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At least 19 recordsLinked to original sources

PubMed2026

New form of HPV18 L1 found in native virions contributes to virion stability and infectivity.

UNLABELLED: Current dogma states that papillomavirus virions consist of only one form of the L1 major capsid protein. Human papillomaviruses (HPVs) have been studied using recombinant particles, thereby bypassing the need to grow the virus in a model of differentiating epithelium. Expression vectors engineered to produce L1 utilize a consensus methionine found in many HPV types. However, HPV18 has two additional in-frame methionine residues located 61 and 26 amino acids upstream of the consensus methionine. These methionine residues are excluded from the L1 expression vector used to create the recombinant virus. On the other hand, HPV produced from organotypic raft culture allows virions to be assembled in a differentiating epithelium in the presence of its native promoters and complete genome. Here, we have utilized this system to show that the wild-type HPV18, produced in a more natural setting, translates a larger form of L1 from the upstream methionine, which is 61 amino acid residues upstream from the consensus methionine, and a smaller form from the consensus methionine, with both sizes assembled in the newly formed virion. Ablation of the upstream methionine residue at position 61 altered the virion capsid conformation, and also decreased virion stability and infectivity. IMPORTANCE: The present study investigates whether the papillomavirus virions contain more than one isoform of the L1 major capsid protein. By using organotypic raft cultures that mimic the naturally differentiating epithelium, we revealed that wild-type HPV18 expresses and incorporates two distinct L1 isoforms into the virion, including a previously unrecognized longer form translated from an upstream methionine. Our study has further shown that disruption of the upstream methionine impairs virion stability and infectivity, demonstrating that the longer L1 isoform contributes to proper capsid architecture and viral function. These findings offer a new understanding of HPV capsid biology and have significant implications for the future development of HPV vaccines, diagnostics, and antiviral therapies.

HPV18↗

PubMed2024

A RNA Dodecahedral Cage Inside a Human Virus Plays a Dual Biological Role in Virion Assembly and Genome Release Control.

Human rhinoviruses (RV) are among the most frequent human pathogens. As major causative agents of common colds they originate serious socioeconomic problems and huge expenditure every year, and they also exacerbate severe respiratory diseases. No anti-rhinoviral drugs or vaccines are available so far. Antiviral drug design may benefit from an understanding of the role during the infectious cycle of the interactions in the virion between the capsid and the viral nucleic acid. The genomic RNA inside the human RV virion forms a dodecahedral cage made of 30 double-stranded RNA elements that interact with equivalent sites at the capsid inner wall. RNA dodecahedral cages also occur in distantly related insect and plant viruses. However, the functional role(s) of the interactions between any dodecahedral cage and the capsid remained to be established. Here we describe an extensive structure-function mutational analysis of the capsid-RNA dodecahedral cage interface in the RV virion, to dissect the role of the interactions between the capsid and the cage-forming RNA duplexes in: (i) infection by RV; (ii) virus biological fitness; (iii) virion assembly; (iv) virion stability; and (v) viral RNA uncoating. The results reveal that the capsid-bound dsRNA dodecahedral cage in the human RV virion is a multifunctional structural element. Two structurally overlapping subsets of RNA duplex-capsid interactions promote virus infectivity and biological fitness by respectively facilitating virion assembly or restraining the untimely, unproductive uncoating of the viral RNA genome. These results provide new insights into virion morphogenesis and genome uncoating, and have implications for antiviral drug design.

RNA, Viral↗

PubMed2026

The Bunyamwera orthobunyavirus Gc glycoprotein head and stalk drives an infectious virion assembly pathway specific for the insect host.

