RESUMEN
Herpes simplex virus 1 (HSV-1) requires seven proteins to package its genome through a vertex in its capsid, one of which is the portal protein, pUL6. The portal protein is also thought to facilitate assembly of the procapsid. While the portal has been visualized in mature capsids, we aimed to elucidate its role in the assembly and maturation of procapsids using cryo-electron tomography (cryoET). We identified the portal vertex in individual procapsids, calculated a subtomogram average, and compared that with the portal vertex in empty mature capsids (A-capsids). The resulting maps show the portal on the interior surface with its narrower end facing outwards, while maintaining close contact with the capsid shell. In the procapsid, the portal is embedded in the underlying scaffold, suggesting that assembly involves a portal-scaffold complex. During maturation, the capsid shell angularizes with a corresponding outward movement of the vertices. We found that in A-capsids, the portal translocates outward further than the adjacent capsomers and strengthens its contacts with the capsid shell. Our methodology also allowed us to determine the number of portal vertices in each capsid, with most having one per capsid, but some none or two, and rarely three. The predominance of a single portal per capsid supports facilitation of the assembly of the procapsid.IMPORTANCE Herpes simplex virus 1 (HSV-1) infects a majority of humans, causing mostly mild disease but in some cases progressing toward life-threatening encephalitis. Understanding the life cycle of the virus is important to devise countermeasures. Production of the virion starts with the assembly of an icosahedral procapsid, which includes DNA packaging proteins at a vertex, one of which is the dodecameric portal protein. The procapsid then undergoes maturation and DNA packaging through the portal, driven by a terminase complex. We used cryo-electron tomography to visualize the portal in procapsids and compare them to mature empty capsids. We found the portal located inside one vertex interacting with the scaffold protein in the procapsid. On maturation, the scaffold is cleaved and dissociates, the capsid angularizes, and the portal moves outward, interacting closely with the capsid shell. These transformations may provide a basis for the development of drugs to prevent HSV-1 infections.
Asunto(s)
Cápside/metabolismo , Cápside/ultraestructura , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Herpesvirus Humano 1/ultraestructura , Proteínas Virales/metabolismo , Ensamble de Virus , Proteínas de la Cápside/genética , Herpesvirus Humano 1/metabolismoRESUMEN
"Giant" phages have genomes of >200 kbp, confined in correspondingly large capsids whose assembly and maturation are still poorly understood. Nevertheless, the first assembly product is likely to be, as in other tailed phages, a procapsid that subsequently matures and packages the DNA. The associated transformations include the cleavage of many proteins by the phage-encoded protease, as well as the thinning and angularization of the capsid. We exploited an amber mutation in the viral protease gene of the Salmonella giant phage SPN3US, which leads to the accumulation of a population of capsids with distinctive properties. Cryo-electron micrographs reveal patterns of internal density different from those of the DNA-filled heads of virions, leading us to call them "mottled capsids". Reconstructions show an outer shell with T = 27 symmetry, an embellishment of the HK97 prototype composed of the major capsid protein, gp75, which is similar to some other giant viruses. The mottled capsid has a T = 1 inner icosahedral shell that is a complex network of loosely connected densities composed mainly of the ejection proteins gp53 and gp54. Segmentation of this inner shell indicated that a number of densities (~12 per asymmetric unit) adopt a "twisted hook" conformation. Large patches of a proteinaceous tetragonal lattice with a 67 Å repeat were also present in the cell lysate. The unexpected nature of these novel inner shell and lattice structures poses questions as to their functions in virion assembly.
