VP40 dimer traffics to the cellular membrane. Once there, electrostatic interactions trigger rearrangement of the polypeptide into a linear hexamer. These hexamers construct a multilayered, filamentous matrix structure that is critical for budding and resembles tomograms of authentic virions. A third structure of VP40, formed by a different rearrangement, is not involved in virus assembly but instead uniquely binds RNA to regulate viral transcription inside infected cells. These results provide a functional model for ebolavirus matrix assembly and the other roles of VP40 in the virus life cycle and demonstrate how a single wild-type, unmodified polypeptide can assemble into different structures for different functions.

Architecture and self-assembly of the jumbo bacteriophage nuclear shell.

Bacteria encode myriad defences that target the genomes of infecting bacteriophage, including restriction-modification and CRISPR-Cas systems1. In response, one family of large bacteriophages uses a nucleus-like compartment to protect its replicating genomes by excluding host defence factors2-4. However, the principal composition and structure of this compartment remain unknown. Here we find that the bacteriophage nuclear shell assembles primarily from one protein, which we name chimallin (ChmA). Combining cryo-electron tomography of nuclear shells in bacteriophage-infected cells and cryo-electron microscopy of a minimal chimallin compartment in vitro, we show that chimallin self-assembles as a flexible sheet into closed micrometre-scale compartments. The architecture and assembly dynamics of the chimallin shell suggest mechanisms for its nucleation and growth, and its role as a scaffold for phage-encoded factors mediating macromolecular transport, cytoskeletal interactions, and viral maturation.

Giant variations in giant virus genome packaging.

Giant viruses (Nucleocytoviricota) have a largely conserved lifecycle, yet how they cram their large genomes into viral capsids is mostly unknown. The major capsid protein and the packaging ATPase (pATPase) comprise a highly conserved morphogenesis module in giant viruses, yet some giant viruses dispense with an icosahedral capsid, and others encode multiple versions of pATPases, including conjoined ATPase doublets, or encode none. Some giant viruses have acquired DNA-condensing proteins to compact their genomes, including sheath-like structures encasing folded DNA or densely packed viral nucleosomes that show a resemblance to eukaryotic nucleosomes at the telomeres. Here, we review what is known and unknown about these ATPases and condensing proteins, and place these variations in the context of viral lifecycles.

Herpesviruses assimilate kinesin to produce motorized viral particles.

Neurotropic alphaherpesviruses initiate infection in exposed mucosal tissues and, unlike most viruses, spread rapidly to sensory and autonomic nerves where life-long latency is established1. Recurrent infections arise sporadically from the peripheral nervous system throughout the life of the host, and invasion of the central nervous system may occur, with severe outcomes2. These viruses directly recruit cellular motors for transport along microtubules in nerve axons, but how the motors are manipulated to deliver the virus to neuronal nuclei is not understood. Here, using herpes simplex virus type I and pseudorabies virus as model alphaherpesviruses, we show that a cellular kinesin motor is captured by virions in epithelial cells, carried between cells, and subsequently used in neurons to traffic to nuclei. Viruses assembled in the absence of kinesin are not neuroinvasive. The findings explain a critical component of the alphaherpesvirus neuroinvasive mechanism and demonstrate that these viruses assimilate a cellular protein as an essential proviral structural component. This principle of viral assimilation may prove relevant to other virus families and offers new strategies to combat infection.

Hijacking the Hijackers: Escherichia coli Pathogenicity Islands Redirect Helper Phage Packaging for Their Own Benefit.

Phage-inducible chromosomal islands (PICIs) represent a novel and universal class of mobile genetic elements, which have broad impact on bacterial virulence. In spite of their relevance, how the Gram-negative PICIs hijack the phage machinery for their own specific packaging and how they block phage reproduction remains to be determined. Using genetic and structural analyses, we solve the mystery here by showing that the Gram-negative PICIs encode a protein that simultaneously performs these processes. This protein, which we have named Rpp (for redirecting phage packaging), interacts with the phage terminase small subunit, forming a heterocomplex. This complex is unable to recognize the phage DNA, blocking phage packaging, but specifically binds to the PICI genome, promoting PICI packaging. Our studies reveal the mechanism of action that allows PICI dissemination in nature, introducing a new paradigm in the understanding of the biology of pathogenicity islands and therefore of bacterial pathogen evolution.