The Orthobunyavirus genus of arthropod-borne segmented RNA viruses comprises important pathogens including the human-infecting Oropouche virus and ruminant-infecting Schmallenberg virus (SBV). The prototypical Bunyamwera orthobunyavirus (BUNV) possesses envelope-embedded glycoprotein Gn-Gc tripodal spikes, of which the ectodomains mediate virus entry, while endodomains interact with nucleoprotein (NP) enwrapped genome segments driving virion assembly. Interestingly, BUNV Gc head/stalk domains are redundant for virus growth in mammalian cells, consistent with isolations of SBV from ruminants bearing head/stalk deletions. However, these domains appear strictly maintained in orthobunyavirus isolations from arthropods in nature. To investigate the molecular mechanism that underlines this discrepancy, we compared the multiplication characteristics of wildtype BUNV (BUNV-WT) with a Gc head/stalk deleted BUNV (BUNV-∆7). In mammalian cells BUNV-WT and BUNV-∆7 grew to equivalent titres, whereas in insect cells BUNV-∆7 titres were 1000-fold lower and strikingly produced no virions following blood meal infection of Aedes mosquitoes. To understand this insect-specific restriction in virion production, we showed the intracellular abundance of BUNV-WT and BUNV-∆7 Gc and NP components were equivalent, suggesting the deletion impacted post-translational stages of the infection cycle. To explore this, we investigated Gc and ∆7-Gc interactions during BUNV-WT and BUNV-∆7 infections of both insect and mammalian cells by co-immunoprecipitation and multiplex mass spectrometry, revealing ∆7-Gc exhibited markedly reduced NP interactions in insect cells, potentially indicating reduced segment interactions during assembly. We hypothesize that the Gc head/stalk performs an insect cell-specific role in segment recruitment during virion formation, and that maintenance in nature of full-length Gc is due to this essential role in the insect host.

Animals↗

PubMed2025

Interstrain Recombinants of Human Cytomegalovirus Reveal Complex Genetic Correlates and Epistasis Influencing Glycoprotein Display, Virion Infectivity and Spread Characteristics.

Most of the nucleotide diversity in the human cytomegalovirus (HCMV) genome is due to approximately 17 genes with 2-14 alleles each. These allelic genes are interspersed among longer stretches of highly conserved sequences with signatures of extensive recombination that would shuffle the allelic genes into a vast number of allelic haplotypes. Bacterial artificial chromosome clones derived from 3 independent clinical isolates (TB40/e (TB), TR and Merlin (ME)) display dramatic differences in the abundance of entry-mediating glycoproteins gH/gL/gO and gH/gL/UL128-131, virion infectivity and efficiency of cell-free and cell-to-cell modes of spread. Of these, TB and ME are the most phenotypically different and share only 2 of the 17 allelic genes. A set of recombinant HCMV was generated by coinfecting cells with TB and ME and restriction fragment length polymorphism (RFLP) analyses demonstrated complex crossover patterns. Most recombinants were either "TB-like" with much more gH/gL/gO than gH/gL/UL128-131, or "ME-like" with much more gH/gL/UL128-131. This correlated with a TB or ME UL128 sequence, consistent with a G/T polymorphism affecting UL128 pre-mRNA splicing. One recombinant had a gH/gL/gO:gH/gL/UL128-131 ratio of 0.8, suggesting genetic determinants beyond UL128. Virion infectivity correlated with TB versus ME-like glycoprotein display, but intragroup variability indicated additional factors and variability in spread efficiency and the contribution of cell-free and cell-to-cell spread modes indicated an influence of characteristics beyond virion infectivity. Results suggest that the relationships among these three phenotypes are not strictly causal and that all three phenotypes are genetically complex and influenced by epistasis among polymorphic loci across the genome.

Journal Article↗

PubMed2026

RNA-repelling Anionic Clusters in Human Rhinovirus Cooperate with Cationic Residues to Promote Virion Assembly and Restrain RNA Release.