Asunto(s)
Cápside/metabolismo , Virus Gigantes/fisiología , Fagos de Salmonella/fisiología , Ensamble de Virus , Cápside/ultraestructura , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Microscopía por Crioelectrón , Empaquetamiento del ADN , Genoma Viral , Virus Gigantes/genética , Virus Gigantes/ultraestructura , Salmonella/virología , Fagos de Salmonella/genética , Fagos de Salmonella/ultraestructura , Virión/genética , Virión/fisiología , Virión/ultraestructuraRESUMEN
The 240-kb Salmonella phage SPN3US genome encodes 264 gene products, many of which are functionally uncharacterized. We have previously used mass spectrometry to define the proteomes of wild-type and mutant forms of the SPN3US virion. In this study, we sought to determine whether this technique was suitable for the characterization of the SPN3US proteome during liquid infection. Mass spectrometry of SPN3US-infected cells identified 232 SPN3US and 1,994 Salmonella proteins. SPN3US proteins with related functions, such as proteins with roles in DNA replication, transcription, and virion formation, were coordinately expressed in a temporal manner. Mass spectral counts showed the four most abundant SPN3US proteins to be the major capsid protein, two head ejection proteins, and the functionally unassigned protein gp22. This high abundance of gp22 in infected bacteria contrasted with its absence from mature virions, suggesting that it might be the scaffold protein, an essential head morphogenesis protein yet to be identified in giant phages. We identified homologs to SPN3US gp22 in 45 related giant phages, including ÏKZ, whose counterpart is also abundant in infected bacteria but absent in the virion. We determined the ÏKZ counterpart to be cleaved in vitro by its prohead protease, an event that has been observed to promote head maturation of some other phages. Our findings are consistent with a scaffold protein assignment for SPN3US gp22, although direct evidence is required for its confirmation. These studies demonstrate the power of mass spectral analyses for facilitating the acquisition of new knowledge into the molecular events of viral infection.IMPORTANCE "Giant" phages with genomes >200 kb are being isolated in increasing numbers from a range of environments. With hosts such as Salmonella enterica, Pseudomonas aeruginosa, and Erwinia amylovora, these phages are of interest for phage therapy of multidrug-resistant pathogens. However, our understanding of how these complex phages interact with their hosts is impeded by the proportion (â¼80%) of their gene products that are functionally uncharacterized. To develop the repertoire of techniques for analysis of phages, we analyzed a liquid infection of Salmonella phage SPN3US (240-kb genome) using third-generation mass spectrometry. We observed the temporal production of phage proteins whose genes collectively represent 96% of the SPN3US genome. These findings demonstrate the sensitivity of mass spectrometry for global proteomic profiling of virus-infected cells, and the identification of a candidate for a major head morphogenesis protein will facilitate further studies into giant phage head assembly.
Asunto(s)
Virus Gigantes/genética , Glicoproteínas/genética , Proteoma/análisis , Fagos de Salmonella/genética , Salmonella typhimurium/virología , Proteínas Virales/genética , ADN Viral/genética , Perfilación de la Expresión Génica , Genoma Viral/genética , Espectrometría de Masas , Pseudomonas aeruginosa/virologíaRESUMEN
Many viruses migrate between different cellular compartments for successive stages of assembly. The HSV-1 capsid assembles in the nucleus and then transfers into the cytoplasm. First, the capsid buds through the inner nuclear membrane, becoming coated with nuclear egress complex (NEC) protein. This yields a primary enveloped virion (PEV) whose envelope fuses with the outer nuclear membrane, releasing the capsid into the cytoplasm. We investigated the associated molecular mechanisms by isolating PEVs from US3-null-infected cells and imaging them by cryo-electron microscopy and tomography. (pUS3 is a viral protein kinase in whose absence PEVs accumulate in the perinuclear space.) Unlike mature extracellular virions, PEVs have very few glycoprotein spikes. PEVs are ~20% smaller than mature virions, and the little space available between the capsid and the NEC layer suggests that most tegument proteins are acquired later in the egress pathway. Previous studies have proposed that NEC is organized as hexamers in honeycomb arrays in PEVs, but we find arrays of heptameric rings in extracts from US3-null-infected cells. In a PEV, NEC contacts the capsid predominantly via the pUL17/pUL25 complexes which are located close to the capsid vertices. Finally, the NEC layer dissociates from the capsid as it leaves the nucleus, possibly in response to pUS3-mediated phosphorylation. Overall, nuclear egress emerges as a process driven by a program of multiple weak interactions.IMPORTANCE On its maturation pathway, the newly formed HSV-1 nucleocapsid must traverse the nuclear envelope, while respecting the integrity of that barrier. Nucleocapsids (125 nm in diameter) are too large to pass through the nuclear pore complexes that conduct most nucleocytoplasmic traffic. It is now widely accepted that the process involves envelopment/de-envelopment of a key intermediate-the primary enveloped virion. In wild-type infections, PEVs are short-lived, which has impeded study. Using a mutant that accumulates PEVs in the perinuclear space, we were able to isolate PEVs in sufficient quantity for structural analysis by cryo-electron microscopy and tomography. The findings not only elucidate the maturation pathway of an important human pathogen but also have implications for cellular processes that involve the trafficking of large macromolecular complexes.
