Myxococcus xanthus encapsulin cargo protein EncD is a flavin-binding protein with ferric reductase activity.

Encapsulins are protein nanocompartments that regulate cellular metabolism in several bacteria and archaea. Myxococcus xanthus encapsulins protect the bacterial cells against oxidative stress by sequestering cytosolic iron. These encapsulins are formed by the shell protein EncA and three cargo proteins: EncB, EncC, and EncD. EncB and EncC form rotationally symmetric decamers with ferroxidase centers (FOCs) that oxidize Fe+2 to Fe+3 for iron storage in mineral form. However, the structure and function of the third cargo protein, EncD, have yet to be determined. Here, we report the x-ray crystal structure of EncD in complex with flavin mononucleotide. EncD forms an α-helical hairpin arranged as an antiparallel dimer, but unlike other flavin-binding proteins, it has no ß-sheet, showing that EncD and its homologs represent a unique class of bacterial flavin-binding proteins. The cryo-EM structure of EncA-EncD encapsulins confirms that EncD binds to the interior of the EncA shell via its C-terminal targeting peptide. With only 100 amino acids, the EncD α-helical dimer forms the smallest flavin-binding domain observed to date. Unlike EncB and EncC, EncD lacks a FOC, and our biochemical results show that EncD instead is a NAD(P)H-dependent ferric reductase, indicating that the M. xanthus encapsulins act as an integrated system for iron homeostasis. Overall, this work contributes to our understanding of bacterial metabolism and could lead to the development of technologies for iron biomineralization and the production of iron-containing materials for the treatment of various diseases associated with oxidative stress.

Defined extracellular matrix compositions support stiffness-insensitive cell spreading and adhesion signaling.

Integrin-dependent adhesion to the extracellular matrix (ECM) mediates mechanosensing and signaling in response to altered microenvironmental conditions. In order to provide tissue- and organ-specific cues, the ECM is composed of many different proteins that temper the mechanical properties and provide the necessary structural diversity. Despite most human tissues being soft, the prevailing view from predominantly in vitro studies is that increased stiffness triggers effective cell spreading and activation of mechanosensitive signaling pathways. To address the functional coupling of ECM composition and matrix rigidity on compliant substrates, we developed a matrix spot array system to screen cell phenotypes against different ECM mixtures on defined substrate stiffnesses at high resolution. We applied this system to both cancer and normal cells and surprisingly identified ECM mixtures that support stiffness-insensitive cell spreading on soft substrates. Employing the motor-clutch model to simulate cell adhesion on biochemically distinct soft substrates, with varying numbers of available ECM-integrin-cytoskeleton (clutch) connections, we identified conditions in which spreading would be supported on soft matrices. Combining simulations and experiments, we show that cell spreading on soft is supported by increased clutch engagement on specific ECM mixtures and even augmented by the partial inhibition of actomyosin contractility. Thus, "stiff-like" spreading on soft is determined by a balance of a cell's contractile and adhesive machinery. This provides a fundamental perspective for in vitro mechanobiology studies, identifying a mechanism through which cells spread, function, and signal effectively on soft substrates.

TrackMate 7: integrating state-of-the-art segmentation algorithms into tracking pipelines.

TrackMate is an automated tracking software used to analyze bioimages and is distributed as a Fiji plugin. Here, we introduce a new version of TrackMate. TrackMate 7 is built to address the broad spectrum of modern challenges researchers face by integrating state-of-the-art segmentation algorithms into tracking pipelines. We illustrate qualitatively and quantitatively that these new capabilities function effectively across a wide range of bio-imaging experiments.

Insights into a viral motor: the structure of the HK97 packaging termination assembly.

Double-stranded DNA viruses utilise machinery, made of terminase proteins, to package viral DNA into the capsid. For cos bacteriophage, a defined signal, recognised by small terminase, flanks each genome unit. Here we present the first structural data for a cos virus DNA packaging motor, assembled from the bacteriophage HK97 terminase proteins, procapsids encompassing the portal protein, and DNA containing a cos site. The cryo-EM structure is consistent with the packaging termination state adopted after DNA cleavage, with DNA density within the large terminase assembly ending abruptly at the portal protein entrance. Retention of the large terminase complex after cleavage of the short DNA substrate suggests that motor dissociation from the capsid requires headful pressure, in common with pac viruses. Interestingly, the clip domain of the 12-subunit portal protein does not adhere to C12 symmetry, indicating asymmetry induced by binding of the large terminase/DNA. The motor assembly is also highly asymmetric, showing a ring of 5 large terminase monomers, tilted against the portal. Variable degrees of extension between N- and C-terminal domains of individual subunits suggest a mechanism of DNA translocation driven by inter-domain contraction and relaxation.