Hierarchical assembly is more robust than egalitarian assembly in synthetic capsids.

Self-assembly of complex and functional materials remains a grand challenge in soft material science. Efficient assembly depends on a delicate balance between thermodynamic and kinetic effects, requiring fine-tuning affinities and concentrations of subunits. By contrast, we introduce an assembly paradigm that allows large error-tolerance in the subunit affinity and helps avoid kinetic traps. Our combined experimental and computational approach uses a model system of triangular subunits programmed to assemble into T = 3 icosahedral capsids comprising 60 units. The experimental platform uses DNA origami to create monodisperse colloids whose three-dimensional geometry is controlled to nanometer precision, with two distinct bonds whose affinities are controlled to kBT precision, quantified in situ by static light scattering. The computational model uses a coarse-grained representation of subunits, short-ranged potentials, and Langevin dynamics. Experimental observations and modeling reveal that when the bond affinities are unequal, two distinct hierarchical assembly pathways occur, in which the subunits first form dimers in one case and pentamers in another. These hierarchical pathways produce complete capsids faster and are more robust against affinity variation than egalitarian pathways, in which all binding sites have equal strengths. This finding suggests that hierarchical assembly may be a general engineering principle for optimizing self-assembly of complex target structures.

SRPK2 Mediates HBV Core Protein Phosphorylation and Capsid Assembly via Docking Interaction.

Members of the serine-arginine protein kinase (SRPK) family, SRPK1 and SRPK2, phosphorylate the hepatitis B core protein (Cp) and are crucial for pregenomic RNA encapsidation during viral nucleocapsid assembly. Among them, SRPK2 exhibits higher kinase activity toward Cp. In this study, we identified Cp sites that are phosphorylated by SRPK2 and demonstrated that the kinase utilizes an SRPK-specific docking groove to interact with and regulate the phosphorylation of the C-terminal arginine rich domain of Cp. We determined that direct interaction between the docking groove of SRPK2 and unphosphorylated Cp inhibited premature viral capsid assembly in vitro, whereas the phosphorylation of the viral protein reactivated the process. Pull-down assays together with the new cryo-electron microscopy structure of the HBV capsid in complex with SRPK2 revealed that the kinases decorate the surface of the viral capsid by interacting with the C-terminal domain of Cp, underscoring the importance of the docking interaction in regulating capsid assembly and pregenome packaging. Moreover, SRPK2-knockout in HepG2 cells suppressed Cp phosphorylation, indicating that SRPK2 is an important cellular kinase for HBV life cycle.

Human cytomegalovirus deploys molecular mimicry to recruit VPS4A to sites of virus assembly.

The AAA-type ATPase VPS4 is recruited by proteins of the endosomal sorting complex required for transport III (ESCRT-III) to catalyse membrane constriction and membrane fission. VPS4A accumulates at the cytoplasmic viral assembly complex (cVAC) of cells infected with human cytomegalovirus (HCMV), the site where nascent virus particles obtain their membrane envelope. Here we show that VPS4A is recruited to the cVAC via interaction with pUL71. Sequence analysis, deep-learning structure prediction, molecular dynamics and mutagenic analysis identify a short peptide motif in the C-terminal region of pUL71 that is necessary and sufficient for the interaction with VPS4A. This motif is predicted to bind the same groove of the N-terminal VPS4A Microtubule-Interacting and Trafficking (MIT) domain as the Type 2 MIT-Interacting Motif (MIM2) of cellular ESCRT-III components, and this viral MIM2-like motif (vMIM2) is conserved across ß-herpesvirus pUL71 homologues. However, recruitment of VPS4A by pUL71 is dispensable for HCMV morphogenesis or replication and the function of the conserved vMIM2 during infection remains enigmatic. VPS4-recruitment via a vMIM2 represents a previously unknown mechanism of molecular mimicry in viruses, extending previous observations that herpesviruses encode proteins with structural and functional homology to cellular ESCRT-III components.