Asunto(s)
Herpesvirus Humano 1/fisiología , Virión/fisiología , Liberación del Virus , Animales , Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Núcleo Celular , Chlorocebus aethiops , Microscopía por Crioelectrón , Herpesvirus Humano 1/genética , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Fosforilación , Células Vero , Proteínas Virales/genética , Proteínas Virales/metabolismo , Ensamble de VirusRESUMEN
UNLABELLED: The herpes simplex virus 1 (HSV-1) capsid is a huge assembly, â¼1,250 Å in diameter, and is composed of thousands of protein subunits with a combined mass of â¼200 MDa, housing a 100-MDa genome. First, a procapsid is formed through coassembly of the surface shell with an inner scaffolding shell; then the procapsid matures via a major structural transformation, triggered by limited proteolysis of the scaffolding proteins. Three mature capsids are found in the nuclei of infected cells. A capsids are empty, B capsids retain a shrunken scaffolding shell, and C capsids-which develop into infectious virions-are filled with DNA and ostensibly have expelled the scaffolding shell. The possible presence of other internal proteins in C capsids has been moot as, in cryo-electron microscopy (cryo-EM), they would be camouflaged by the surrounding DNA. We have used bubblegram imaging to map internal proteins in all four capsids, aided by the discovery that the scaffolding protein is exceptionally prone to radiation-induced bubbling. We confirmed that this protein forms thick-walled inner shells in the procapsid and the B capsid. C capsids generate two classes of bubbles: one occupies positions beneath the vertices of the icosahedral surface shell, and the other is distributed throughout its interior. A likely candidate is the viral protease. A subpopulation of C capsids bubbles particularly profusely and may represent particles in which expulsion of scaffold and DNA packaging are incomplete. Based on the procapsid structure, we propose that the axial channels of hexameric capsomers afford the pathway via which the scaffolding protein is expelled. IMPORTANCE: In addition to DNA, capsids of tailed bacteriophages and their distant relatives, herpesviruses, contain internal proteins. These proteins are often essential for infectivity but are difficult to locate within the virion. A novel adaptation of cryo-EM based on detecting gas bubbles generated by radiation damage was used to localize internal proteins of HSV-1, yielding insights into how capsid maturation is regulated. The scaffolding protein, which forms inner shells in the procapsid and B capsid, is exceptionally bubbling-prone. In the mature DNA-filled C capsid, a previously undetected protein was found to underlie the icosahedral vertices: this is tentatively assigned as a storage form of the viral protease. We also observed a capsid species that appears to contain substantial amounts of scaffolding protein as well as DNA, suggesting that DNA packaging and expulsion of the scaffolding protein are coupled processes.
Asunto(s)
Proteínas de la Cápside/química , Cápside/química , Cápside/ultraestructura , Herpesvirus Humano 1/ultraestructura , Cápside/metabolismo , Microscopía por Crioelectrón/instrumentación , Microscopía por Crioelectrón/métodos , Empaquetamiento del ADN , Herpesvirus Humano 1/química , Virión , Ensamble de VirusRESUMEN
UNLABELLED: The herpes simplex virus 1 (HSV-1) capsid is a massive particle (~200 MDa; 1,250-Å diameter) with T=16 icosahedral symmetry. It initially assembles as a procapsid with ~4,000 protein subunits of 11 different kinds. The procapsid undergoes major changes in structure and composition as it matures, a process driven by proteolysis and expulsion of the internal scaffolding protein. Assembly also relies on an external scaffolding protein, the triplex, an α2ß heterotrimer that coordinates neighboring capsomers in the procapsid and becomes a stabilizing clamp in the mature capsid. To investigate the mechanisms that regulate its assembly, we developed a novel isolation procedure for the metastable procapsid and collected a large set of cryo-electron microscopy data. In addition to procapsids, these preparations contain maturation intermediates, which were distinguished by classifying the images and calculating a three-dimensional reconstruction for each class. Appraisal of the procapsid structure led to a new model for assembly; in it, the protomer (assembly unit) consists of one triplex, surrounded by three major capsid protein (MCP) subunits. The model exploits the triplexes' departure from 3-fold symmetry to explain the highly skewed MCP hexamers, the triplex orientations at each 3-fold site, and the T=16 architecture. These observations also yielded new insights into maturation. IMPORTANCE: This paper addresses the molecular mechanisms that govern the self-assembly of large, structurally complex, macromolecular particles, such as the capsids of double-stranded DNA viruses. Although they may consist of thousands of protein subunits of many different kinds, their assembly is precise, ranking them among the largest entities in the biosphere whose structures are uniquely defined to the atomic level. Assembly proceeds in two stages: formation of a precursor particle (procapsid) and maturation, during which major changes in structure and composition take place. Our analysis of the HSV procapsid by cryo-electron microscopy suggests a hierarchical pathway in which multisubunit "protomers" are the building blocks of the procapsid but their subunits are redistributed into different subcomplexes upon being incorporated into a nascent procapsid and are redistributed again in maturation. Assembly is a highly virus-specific process, making it a potential target for antiviral intervention.