Structural basis of DNA packaging by a ring-type ATPase from an archetypal viral system.

Many essential cellular processes rely on substrate rotation or translocation by a multi-subunit, ring-type NTPase. A large number of double-stranded DNA viruses, including tailed bacteriophages and herpes viruses, use a homomeric ring ATPase to processively translocate viral genomic DNA into procapsids during assembly. Our current understanding of viral DNA packaging comes from three archetypal bacteriophage systems: cos, pac and phi29. Detailed mechanistic understanding exists for pac and phi29, but not for cos. Here, we reconstituted in vitro a cos packaging system based on bacteriophage HK97 and provided a detailed biochemical and structural description. We used a photobleaching-based, single-molecule assay to determine the stoichiometry of the DNA-translocating ATPase large terminase. Crystal structures of the large terminase and DNA-recruiting small terminase, a first for a biochemically defined cos system, reveal mechanistic similarities between cos and pac systems. At the same time, mutational and biochemical analyses indicate a new regulatory mechanism for ATPase multimerization and coordination in the HK97 system. This work therefore establishes a framework for studying the evolutionary relationships between ATP-dependent DNA translocation machineries in double-stranded DNA viruses.

High-resolution asymmetric structure of a Fab-virus complex reveals overlap with the receptor binding site.

Canine parvovirus is an important pathogen causing severe diseases in dogs, including acute hemorrhagic enteritis, myocarditis, and cerebellar disease. Overlap on the surface of parvovirus capsids between the antigenic epitope and the receptor binding site has contributed to cross-species transmission, giving rise to closely related variants. It has been shown that Mab 14 strongly binds and neutralizes canine but not feline parvovirus, suggesting this antigenic site also controls species-specific receptor binding. To visualize the conformational epitope at high resolution, we solved the cryogenic electron microscopy (cryo-EM) structure of the Fab-virus complex. We also created custom software, Icosahedral Subparticle Extraction and Correlated Classification, to solve a Fab-virus complex with only a few Fab bound per capsid and visualize local structures of the Fab-bound and -unbound antigenic sites extracted from the same complex map. Our results identified the antigenic epitope that had significant overlap with the receptor binding site, and the structures revealed that binding of Fab induced conformational changes to the virus. We were also able to assign the order and position of attached Fabs to allow assessment of complementarity between the Fabs bound to different positions. This approach therefore provides a method for using cryo-EM to investigate complementarity of antibody binding.

LEGACY: Phase 2a Trial to Evaluate the Safety, Pharmacokinetics, and Pharmacodynamic Effects of the Anti-EL (Endothelial Lipase) Antibody MEDI5884 in Patients With Stable Coronary Artery Disease.

OBJECTIVE: Functional HDL (high-density lipoprotein) particles that facilitate cholesterol efflux may be cardioprotective. EL (endothelial lipase) hydrolyzes phospholipids promoting catabolism of HDL and subsequent renal excretion. MEDI5884 is a selective, humanized, monoclonal, EL-neutralizing antibody. We sought to determine the safety, pharmacokinetics, and pharmacodynamic effects of multiple doses of MEDI5884 in patients with stable coronary artery disease. Approach and Results: LEGACY was a phase 2a, double-blind, placebo-controlled, parallel-design trial that randomized 132 patients with stable coronary artery disease receiving high-intensity statin therapy to 3 monthly doses of 1 of 5 dose levels of MEDI5884 (50, 100, 200, 350, or 500 mg SC) or matching placebo. The primary end point was the safety and tolerability of MEDI5884 through the end of the study (day 151). Additional end points included change in HDL cholesterol and cholesterol efflux from baseline to day 91, hepatic uptake of cholesterol at day 91, changes in various other lipid parameters. The incidence of adverse events was similar between the placebo and MEDI5884 groups. In a dose-dependent manner, MEDI5884 increased HDL cholesterol up to 51.4% (P<0.0001) and global cholesterol efflux up to 26.2% ([95% CI, 14.3-38.0] P<0.0001). MEDI5884 increased HDL particle number up to 14.4%. At the highest dose tested, an increase in LDL (low-density lipoprotein) cholesterol up to 28.7% (P<0.0001) and apoB (apolipoprotein B) up to 13.1% (P=0.04) was observed with MEDI5884. However, at the potential target doses for future studies, there was no meaningful increase in LDL cholesterol or apoB. CONCLUSIONS: Inhibition of EL by MEDI5884 increases the quantity and quality of functional HDL in patients with stable coronary artery disease on high-intensity statin therapy without an adverse safety signal at the likely dose to be used. These data support further clinical investigation. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03351738.