Research on virus nucleic acid-protein interactions is important to understand infection and guide antiviral drug design. In previous studies we showed that the human rhinovirus (RV) genomic RNA is organized as a dodecahedral cage formed by 30 RNA duplex elements anchored to capsid concavities. We showed also that capsid-RNA duplex interactions include conserved tryptophans, neutral polar residues, and many positively charged residues that promote virion assembly and restrain RNA release by stabilizing the negatively charged RNA duplex structure. The present study expands our understanding of the capsid-RNA duplex interface in RV by addressing the structural and functional roles of conserved patches of negatively charged capsid residues interposed between each RNA duplex and its binding site at the capsid inner surface. The initial hypothesis was that electrostatic repulsion between anionic residues and RNA phosphates would lead to functional effects opposite to those previously found for cationic residues that can electrostatically attract RNA phosphates. In fact, those anionic residues do not oppose, but act together with cationic residues at the RNA duplex binding sites to promote virion assembly and restrain RNA release. Cryogenic electron microscopy analysis showed that negatively charged residues at the capsid-RNA duplex interfaces have a different structural role than positively charged residues, even though they all play similar functional roles. A tentative model is discussed to explain the functional effects of the complex distribution of negative and positive electrostatic potential found at capsid-RNA duplex interfaces in RV.

capsid↗

PubMed2025

A Quantitative Real-Time PCR Assay for Measuring Poxvirus Replication and Cell Binding.

Quantitative real-time PCR (qPCR) is a fast and reliable method to quantify viral genomes as a surrogate to titering on monolayers of cells for measuring virus replication. Whether it be for determining the number of virions released, the total number of genomes produced during infection, or the number of virions bound to a cell, qPCR assays can be adapted to quickly enumerate total viral genomes in a broad range of experiments comparing virus replication under different conditions. In addition, qPCR offers several advantages compared to plaque assays including time, linearity over 9 logs, and scalability from tens-to-hundreds of samples, depending on the qPCR machine. Here we describe a qPCR assay for quantifying vaccinia virus' dsDNA genome that can be used to determine the total number of virions produced. Furthermore, we describe a straightforward protocol for a cell-binding assay that is sensitive enough to use with small concentrations of inoculating virions. This protocol is suitable for measuring the cell-binding ability of mutations that affect virus production and infectivity.

Virus Replication↗

PubMed2024

Cryo-electron tomography reveals coupled flavivirus replication, budding and maturation.

Flaviviruses replicate their genomes in replication organelles (ROs) formed as bud-like invaginations on the endoplasmic reticulum (ER) membrane, which also functions as the site for virion assembly. While this localization is well established, it is not known to what extent viral membrane remodeling, genome replication, virion assembly, and maturation are coordinated. Here, we imaged tick-borne flavivirus replication in human cells using cryo-electron tomography. We find that the RO membrane bud is shaped by a combination of a curvature-establishing coat and the pressure from intraluminal template RNA. A protein complex at the RO base extends to an adjacent membrane, where immature virions bud. Naturally occurring furin site variants determine whether virions mature in the immediate vicinity of ROs. We further visualize replication in mouse brain tissue by cryo-electron tomography. Taken together, these findings reveal a close spatial coupling of flavivirus genome replication, budding, and maturation.

Journal Article↗

PubMed2026

Systematic profiling of nudivirus-like genes reveals conserved and differentiated roles in a domesticated endogenous virus.

Cotesia vestalis bracovirus (CvBV) is a type of domesticated endogenous virus (DEV) derived from ancestral nudiviruses that is integrated into the genome of the parasitoid wasp Cotesia vestalis. The CvBV proviral genome is composed of two distinct components: one encoding genes associated with virion morphogenesis and assembly, and the other harboring virulence genes that are excised, circularized, and packaged into virions. CvBV replication and particle assembly occur exclusively in the ovaries of female wasps. While prior studies have largely focused on the function of virulence genes during parasitization, the molecular mechanisms underlying CvBV replication and assembly remain poorly understood. Here, we identified 71 nudivirus-like genes in the C. vestalis genome through integrated transcriptomic and proteomic analyses. Using gene silencing and microscopy-based imaging approaches, we functionally characterized 24 key genes involved in DNA replication (helicase, integrase-1, and integrase-2), transcriptional regulation (p47, lef-5, and lef-9), capsid formation (vp39, PmV, HzNVorf9-1, HzNVorf9-2, HzNVorf106, 38k, 27b, and K425_459), envelope formation (11k, 17a-1, 35a-1, 35a-2, and K425_461), virion assembly (vlf-1, HzNVorf140-1, and HzNVorf140-2), and viral infectivity (pif-0 and vp91). Although the functions of most nudivirus-like genes are generally conserved among baculoviruses, nudiviruses, and bracoviruses, lef-5, K425_459, 11k, and vp91 appear to have undergone functional divergence relative to their homologs in baculoviruses, nudiviruses, and Microplitis demolitor bracovirus, highlighting lineage-specific adaptations in CvBV. Collectively, our work provides a molecular framework for understanding CvBV assembly and serves as a valuable resource for investigating bracovirus evolution.