Asunto(s)
Cápside/metabolismo , Simplexvirus/fisiología , Ensamble de Virus , Cápside/ultraestructura , Microscopía por Crioelectrón , Modelos Moleculares , Multimerización de Proteína , Simplexvirus/ultraestructuraRESUMEN
A previous study used a PortaCount Plus to measure the ratio of particle concentrations outside (C out) to inside (C in) of filtering facepiece respirators (FFRs) worn by test subjects and calculated the total inward leakage (TIL) (C in/C out) to evaluate the reproducibility of the TIL test method between two different National Institute for Occupational Safety and Health laboratories (Laboratories 1 and 2) at the Pittsburgh Campus. The purpose of this study is to utilize the originally obtained PortaCount C out/C in ratio as a measure of protection factor (PF) and evaluate the influence of particle distribution and filter efficiency. PFs were obtained for five N95 model FFRs worn by 35 subjects for three donnings (5 models × 35 subjects × 3 donnings) for a total of 525 tests in each laboratory. The geometric mean of PFs, geometric standard deviation (GSD), and the 5th percentile values for the five N95 FFR models were calculated for the two laboratories. Filter efficiency was obtained by measuring the penetration for four models (A, B, C, and D) against Laboratory 2 aerosol using two condensation particle counters. Particle size distribution, measured using a Scanning Mobility Particle Sizer, showed a mean count median diameter (CMD) of 82 nm in Laboratory 1 and 131 nm in Laboratory 2. The smaller CMD showed relatively higher concentration of nanoparticles in Laboratory 1 than in Laboratory 2. Results showed that the PFs and 5th percentile values for two models (B and E) were larger than other three models (A, C, and D) in both laboratories. The PFs and 5th percentile values of models B and E in Laboratory 1 with a count median diameter (CMD) of 82 nm were smaller than in Laboratory 2 with a CMD of 131 nm, indicating an association between particle size distribution and PF. The three lower efficiency models (A, C, and D) showed lower PF values than the higher efficiency model B showing the influence of filter efficiency on PF value. Overall, the data show that particle size distribution and filter efficiency influence the PFs and 5th percentile values. The PFs and 5th percentile values decreased with increasing nanoparticle concentration (from CMD of 131 to 82 nm) indicating lower PFs for aerosol distribution within nanoparticle size range (<100 nm). Further studies on the relationship between particle size distribution and PF are needed to better understand the respiratory protection against nanoparticles.
Asunto(s)
Aerosoles/análisis , Contaminantes Ocupacionales del Aire/análisis , Filtración/instrumentación , Nanopartículas/análisis , Dispositivos de Protección Respiratoria/normas , Diseño de Equipo/normas , Filtración/normas , Humanos , Exposición por Inhalación/análisis , Exposición por Inhalación/prevención & control , Ensayo de Materiales/métodos , National Institute for Occupational Safety and Health, U.S. , Exposición Profesional/prevención & control , Tamaño de la Partícula , Reproducibilidad de los Resultados , Estados UnidosRESUMEN
National Institute for Occupational Safety and Health (NIOSH) certified particulate respirators need to be properly fit tested before use to ensure workers' respiratory protection. However, the effectiveness of American National Standards Institute-/Occupational Safety and Health Administration (ANSI-/OSHA)-accepted fit tests for particulate respirators in predicting actual workplace protection provided to workers is lacking. NIOSH addressed this issue by evaluating the fit of half-mask particulate filtering respirators as a component of a program designed to add total inward leakage (TIL) requirements for all respirators to Title 42 Code of Federal Regulations Part 84. Specifically, NIOSH undertook a validation study to evaluate the reproducibility of the TIL test procedure between two laboratories. A PortaCount® was used to measure the TIL of five N95 model filtering facepiece respirators (FFRs) on test subjects in two different laboratories. Concurrently, filter efficiency for four of the five N95 FFR models was measured using laboratory aerosol as well as polydisperse NaCl aerosol employed for NIOSH particulate respirator certification. Results showed that two N95 models passed the TIL tests at a rate of ~80-85% and ~86-94% in the two laboratories, respectively. However, the TIL passing rate for the other three N95 models was 0-5.7% in both laboratories combined. Good agreement (≥83%) of the TIL data between the two laboratories was obtained. The three models that had relatively lower filter efficiency for laboratory aerosol as well as for NaCl aerosol showed relatively low TIL passing rates in both laboratories. Of the four models tested for penetration, one model with relatively higher efficiency showed a higher passing rate for TIL tests in both laboratories indicating that filter efficiency might influence TIL. Further studies are needed to better understand the implications of the data in the workplace.