Capsid expansion of bacteriophage T5 revealed by high resolution cryoelectron microscopy.

The large (90-nm) icosahedral capsid of bacteriophage T5 is composed of 775 copies of the major capsid protein (mcp) together with portal, protease, and decoration proteins. Its assembly is a regulated process that involves several intermediates, including a thick-walled round precursor prohead that expands as the viral DNA is packaged to yield a thin-walled and angular mature capsid. We investigated capsid maturation by comparing cryoelectron microscopy (cryo-EM) structures of the prohead, the empty expanded capsid both with and without decoration protein, and the virion capsid at a resolution of 3.8 Å for the latter. We detail the molecular structure of the mcp, its complex pattern of interactions, and their evolution during maturation. The bacteriophage T5 mcp is a variant of the canonical HK97-fold with a high level of plasticity that allows for the precise assembly of a giant macromolecule and the adaptability needed to interact with other proteins and the packaged DNA.

A packing for A-form DNA in an icosahedral virus.

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

Coronavirus Disease 2019 (COVID-19) Pharmacologic Treatments for Children: Research Priorities and Approach to Pediatric Studies.

Clinical trials of pharmacologic treatments of coronavirus disease 2019 (COVID-19) are being rapidly designed and implemented in adults. Children are often not considered during development of novel treatments for infectious diseases until very late. Although children appear to have a lower risk compared with adults of severe COVID-19 disease, a substantial number of children globally will benefit from pharmacologic treatments. It will be reasonable to extrapolate efficacy of most treatments from adult trials to children. Pediatric trials should focus on characterizing a treatment's pharmacokinetics, optimal dose, and safety across the age spectrum. These trials should use an adaptive design to efficiently add or remove arms in what will be a rapidly evolving treatment landscape, and should involve a large number of sites across the globe in a collaborative effort to facilitate efficient implementation. All stakeholders must commit to equitable access to any effective, safe treatment for children everywhere.

Role of the Herpes Simplex Virus CVSC Proteins at the Capsid Portal Vertex.

The packaging of DNA into preformed capsids is a critical step during herpesvirus infection. For herpes simplex virus, this process requires the products of seven viral genes: the terminase proteins pUL15, pUL28, and pUL33; the capsid vertex-specific component (CVSC) proteins pUL17 and pUL25; and the portal proteins pUL6 and pUL32. The pUL6 portal dodecamer is anchored at one vertex of the capsid by interactions with the adjacent triplexes as well as helical density attributed to the pUL17 and pUL25 subunits of the CVSC. To define the roles and structures of the CVSC proteins in virus assembly and DNA packaging, we isolated a number of recombinant viruses expressing pUL25, pUL17, and pUL36 fused with green or red fluorescent proteins as well as viruses with specific deletions in the CVSC genes. Biochemical and structural studies of these mutants demonstrated that (i) four of the helices in the CVSC helix bundle can be attributed to two copies each of pUL36 and pUL25, (ii) pUL17 and pUL6 are required for capsid binding of the terminase complex in the nucleus, (iii) pUL17 is important for determining the site of the first cleavage reaction generating replicated genomes with termini derived from the long-arm component of the herpes simplex virus 1 (HSV-1) genome, (iv) pUL36 serves no direct role in cleavage/packaging, (v) cleavage and stable packaging of the viral genome involve an ordered interaction of the terminase complex and pUL25 with pUL17 at the portal vertex, and (vi) packaging of the viral genome results in a dramatic displacement of the portal.IMPORTANCE Herpes simplex virus 1 (HSV-1) is the causative agent of several pathologies ranging in severity from the common cold sore to life-threatening encephalitic infection. A critical step during productive HSV-1 infection is the cleavage and packaging of replicated, concatemeric viral DNA into preformed capsids. A key knowledge gap is how the capsid engages the replicated viral genome and the subsequent packaging of a unit-length HSV genome. Here, biochemical and structural studies focused on the unique portal vertex of wild-type HSV and packaging mutants provide insights into the mechanism of HSV genome packaging. The significance of our research is in identifying the portal proteins pUL6 and pUL17 as key viral factors for engaging the terminase complex with the capsid and the subsequent cleavage, packaging, and stable incorporation of the viral genome in the HSV-1 capsid.