Animals↗

PubMed2026

Refining a giant virus lineage: a novel order unifying Mamonoviridae and "Manesviridae," unveiled by the discovery of furtivovirus.

UNLABELLED: The evolutionary origins and taxonomic framework of giant viruses related to the family Mamonoviridae and its relative group, including clandestinovirus, remain unclassified due to gaps in genome size and host range between these two groups. This study aimed to address this gap by integrating our newly isolated virus with publicly available metagenome-assembled genomes (MAGs) to construct a more robust phylogenetic framework. Here, we report the isolation and characterization of a new giant virus, furtivovirus, using the unicellular amoeba Vermamoeba vermiformis as a host. Furtivovirus has a genome of approximately 560 kbp and shares key features with its closest relative, clandestinovirus. Ultrastructural analysis revealed a unique host-nucleus-dependent replication strategy characterized by the breakdown of the nuclear membrane and the packaging of nascent virions directly within the nucleoplasm, distinguishing it from canonical cytoplasmic virion factories. Comprehensive phylogenetic and comparative genomic analyses of shared orthologous groups and nucleocytovirus marker proteins revealed that furtivovirus, clandestinovirus, ushikuvirus, and usurpativirus form a distinct monophyletic clade, for which we propose a new family, "Manesviridae." Further analysis using amino acid-based similarity metrics of Nucleocytoviricota viral genomes, including established MAGs, demonstrated that this new family is robustly placed as a sister group to the family Mamonoviridae. This study elucidated the evolutionary relationships between viruses with large and small genomes that possess similar virion sizes within this lineage. Based on this cumulative evidence, we propose the establishment of a new order to unify these two families, thereby expanding their diversity and clarifying the evolutionary history of this branch within Nucleocytoviricota. IMPORTANCE: Giant viruses challenge our traditional understanding of viral evolution, raising the question of how a single related group can diverge to infect different hosts while evolving into vastly different genome sizes and replication strategies. The family Mamonoviridae and its relatives epitomize this evolutionary divergence: one group possesses massive genomes, whereas the other has genomes that are less than half their size. The discovery of furtivovirus and its unique nucleoplasm-dependent replication cycle provides a critical biological context for this genomic disparity. Through deep comparative genomic analysis, we demonstrated that these seemingly disparate lineages share a cohesive evolutionary origin that is distinct from other established orders. This finding highlights the complexity of genome evolution, demonstrating that giant viruses can expand their overall genome size to adapt to uncertain environments while reducing their core essential genes, thereby providing new insights into the evolutionary pressures that shape the diversity of the virosphere.

Giant Viruses↗

PubMed2025

Induction of tunnelling nanotube-like structures by influenza A viruses requires the onset of apoptosis.

As well as spreading through virions, influenza A viruses (IAVs) can evade antiviral drugs and neutralising antibodies by spreading directly from cell to cell. In cell culture this can occur by the induction of intercellular membrane connections known as tunnelling nanotube-like structures (TLSs), which are capable of trafficking the viral genome between cells. Here, we showed that TLSs are formed by IAV infected cells in vivo, and then used in vitro models to ask how IAVs induce their formation. We found that TLS formation is not induced by cytokine signalling from infected to uninfected cells, but induction does require intracellular IAV replication. IAV replication can form filamentous virions which have structural similarities to TLSs, but we found that TLS induction is independent of virion morphology. We therefore looked at the intracellular responses to infection and found that the induction of TLSs correlated with the induction of apoptosis. Furthermore, the ability of IAVs to drive TLS formation can be modulated by chemically inhibiting, or inducing apoptosis. Finally, we found that inhibiting apoptosis, which prevents IAVs from inducing TLSs, lead to a significant reduction in the ability of IAVs to directly spread between cells. Our results, which suggest that IAVs can control their ability to spread directly from cell to cell by driving infected cells into apoptosis, identifies a new way in which a virus can manipulate its host to evade antiviral immune responses.