Asunto(s)
Contaminantes Ocupacionales del Aire/análisis , Falla de Equipo , Filtración/instrumentación , Exposición por Inhalación/prevención & control , Máscaras/normas , Ensayo de Materiales/métodos , Dispositivos de Protección Respiratoria/normas , Aerosoles , Diseño de Equipo/normas , Humanos , Exposición por Inhalación/análisis , National Institute for Occupational Safety and Health, U.S. , Exposición Profesional/prevención & control , Salud Laboral , Tamaño de la Partícula , Reproducibilidad de los Resultados , Estados UnidosRESUMEN
A growing number of reports indicate the frequent presence of DNA sequences and gene products of human cytomegalovirus in various tumors as compared to adjacent normal tissues, the brain tumors being studied most intensely. The mechanisms underlying the tropism of human cytomegalovirus to the tumor cells or to the cells of tumor origin, as well as the role of the host's genetic background in virus-associated oncogenesis are not well understood. It is also not clear why cytomegalovirus can be detected in many but not in all tumor specimens. Our in silico prediction results indicate that microRNA-34a may be involved in replication of some human DNA viruses by targeting and downregulating the genes encoding a diverse group of proteins, such as platelet-derived growth factor receptor-alpha, complement component receptor 2, herpes simplex virus entry mediators A, B, and C, and CD46. Notably, while their functions vary, these surface molecules have one feature in common: they serve as cellular entry receptors for human DNA viruses (cytomegalovirus, Epstein-Barr virus, human herpes virus 6, herpes simplex viruses 1 and 2, and adenoviruses) that are either proven or suspected to be linked with malignancies. MicroRNA-34a is strictly dependent on its transcriptional activator tumor suppressor protein p53, and both p53 and microRNA-34a are frequently mutated or downregulated in various cancers. We hypothesize that p53-microRNA-34a axis may alter susceptibility of cells to infection with some viruses that are detected in tumors and either proven or suspected to be associated with tumor initiation and progression.
Asunto(s)
Fusión Celular , Endocitosis , Herpesviridae/fisiología , MicroARNs/fisiología , Proteína p53 Supresora de Tumor/fisiología , HumanosRESUMEN
Previous studies have shown a sampling probe bias for measuring fit factors (FFs) in respirator facepieces. This study was conducted to evaluate three sampling probes for fit testing NIOSH-certified N95 filtering facepiece respirators (FFRs). Two phases of fit test experiments were conducted incorporating 'side-by-side' probe mounting: (i) flush probe versus deep probe and (ii) flush probe versus disc probe. Seven test subjects in Phase 1 and six subjects in Phase 2 were fit tested with one to three N95 FFR models for a total of 10 subject/FFR model combinations for each phase. For each experimental condition, induced faceseal leakage (IFSL) through an induced leak was measured using a PORTACOUNT® Plus model 8020A Respirator Fit Tester with a model 8095 N95-Companion™ accessory. For Phase 1, the mean IFSL of all flush probe measurements (3.6%) was significantly greater than (P < 0.05) the mean IFSL of all deep probe measurements (3.3%). For Phase 2, the mean IFSL of all flush probe measurements (8.5%) was not significantly greater than (P > 0.05) the mean IFSL of all disc probe measurements (8.3%). Results indicate that some leak site and subject/FFR model/leak site combination comparisons (flush probe versus deep probe or flush probe versus disc probe) were statistically different (P < 0.05). The overall mean IFSL for subject/FFR model/leak site combinations differed by 14 and 4% for the flush probe versus deep probe and the flush probe versus disc probe, respectively; however, from a practical standpoint, there is little difference between the flush probe tests compared with the deep probe or disc probe tests. Overall, IFSL measured using the flush probe is higher (resulting in a more conservative measure of faceseal leakage) compared with either the deep probe or disc probe. The more conservative results obtained using the flush probe provide support for its common usage for fit testing cup-shaped FFRs in the USA and potential use for fit testing FFRs in Europe.
Asunto(s)
Filtración/instrumentación , Ensayo de Materiales/métodos , Dispositivos de Protección Respiratoria/normas , Adolescente , Adulto , Diseño de Equipo/normas , Femenino , Filtración/normas , Humanos , Masculino , Persona de Mediana Edad , National Institute for Occupational Safety and Health, U.S. , Exposición Profesional/análisis , Estados Unidos , Adulto JovenRESUMEN
Herpes simplex virus 1 (HSV-1) was shown to contain catalase, an enzyme able to detoxify hydrogen peroxide by converting it to water and oxygen. Studies with a catalase inhibitor indicated that virus-associated catalase can have a role in protecting the virus from oxidative inactivation. HSV-1 was found to be more sensitive to killing by hydrogen peroxide in the presence of a catalase inhibitor than in its absence. The results suggest a protective role for catalase during the time HSV-1 spends in the oxidizing environment outside a host cell.