Mass Incarceration and Children's Health: A State-Level Analysis of Adverse Birth Outcomes and Infant, Child, and Teen Mortality.

Children's health indicators such as mortality and adverse birth outcomes are poorer in the United States than in comparable nations. These measures also show racial inequities within the United States, with Black children experiencing the highest levels. Mass incarceration may partially explain these findings. High incarceration rates can disrupt community functioning, influencing behavior and health. The purpose of the current study was to conduct a macro (state)-level analysis examining whether yearly state incarceration rates predict health outcomes including infant, child, and teen mortality as well as preterm birth and low birth weight. It was hypothesized that prior year incarceration rates would show positive relationships with all outcomes and that relationships would be stronger for Black than for white children. Yearly state-level panel data were gathered from 1990 to 2017. Weighted least squares regression used states' prior year incarceration rates to predict child health outcomes, using controls for overall state effects and year-to-year effects. Time-varying covariates such as state unemployment rate were also included to address the possibility of spurious relationships. Results indicated that as hypothesized, incarceration rates positively predicted infant mortality, child mortality (for Black children only), preterm births, and low-weight births. Relationships tended to be stronger for Black than for white children.

Should Virus Capsids Assemble Perfectly? Theory and Observation of Defects.

Although published structural models of viral capsids generally exhibit a high degree of regularity or symmetry, structural defects might be expected because of the fluctuating environment in which capsids assemble and the requirement of some capsids for disassembly before genome delivery. Defective structures are observed in computer simulations, and are evident in single-particle cryoelectron microscopy studies. Here, we quantify the conditions under which defects might be expected, using a statistical mechanics model allowing for ideal, defective, and vacant sites. The model displays a threshold in affinity parameters below which there is an appreciable population of defective capsids. Even when defective sites are not allowed, there is generally some population of vacancies. Analysis of single particles in cryoelectron microscopy micrographs yields a confirmatory ≳15% of defective particles. Our findings suggest structural heterogeneity in virus capsids may be under-appreciated, and also points to a nontraditional strategy for assembly inhibition.

Mitochondrial fatty acid oxidation and the electron transport chain comprise a multifunctional mitochondrial protein complex.

Three mitochondrial metabolic pathways are required for efficient energy production in eukaryotic cells: the electron transfer chain (ETC), fatty acid ß-oxidation (FAO), and the tricarboxylic acid cycle. The ETC is organized into inner mitochondrial membrane supercomplexes that promote substrate channeling and catalytic efficiency. Although previous studies have suggested functional interaction between FAO and the ETC, their physical interaction has never been demonstrated. In this study, using blue native gel and two-dimensional electrophoreses, nano-LC-MS/MS, immunogold EM, and stimulated emission depletion microscopy, we show that FAO enzymes physically interact with ETC supercomplexes at two points. We found that the FAO trifunctional protein (TFP) interacts with the NADH-binding domain of complex I of the ETC, whereas the electron transfer enzyme flavoprotein dehydrogenase interacts with ETC complex III. Moreover, the FAO enzyme very-long-chain acyl-CoA dehydrogenase physically interacted with TFP, thereby creating a multifunctional energy protein complex. These findings provide a first view of an integrated molecular architecture for the major energy-generating pathways in mitochondria that ensures the safe transfer of unstable reducing equivalents from FAO to the ETC. They also offer insight into clinical ramifications for individuals with genetic defects in these pathways.

Unique expansion of IL-21+ Tfh and Tph cells under control of ICOS identifies Sjögren's syndrome with ectopic germinal centres and MALT lymphoma.