Apoptosis↗

PubMed2025

SARS-CoV-2 remodels the Golgi apparatus to facilitate viral assembly and secretion.

The COVID-19 pandemic is caused by the enveloped virus SARS-CoV-2. Despite extensive investigation, the molecular mechanisms for its assembly and secretion remain largely elusive. Here, we show that SARS-CoV-2 infection induces global alterations of the host endomembrane system, including dramatic Golgi fragmentation. SARS-CoV-2 virions are enriched in the fragmented Golgi. Blocking endoplasmic reticulum (ER) to Golgi trafficking dramatically inhibits SARS-CoV-2 assembly and secretion without reducing viral genome replication. Significantly, SARS-CoV-2 infection down-regulates GRASP55 but up-regulates TGN46 protein levels. Surprisingly, GRASP55 expression reduces both viral secretion and spike number on each virion without affecting viral entry, while GRASP55 depletion displays opposite effects. In contrast, TGN46 depletion only inhibits viral secretion without affecting spike incorporation into virions. Taken together, we show that SARS-CoV-2 alters Golgi structure and function to modulate viral assembly and secretion, highlighting the Golgi as a potential therapeutic target for blocking SARS-CoV-2 infection.

Golgi Apparatus↗

PubMed2027

Overview of Chikungunya Virus Epidemiology, Biology, and Pathogenesis.

Chikungunya virus (CHIKV), an arthropod-borne alphavirus within the Togaviridae family, is transmitted primarily by Aedes aegypti and Aedes albopictus. The virus causes an acute febrile illness characterized by severe, often bilateral polyarthralgia, with potential progression to chronic musculoskeletal pain and rare systemic complications involving cardiovascular and neurological systems. CHIKV exhibits a spherical, enveloped virion (~70 nm) with T = 4 icosahedral symmetry, incorporating E1/E2 glycoprotein heterodimers that mediate receptor binding and membrane fusion. Its positive-sense RNA genome (~11.8 kb) encodes nonstructural proteins for replication and structural proteins for virion assembly. Replication occurs in cytoplasmic spherules, involving synthesis of genomic and subgenomic RNAs, followed by glycoprotein maturation and budding at the plasma membrane. Epidemiologically, CHIKV has expanded beyond Africa and Asia, with major outbreaks driven by adaptive mutations enhancing transmission via A. albopictus. Since introduction to the Americas in 2013, the global incidence remains high, with >180,000 confirmed cases reported in 2025. Preventive strategies rely on vector control and vaccination; VLP-based vaccines (e.g., Vimkunya) show promise, while live-attenuated formulations face safety concerns. No licensed antivirals exist; current management is supportive, though investigational therapies targeting viral replication and immune modulation are under development.

Chikungunya virus↗

PubMed2026

Construction of an infectious clone of Spodoptera frugiperda densovirus and its biological characteristics.

Densoviruses are highly pathogenic to their insect hosts and have great potential for biocontrol. Spodoptera frugiperda densovirus (SfDV) was isolated from diseased larvae of Spodoptera frugiperda, while its biological functions remain unclear. Herein, we successfully constructed an infectious clone of SfDV. The S. frugiperda larvae transfected with the infectious clone exhibited anorexia, stunted growth, and reduced activity. Histopathological analysis further showed that the epidermis, fat body and trachea were infected instead of muscle and midgut tissues. Transmission electron microscopy (TEM) revealed that numerous virions of about 22 nm were distributed within both the nucleoplasm and cytoplasm of epidermal cells. Moreover, many virions were also found contained within vesicles in the cytoplasm. The replication kinetics of the rescued SfDV (rSfDV) was similar to that of the parental SfDV. The median lethal dose (LD50) and median lethal time (LT50) values of rSfDV were 6.63 × 107 viral genome copies (vgc), 5.23 d, respectively, which were also comparable to those of the parental SfDV. Taken together, the infectious clone of SfDV provides an important tool for further exploring the genome function, pathogenesis, and interactions with its hosts.