Asunto(s)
Catalasa/metabolismo , Desinfectantes/metabolismo , Desinfectantes/toxicidad , Herpesvirus Humano 1/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/toxicidad , Proteínas Virales/metabolismo , Animales , Chlorocebus aethiops , Inhibidores Enzimáticos/metabolismo , Viabilidad Microbiana/efectos de los fármacos , Oxígeno/metabolismo , Células Vero , Carga Viral , Inactivación de Virus/efectos de los fármacos , Agua/metabolismoRESUMEN
Initiation of infection by herpes family viruses involves a step in which most of the virus tegument becomes detached from the capsid. Detachment takes place in the host cell cytosol near the virus entry site and it is followed by dispersal of tegument proteins and disappearance of the tegument as a distinct entity. Here we describe the results of experiments designed to test the idea that the reducing environment of the cytosol may contribute to tegument detachment and disassembly. Non-ionic detergent was used to remove the membrane of purified herpes simplex virus under control and reducing conditions. The effects on the tegument were then examined by SDS-PAGE and electron microscopy. Protein analysis demonstrated that most major tegument proteins were removed under both oxidizing and reducing conditions except for UL49 which required a reducing environment. It is proposed therefore that the reducing conditions in the cytosol are involved in removal of UL49 protein. Electron microscopic analysis revealed that capsids produced under oxidizing conditions contained a coating of protein that was absent in reduced virions and which correlated uniquely with the presence of UL49. This capsid-associated layer is suggested to be the location of UL49 in the extracted virion.
Asunto(s)
Cápside/metabolismo , Citosol/química , Herpesviridae/fisiología , Proteínas Estructurales Virales/metabolismo , Internalización del Virus , Electroforesis en Gel de Poliacrilamida , Herpesviridae/ultraestructura , Microscopía Electrónica , Oxidación-ReducciónRESUMEN
In the final stages of the herpes simplex virus 1 (HSV-1) life cycle, a viral nucleocapsid buds into a vesicle of trans-Golgi network (TGN)/endosome origin, acquiring an envelope and an outer vesicular membrane. The virus-containing vesicle then traffics to the plasma membrane where it fuses, exposing a mature virion. Although the process of directed egress has been studied in polarized epithelial cell lines, less work has been done in nonpolarized cell types. In this report, we describe a study of HSV-1 egress as it occurs in nonpolarized cells. The examination of infected Vero cells by electron, confocal, and total internal reflection fluorescence (TIRF) microscopy revealed that HSV-1 was released at specific pocket-like areas of the plasma membrane that were found along the substrate-adherent surface and cell-cell-adherent contacts. Both the membrane composition and cytoskeletal structure of egress sites were found to be modified by infection. The plasma membrane at virion release sites was heavily enriched in viral glycoproteins. Small glycoprotein patches formed early in infection, and virus became associated with these areas as they expanded. Glycoprotein-rich areas formed independently from virion trafficking as confirmed by the use of a UL25 mutant with a defect in capsid nuclear egress. The depolymerization of the cytoskeleton indicated that microtubules were important for the trafficking of virions and glycoproteins to release sites. In addition, the actin cytoskeleton was found to be necessary for maintaining the integrity of egress sites. When actin was depolymerized, the glycoprotein concentrations dispersed across the membrane, as did the surface-associated virus. Lastly, viral glycoprotein E appeared to function in a different manner in nonpolarized cells compared to previous studies of egress in polarized epithelial cells; the total amount of virus released at egress sites was slightly increased in infected Vero cells when gE was absent. However, gE was important for egress site formation, as Vero cells infected with gE deletion mutants formed glycoprotein patches that were significantly reduced in size. The results of this study are interpreted to indicate that the egress of HSV-1 in Vero cells is directed to virally induced, specialized egress sites that form along specific areas of the cell membrane.
Asunto(s)
Membrana Celular/virología , Herpesvirus Humano 1/fisiología , Interacciones Huésped-Patógeno , Liberación del Virus , Animales , Chlorocebus aethiops , Citoesqueleto/metabolismo , Glicoproteínas/metabolismo , Herpesvirus Humano 1/crecimiento & desarrollo , Microscopía Confocal , Microscopía Electrónica , Microscopía Fluorescente , Células Vero , Proteínas Virales/metabolismoRESUMEN
Herpesviruses have an icosahedral nucleocapsid surrounded by an amorphous tegument and a lipoprotein envelope. The tegument comprises at least 20 proteins destined for delivery into the host cell. As the tegument does not have a regular structure, the question arises of how its proteins are recruited. The herpes simplex virus 1 (HSV-1) tegument is known to contact the capsid at its vertices, and two proteins, UL36 and UL37, have been identified as candidates for this interaction. We show that the interaction is mediated exclusively by UL36. HSV-1 nucleocapsids extracted from virions shed their UL37 upon incubation at 37°C. Cryo-electron microscopy (cryo-EM) analysis of capsids with and without UL37 reveals the same penton-capping density in both cases. As no other tegument proteins are retained in significant amounts, it follows that this density feature (â¼100 kDa) represents the ordered portion of UL36 (336 kDa). It binds between neighboring UL19 protrusions and to an adjacent UL17 molecule. These observations support the hypothesis that UL36 plays a major role in the tegumentation of the virion, providing a flexible scaffold to which other tegument proteins, including UL37, bind. They also indicate how sequential conformational changes in the maturing nucleocapsid control the ordered binding, first of UL25/UL17 and then of UL36.