OBJECTIVES: To explore the relevance of T-follicular-helper (Tfh) and pathogenic peripheral-helper T-cells (Tph) in promoting ectopic lymphoid structures (ELS) and B-cell mucosa-associated lymphoid tissue (MALT) lymphomas (MALT-L) in Sjögren's syndrome (SS) patients. METHODS: Salivary gland (SG) biopsies with matched peripheral blood were collected from four centres across the European Union. Transcriptomic (microarray and quantitative PCR) analysis, FACS T-cell immunophenotyping with intracellular cytokine detection, multicolor immune-fluorescence microscopy and in situ hybridisation were performed to characterise lesional and circulating Tfh and Tph-cells. SG-organ cultures were used to investigate functionally the blockade of T-cell costimulatory pathways on key proinflammatory cytokine production. RESULTS: Transcriptomic analysis in SG identified Tfh-signature, interleukin-21 (IL-21) and the inducible T-cell co-stimulator (ICOS) costimulatory pathway as the most upregulated genes in ELS+SS patients, with parotid MALT-L displaying a 400-folds increase in IL-21 mRNA. Peripheral CD4+CXC-motif chemokine receptor 5 (CXCR5)+programmed cell death protein 1 (PD1)+ICOS+ Tfh-like cells were significantly expanded in ELS+SS patients, were the main producers of IL-21, and closely correlated with circulating IgG and reduced complement C4. In the SG, lesional CD4+CD45RO+ICOS+PD1+ cells selectively infiltrated ELS+ tissues and were aberrantly expanded in parotid MALT-L. In ELS+SG and MALT-L parotids, conventional CXCR5+CD4+PD1+ICOS+Foxp3- Tfh-cells and a uniquely expanded population of CXCR5-CD4+PD1hiICOS+Foxp3- Tph-cells displayed frequent IL-21/interferon-γ double-production but poor IL-17 expression. Finally, ICOS blockade in ex vivo SG-organ cultures significantly reduced the production of IL-21 and inflammatory cytokines IL-6, IL-8 and tumour necrosis factor-α (TNF-α). CONCLUSIONS: Overall, these findings highlight Tfh and Tph-cells, IL-21 and the ICOS costimulatory pathway as key pathogenic players in SS immunopathology and exploitable therapeutic targets in SS.

GDNF/RET signaling in dopamine neurons in vivo.

The glial cell line-derived neurotrophic factor (GDNF) and its canonical receptor Ret can signal both in tandem and separately to exert many vital functions in the midbrain dopamine system. It is known that Ret has effects on maintenance, physiology, protection and regeneration in the midbrain dopamine system, with the physiological functions of GDNF still somewhat unclear. Notwithstanding, Ret ligands, such as GDNF, are considered as promising candidates for neuroprotection and/or regeneration in Parkinson's disease, although data from clinical trials are so far inconclusive. In this review, we discuss the current knowledge of GDNF/Ret signaling in the dopamine system in vivo as well as crosstalk with pathology-associated proteins and their signaling in mammals.

Two Cases Illustrating the Diagnostic Challenge of Pediatric Blastomycosis Presenting as Osteomyelitis.

Blastomyces dermatitidis is a dimorphic fungus endemic to the United States and Canada. Although both Histoplasma and Blastomyces are found in similar geographic regions, Blastomyces is many times more likely to cause dissemination in the immunocompetent host, frequently involving the bone. However, given the indolent nature of this fungal infection and more prevalent bacterial etiologies of osteomyelitis, diagnosis and treatment are often significantly delayed. We review 2 pediatric cases that initially presented with isolated orthopedic symptoms without documented fever or pulmonary complaints, although both had signs of pulmonary infection on imaging. These cases demonstrate the importance of a high level of suspicion as well as appropriate diagnostic workup, including surgical pathology with fungal stains, when evaluating osteomyelitis in patients exposed to a Blastomyces-endemic region.

Combating pancreatic cancer with PI3K pathway inhibitors in the era of personalised medicine.

Pancreatic ductal adenocarcinoma (PDAC) is among the most deadly solid tumours. This is due to a generally late-stage diagnosis of a primarily treatment-refractory disease. Several large-scale sequencing and mass spectrometry approaches have identified key drivers of this disease and in doing so highlighted the vast heterogeneity of lower frequency mutations that make clinical trials of targeted agents in unselected patients increasingly futile. There is a clear need for improved biomarkers to guide effective targeted therapies, with biomarker-driven clinical trials for personalised medicine becoming increasingly common in several cancers. Interestingly, many of the aberrant signalling pathways in PDAC rely on downstream signal transduction through the mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K) pathways, which has led to the development of several approaches to target these key regulators, primarily as combination therapies. The following review discusses the trend of PDAC therapy towards molecular subtyping for biomarker-driven personalised therapies, highlighting the key pathways under investigation and their relationship to the PI3K pathway.