Animals↗

PubMed2026

Infection cycles of viruses of the phylum Nucleocytoviricota.

The phylum Nucleocytoviricota, formerly known as nucleocytoplasmic large DNA viruses (NCLDVs), comprises evolutionarily related viruses with remarkably diverse genome sizes, coding capacities and virion morphologies. These viruses infect hosts across the eukaryotic tree of life, from protists to humans, and are believed to have emerged during the early stages of eukaryotic evolution. How the basic aspects of virus-host interaction have evolved in different lineages and whether they share a conserved infection cycle remain unclear. In this Review, we synthesize the information on the infection cycles of model representatives from the major orders within the phylum, revealing both shared traits and lineage-specific innovations. We compare the information available for the extensively studied poxviruses, asfiviruses, iridoviruses and chloroviruses with insights from the rapidly expanding literature on the mimiviruses, pandoraviruses, marseilleviruses and pithoviruses. We provide an overview of the molecular details underlying the key stages of Nucleocytoviricota infection cycles: entry via membrane fusion, formation of viral factories organized via phase separation, genome replication, virion morphogenesis through a crescent intermediate, and egress. We highlight outstanding questions in the field, unify concepts across traditionally separated research areas, and provide a conceptual framework to guide future cell biology studies on large double-stranded DNA viruses.

DNA Viruses↗

PubMed2025

Characterization of the Kaposi's sarcoma-associated herpesvirus terminase complex component ORF29.

Kaposi's sarcoma-associated herpesvirus (KSHV) belongs to the Gammaherpesvirinae subfamily. During the lytic phase of herpesviruses, viral capsids form in the host cell nucleus, and the replicated viral genome is packaged into these capsids. The herpesviral genome is replicated as a precursor head-to-tail concatemer consisting of tandemly repeated genomic units, each flanked by terminal repeats (TRs). The herpesvirus terminase complex packages a single genomic unit into a capsid by cleaving the TRs in the precursor genome. Although the terminase complexes of alpha- and beta-herpesviruses are well characterized, the KSHV terminase complex is poorly understood. KSHV ORF7, ORF67.5, and ORF29 are thought to be components of this complex. We previously reported that KSHV deficient in either ORF7 or ORF67.5 formed immature, soccer ball-like capsids and failed to cleave the TRs, resulting in decreased virion production. Moreover, ORF7 interacted with both ORF29 and ORF67.5; however, ORF29 and ORF67.5 did not interact with each other. Thus, although ORF7 and ORF67.5 are important for KSHV terminase function, the function of ORF29 remains largely unknown. In this study, we constructed an ORF29-deficient KSHV and analyzed its virological properties. ORF29 was found to be essential for virion production and TR cleavage. Numerous immature, soccer ball-like capsids were observed in cells harboring ORF29-deficient KSHV. The N-terminal region of ORF29 was important for its interaction with ORF7, although the full-length ORF29 was required for effective assembly of the KSHV terminase complex. Furthermore, ORF29 preferentially interacted with itself rather than with ORF7. Thus, our data show that ORF29 functions as a fundamental component of the terminase complex.IMPORTANCEBecause the role of ORF29 in the Kaposi's sarcoma-associated herpesvirus (KSHV) terminase complex remains unknown, we constructed ORF29-deficient KSHV. Our results demonstrated that ORF29 functions as a component of the KSHV terminase and is essential for mature capsid formation, terminal repeat (TR) cleavage, and terminase complex assembly. Moreover, ORF29 strongly interacted with itself. In herpes simplex virus 1 (HSV-1), the terminase complex (comprising UL15, UL28, and UL33) forms a trimer, and six such trimers assemble into a hexameric ring. The HSV-1 genome passes through this ring and undergoes TR cleavage and genome packaging into a capsid. The self-interaction of ORF29 may be involved in the multimerization of the terminase complex or in the formation of the KSHV terminase ring.