Asunto(s)
Proteínas de la Cápside/química , Herpesvirus Humano 1/química , Proteínas Virales/química , Sitios de Unión , Proteínas de la Cápside/metabolismo , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/ultraestructura , Modelos Moleculares , Nucleocápside/química , Nucleocápside/ultraestructura , Proteínas Virales/metabolismo , Virión/químicaRESUMEN
In all herpesviruses, the capsid is icosahedral in shape, composed of 162 capsomers, and assembled in the infected cell nucleus. Once a closed capsid has formed, it is packaged with the virus DNA and transported to the cytoplasm where further morphogenetic events take place. Herpesvirus capsid populations are highly uniform in shape, and this property has made them attractive for structural analysis particularly by cryo electron microscopy followed by three-dimensional image reconstruction. Here we describe what is known about herpesvirus capsid structure and assembly with emphasis on herpes simplex virus and on the contribution of structural studies. The overall analysis has demonstrated that herpesvirus capsids are formed by a pathway resembling that established for dsDNA bacteriophage such as P22 and HK97. For example herpes capsid assembly is found to: (1) involve a scaffolding protein not present in the mature virus; (2) proceed through a fragile, spherical procapsid intermediate; and (3) result in incorporation of a portal complex at a unique capsid vertex.
Asunto(s)
Cápside/química , Cápside/metabolismo , Herpes Simple/virología , Herpesvirus Humano 1/fisiología , Ensamble de Virus , Animales , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Herpesvirus Humano 1/química , Herpesvirus Humano 1/genética , HumanosRESUMEN
OBJECTIVES: The purpose of this study was to evaluate two electrosurgical vessel-sealing devices in biliary surgery. METHODS: Porcine common bile ducts (CBDs) were sealed with two electrosurgical devices, an electrothermal bipolar vessel-sealing device (EBVS) and ultrasonic coagulation shears. Acute study animals underwent surgical bile duct sealing followed by immediate burst pressure testing. Chronic study animals were maintained for 1 week postoperatively and then tested. RESULTS: The seal failure rate in the acute study was 50% for both the EBVS device and shears, and 0% for the laparoscopic surgical clip device used as a control. The latter had significantly higher burst pressures (646.2 ± 281.8 mmHg; P = 0.006) than the EBVS device (97.6 ± 86.6 mmHg) and shears (71.7 ± 89.3 mmHg). No significant difference in burst pressures was noted between the EBVS device and shears (97.6 ± 86.6 mmHg vs. 71.7 ± 89.3 mmHg). In the chronic study, obvious bile leaks occurred in one of four pigs (25%) in the EBVS device subgroup and two of four pigs (50%) in the shears subgroup. The average proximal CBD pressure in seven pigs was 16.1 ± 4.1 mmHg. The average chronic burst pressure in the control subgroup was 1088.0 ± 922.6 mmHg. CONCLUSIONS: Given the high rates of failure of the EBVS device and the shears in consistently sealing biliary ducts, we do not recommend their routine use in biliary surgery.
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Procedimientos Quirúrgicos del Sistema Biliar/instrumentación , Pérdida de Sangre Quirúrgica/prevención & control , Conducto Colédoco/cirugía , Electrocirugia/instrumentación , Hemostasis Quirúrgica/instrumentación , Laparoscopía/instrumentación , Hemorragia Posoperatoria/prevención & control , Equipo Quirúrgico , Animales , Diseño de Equipo , Falla de Equipo , Análisis de Falla de Equipo , Femenino , Ensayo de Materiales , Modelos Animales , Presión , Estrés Mecánico , Porcinos , Factores de TiempoRESUMEN
The tegument of all herpesviruses contains a high-molecular-weight protein homologous to herpes simplex virus (HSV) UL36. This large (3,164 amino acids), essential, and multifunctional polypeptide is located on the capsid surface and present at 100 to 150 copies per virion. We have been testing the idea that UL36 is important for the structural organization of the tegument. UL36 is proposed to bind directly to the capsid with other tegument proteins bound indirectly by way of UL36. Here we report the results of studies carried out with HSV type 1-derived structures containing the capsid but lacking a membrane and depleted of all tegument proteins except UL36 and a second high-molecular-weight protein, UL37. Electron microscopic analysis demonstrated that, compared to capsids lacking a tegument, these capsids (called T36 capsids) had tufts of protein located at the vertices. Projecting from the tufts were thin, variably curved strands with lengths (15 to 70 nm) in some cases sufficient to extend across the entire thickness of the tegument (approximately 50 nm). Strands were sensitive to removal from the capsid by brief sonication, which also removed UL36 and UL37. The findings are interpreted to indicate that UL36 and UL37 are the components of the tufts and of the thin strands that extend from them. The strand lengths support the view that they could serve as organizing features for the tegument, as they have the potential to reach all parts of the tegument. The variably curved structure of the strands suggests they may be flexible, a property that could contribute to the deformable nature of the tegument.