The Apical Region of the Herpes Simplex Virus Major Capsid Protein Promotes Capsid Maturation.

The herpesvirus capsid assembles in the nucleus as an immature procapsid precursor built around viral scaffold proteins. The event that initiates procapsid maturation is unknown, but it is dependent upon activation of the VP24 internal protease. Scaffold cleavage triggers angularization of the shell and its decoration with the VP26 and pUL25 capsid-surface proteins. In both the procapsid and mature angularized capsid, the apical region of the major capsid protein (VP5) is surface exposed. We investigated whether the VP5 apical region contributes to intracellular transport dynamics following entry into primary sensory neurons and also tested the hypothesis that conserved negatively charged amino acids in the apical region contribute to VP26 acquisition. To our surprise, neither hypothesis proved true. Instead, mutation of glutamic acid residues in the apical region delayed viral propagation and induced focal capsid accumulations in nuclei. Examination of capsid morphogenesis based on epitope unmasking, capsid composition, and ultrastructural analysis indicated that these clusters consisted of procapsids. The results demonstrate that, in addition to established events that occur inside the capsid, the exterior capsid shell promotes capsid morphogenesis and maturation.IMPORTANCE Herpesviruses assemble capsids and encapsidate their genomes by a process that is unlike those of other mammalian viruses but is similar to those of some bacteriophage. Many important aspects of herpesvirus morphogenesis remain enigmatic, including how the capsid shell matures into a stable angularized configuration. Capsid maturation is triggered by activation of a protease that cleaves an internal protein scaffold. We report on the fortuitous discovery that a region of the major capsid protein that is exposed on the outer surface of the capsid also contributes to capsid maturation, demonstrating that the morphogenesis of the capsid shell from its procapsid precursor to the mature angularized form is dependent upon internal and external components of the megastructure.

The C Terminus of the Herpes Simplex Virus UL25 Protein Is Required for Release of Viral Genomes from Capsids Bound to Nuclear Pores.

The herpes simplex virus (HSV) capsid is released into the cytoplasm after fusion of viral and host membranes, whereupon dynein-dependent trafficking along microtubules targets it to the nuclear envelope. Binding of the capsid to the nuclear pore complex (NPC) is mediated by the capsid protein pUL25 and the capsid-tethered tegument protein pUL36. Temperature-sensitive mutants in both pUL25 and pUL36 dock at the NPC but fail to release DNA. The uncoating reaction has been difficult to study due to the rapid release of the genome once the capsid interacts with the nuclear pore. In this study, we describe the isolation and characterization of a truncation mutant of pUL25. Live-cell imaging and immunofluorescence studies demonstrated that the mutant was not impaired in penetration of the host cell or in trafficking of the capsid to the nuclear membrane. However, expression of viral proteins was absent or significantly delayed in cells infected with the pUL25 mutant virus. Transmission electron microscopy revealed capsids accumulated at nuclear pores that retained the viral genome for at least 4 h postinfection. In addition, cryoelectron microscopy (cryo-EM) reconstructions of virion capsids did not detect any obvious differences in the location or structural organization for the pUL25 or pUL36 proteins on the pUL25 mutant capsids. Further, in contrast to wild-type virus, the antiviral response mediated by the viral DNA-sensing cyclic guanine adenine synthase (cGAS) was severely compromised for the pUL25 mutant. These results demonstrate that the pUL25 capsid protein has a critical role in releasing viral DNA from NPC-bound capsids.IMPORTANCE Herpes simplex virus 1 (HSV-1) is the causative agent of several pathologies ranging in severity from the common cold sore to life-threatening encephalitic infection. Early steps in infection include release of the capsid into the cytoplasm, docking of the capsid at a nuclear pore, and release of the viral genome into the nucleus. A key knowledge gap is how the capsid engages the NPC and what triggers release of the viral genome into the nucleus. Here we show that the C-terminal region of the HSV-1 pUL25 protein is required for releasing the viral genome from capsids docked at nuclear pores. The significance of our research is in identifying pUL25 as a key viral factor for genome uncoating. pUL25 is found at each of the capsid vertices as part of the capsid vertex-specific component and implicates the importance of this complex for NPC binding and genome release.