Herpesvirus 8, Human↗

PubMed2026

Discovery of acridone analogs as novel entry inhibitors targeting e protein of dengue virus.

The envelope (E) protein of the Dengue virus (DENV) is critical for virion attachment and membrane fusion with the host cell, as well as the release of the viral RNA genome into the cytoplasm. In this study, we describe the design, synthesis, and biological evaluation of novel viral entry inhibitors containing an acridone core. Notably, compound 13e demonstrated potent cellular antiviral activity (IC50 = 8.6 μM and selectivity index = 21.4). Compound 13e was evaluated using several methods, including time-of-addition and virus entry/binding assays, which revealed that it selectively blocked DENV2 infection by inhibiting virion attachment. Furthermore, compound 13e exhibited potent antiviral efficacy, as evidenced by viremia quantification and histopathological analysis results, without causing significant body weight loss or other toxicities. Furthermore, target engagement assay supported the role of compound 13e as an E protein binder, consistent with its function as an entry inhibitor.

Dengue Virus↗

PubMed2026

Insights into Genome Ejection by a Therapeutic phiKMV-like Bacteriophage.

Ar-KM is a phiKMV-like therapeutic bacteriophage used in clinical candidate phage therapy cocktails to treat lung infections caused by P. aeruginosa. Here, we present an integrative structural atlas of Ar-KM proteins using cryo-EM, proteomics, and bioinformatics. From a single purified Ar-KM preparation, we identified three distinct populations: mature DNA-filled virions, open-nozzle particles with ejection proteins extending from the tail, and closed-nozzle empty particles. Near-atomic-resolution reconstructions of all three states enabled us to build atomic models for eleven structural proteins. The mature virion revealed the pre-ejection conformation of three ejection proteins, gp41, gp42, and gp43, homologous to coliphage T7's gp14, gp15, and gp16, respectively. Unlike T7, peptidoglycan hydrolase activity associated with the ejectosome resides in the gp15-like periplasmic tunnel protein gp42, whereas in T7 the lysozyme-like domain is located at the N-terminus of gp16, underscoring the structural plasticity and evolutionary mosaicity of ejection proteins. We further identified a short α-helical factor, gp34, present in eight copies at the mismatched interface between the portal barrel and gp41. Gp34 forms a cage within the nozzle, acting as a molecular wedge that stabilizes the open conformation and permits gp41 to assemble into a hexameric channel during ejection. Evolutionarily, gp34 appears to be an ortholog of the essential gene gp7.3 in phage T7 and is conserved across sequenced phiKMV-like phages. We propose that this protein functions as an ejection protein assembly factor, stabilizing the open nozzle during infection and allowing the coordinated exit of ejection proteins and their assembly into a DNA-ejectosome.

Pseudomonas phages↗

PubMed2022

Atomic model of vesicular stomatitis virus and mechanism of assembly.

Like other negative-strand RNA viruses (NSVs) such as influenza and rabies, vesicular stomatitis virus (VSV) has a three-layered organization: a layer of matrix protein (M) resides between the glycoprotein (G)-studded membrane envelope and the nucleocapsid, which is composed of the nucleocapsid protein (N) and the encapsidated genomic RNA. Lack of in situ atomic structures of these viral components has limited mechanistic understanding of assembling the bullet-shaped virion. Here, by cryoEM and sub-particle reconstruction, we have determined the in situ structures of M and N inside VSV at 3.47 Å resolution. In the virion, N and M sites have a stoichiometry of 1:2. The in situ structures of both N and M differ from their crystal structures in their N-terminal segments and oligomerization loops. N-RNA, N-N, and N-M-M interactions govern the formation of the capsid. A double layer of M contributes to packaging of the helical nucleocapsid: the inner M (IM) joins neighboring turns of the N helix, while the outer M (OM) contacts G and the membrane envelope. The pseudo-crystalline organization of G is further mapped by cryoET. The mechanism of VSV assembly is delineated by the network interactions of these viral components.

Animals↗