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Cápside/ultraestructura , Herpesvirus Humano 1/fisiología , Proteínas Virales/metabolismo , Ensamble de Virus , Animales , Chlorocebus aethiops , Herpesvirus Humano 1/ultraestructura , Microscopía Electrónica de Transmisión , Células Vero , Proteínas Estructurales Virales/metabolismoRESUMEN
Fanconi anaemia (FA) is a recessive genetic disorder characterized by bone marrow failure, birth defects and cancer. Cells from FA patients are particularly defective in removing DNA interstrand crosslinks. We have developed a working chromatography purification scheme for FANCD2, a pivotal player in the FA DNA repair pathway, to facilitate identification of FANCD2 interacting partners. In doing so, at least three distinct FANCD2 subcomplexes were found to be present, designated as large, middle, and small complexes. The small complex is composed of tetramer of FANCD2 polypeptides, which may be the building block for other FANCD2 subcomplexes.
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Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/aislamiento & purificación , Anemia de Fanconi/metabolismo , Cromatografía en Gel/métodos , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/ultraestructura , Células HeLa , Humanos , Microscopía ElectrónicaRESUMEN
BACKGROUND: Coating prosthetic for hernia repair with a patient's own cells could improve biocompatibility by decreasing inflammation and adhesion formation and by increasing tissue ingrowth and resistance to infection. The objective of this study was to prove the feasibility of prosthetic coating with stem cells and to assess its resistance to adhesion formation when implanted in an animal model. METHODS: Adult Lewis rat bone marrow stem cells were harvested and cultured. Stem cells were then implanted on three different prosthetics. The prosthetic with the best stem cell adherence was implanted intraperitoneally into six adult rats. Untreated prosthetic was implanted in control animals (n = 12). After 2 weeks, intra-abdominal adhesions were graded using an adhesion scoring scale by two surgeons who were blinded to the animal group. Data were analyzed using the Wilcoxon rank-sum test. RESULTS: Stem cells demonstrated the best adherence and growth on polyglactin prosthetics. After implantation, the stem cell-coated polyglactin prosthetic had <25% of its surface area covered with adhesions in five (83%) samples, whereas the control polyglactin group had only one sample (8.3%) with <25% adhesions, and seven of its samples (58.3%) had >50% surface area adhesions (p < 0.05). CONCLUSIONS: The feasibility of hernia prosthetic coating with stem cells was demonstrated. Furthermore, stem-cell coated polyglactin prosthetic exhibited improved biocompatibility by decreasing adhesion formation in an animal model. Further study is needed to determine the factors that promote stem cell adherence to prosthetics and the in vivo prosthetic biomechanics after stem cell coating. This work is underway in our laboratory.
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Materiales Biocompatibles Revestidos , Células Madre Mesenquimatosas/citología , Poliglactina 910 , Prótesis e Implantes , Animales , Células de la Médula Ósea , Adhesión Celular , Proliferación Celular , Células Cultivadas , Herniorrafia , Ratas , Ratas Endogámicas Lew , Mallas Quirúrgicas , Adherencias Tisulares/patología , Ingeniería de TejidosRESUMEN
The herpes simplex virus type 1 UL25 protein is one of seven viral proteins that are required for DNA cleavage and packaging. Together with UL17, UL25 forms part of an elongated molecule referred to as the C-capsid-specific component (CCSC). Five copies of the CCSC are located at each of the capsid vertices on DNA-containing capsids. To study the conformation of UL25 as it is folded on the capsid surface, we identified the sequence recognized by a UL25-specific monoclonal antibody and localized the epitope on the capsid surface by immunogold electron microscopy. The epitope mapped to amino acids 99-111 adjacent to the region of the protein (amino acids 1-50) that is required for capsid binding. In addition, cryo-EM reconstructions of C-capsids in which the green fluorescent protein (GFP) was fused within the N-terminus of UL25 localized the point of contact between UL25 and GFP. The result confirmed the modeled location of the UL25 protein in the CCSC density as the region that is distal to the penton with the N-terminus of UL25 making contact with the triplex one removed from the penton. Immunofluorescence experiments at early times during infection demonstrated that UL25-GFP was present on capsids located within the cytoplasm and adjacent to the nucleus. These results support the view that UL25 is present on incoming capsids with the capsid-binding domain of UL25 located on the surface of the mature DNA-containing capsid.