101 results on '"Condit RC"'
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2. The Brighton Collaboration standardized template for collection of key information for risk/benefit assessment of a Modified Vaccinia Ankara (MVA) vaccine platform.
- Author
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Volkmann A, Williamson AL, Weidenthaler H, Meyer TPH, Robertson JS, Excler JL, Condit RC, Evans E, Smith ER, Kim D, and Chen RT
- Subjects
- Africa, Western, Animals, Canada, Europe, Mice, Vaccinia virus genetics, Ebola Vaccines, Vaccinia
- Abstract
The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) was formed to evaluate the safety and characteristics of live, recombinant viral vector vaccines. The Modified Vaccinia Ankara (MVA) vector system is being explored as a platform for development of multiple vaccines. This paper reviews the molecular and biological features specifically of the MVA-BN vector system, followed by a template with details on the safety and characteristics of an MVA-BN based vaccine against Zaire ebolavirus and other filovirus strains. The MVA-BN-Filo vaccine is based on a live, highly attenuated poxviral vector incapable of replicating in human cells and encodes glycoproteins of Ebola virus Zaire, Sudan virus and Marburg virus and the nucleoprotein of the Thai Forest virus. This vaccine has been approved in the European Union in July 2020 as part of a heterologous Ebola vaccination regimen. The MVA-BN vector is attenuated following over 500 serial passages in eggs, showing restricted host tropism and incompetence to replicate in human cells. MVA has six major deletions and other mutations of genes outside these deletions, which all contribute to the replication deficiency in human and other mammalian cells. Attenuation of MVA-BN was demonstrated by safe administration in immunocompromised mice and non-human primates. In multiple clinical trials with the MVA-BN backbone, more than 7800 participants have been vaccinated, demonstrating a safety profile consistent with other licensed, modern vaccines. MVA-BN has been approved as smallpox vaccine in Europe and Canada in 2013, and as smallpox and monkeypox vaccine in the US in 2019. No signal for inflammatory cardiac disorders was identified throughout the MVA-BN development program. This is in sharp contrast to the older, replicating vaccinia smallpox vaccines, which have a known risk for myocarditis and/or pericarditis in up to 1 in 200 vaccinees. MVA-BN-Filo as part of a heterologous Ebola vaccination regimen (Ad26.ZEBOV/MVA-BN-Filo) has undergone clinical testing including Phase III in West Africa and is currently in use in large scale vaccination studies in Central African countries. This paper provides a comprehensive picture of the MVA-BN vector, which has reached regulatory approvals, both as MVA-BN backbone for smallpox/monkeypox, as well as for the MVA-BN-Filo construct as part of an Ebola vaccination regimen, and therefore aims to provide solutions to prevent disease from high-consequence human pathogens., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors Ariane Volkmann, Heinz Weidenthaler, and Thomas Meyer are employees of Bavarian Nordic., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2021
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3. Leaf turgor loss point shapes local and regional distributions of evergreen but not deciduous tropical trees.
- Author
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Kunert N, Zailaa J, Herrmann V, Muller-Landau HC, Wright SJ, Pérez R, McMahon SM, Condit RC, Hubbell SP, Sack L, Davies SJ, and Anderson-Teixeira KJ
- Subjects
- Colorado, Droughts, Panama, Tropical Climate, Water, Plant Leaves, Trees
- Abstract
The effects of climate change on tropical forests will depend on how diverse tropical tree species respond to drought. Current distributions of evergreen and deciduous tree species across local and regional moisture gradients reflect their ability to tolerate drought stress, and might be explained by functional traits. We measured leaf water potential at turgor loss (i.e. 'wilting point'; π
tlp ), wood density (WD) and leaf mass per area (LMA) on 50 of the most abundant tree species in central Panama. We then tested their ability to explain distributions of evergreen and deciduous species within a 50 ha plot on Barro Colorado Island and across a 70 km rainfall gradient spanning the Isthmus of Panama. Among evergreen trees, species with lower πtlp were associated with drier habitats, with πtlp explaining 28% and 32% of habitat association on local and regional scales, respectively, greatly exceeding the predictive power of WD and LMA. In contrast, πtlp did not predict habitat associations among deciduous species. Across spatial scales, πtlp is a useful indicator of habitat preference for tropical tree species that retain their leaves during periods of water stress, and holds the potential to predict vegetation responses to climate change., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)- Published
- 2021
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4. The Brighton Collaboration standardized template for collection of key information for benefit-risk assessment of viral vector vaccines.
- Author
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Condit RC, Kim D, Robertson JS, Excler JL, Gurwith M, Monath TP, Pavlakis G, Fast PE, Smith J, Smith ER, Chen RT, and Kochhar S
- Subjects
- Animals, Genetic Vectors, Humans, Internet, Risk Assessment, Drug Evaluation, Preclinical standards, Vaccines, Attenuated adverse effects, Viral Vaccines adverse effects
- Abstract
Many of the vaccines under development for COVID-19 involve the use of viral vectors. The Brighton Collaboration Benefit-Risk Assessment of Vaccines by Technology (BRAVATO, formerly the Viral Vector Vaccine Safety Working Group, V3SWG) working group has prepared a standardized template to describe the key considerations for the benefit-risk assessment of viral vector vaccines. This will facilitate key stakeholders to anticipate potential safety issues and interpret or assess safety data. This would also help improve communication and public acceptance of licensed viral vector vaccines., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2020
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5. Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) standardized template for collection of key information for benefit-risk assessment of live-attenuated viral vaccines.
- Author
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Gurwith M, Condit RC, Excler JL, Robertson JS, Kim D, Fast PE, Drew S, Wood D, Klug B, Whelan M, Mallett Moore T, Khuri-Bulos N, Smith ER, Chen RT, and Kochhar S
- Subjects
- COVID-19 Vaccines adverse effects, COVID-19 Vaccines pharmacology, Drug Evaluation, Preclinical methods, Humans, Risk Assessment, Societies, Scientific, Vaccines, Attenuated pharmacology, Viral Vaccines pharmacology, Drug Evaluation, Preclinical standards, Vaccines, Attenuated adverse effects, Viral Vaccines adverse effects
- Abstract
Several live-attenuated viral vaccine candidates are among the COVID-19 vaccines in development. The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) has prepared a standardized template to describe the key considerations for the benefit-risk assessment of live-attenuated viral vaccines. This will help key stakeholders assess potential safety issues and understand the benefit-risk of such vaccines. The standardized and structured assessment provided by the template would also help to contribute to improved communication and support public acceptance of licensed live-attenuated viral vaccines., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2020
- Full Text
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6. The Brighton Collaboration standardized template for collection of key information for benefit-risk assessment of inactivated viral vaccines.
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Kochhar S, Excler JL, Kim D, Robertson JS, Fast PE, Condit RC, Drew S, Wood D, Gurwith M, Klug B, Whelan M, Khuri-Bulos N, Mallett Moore T, Smith ER, and Chen RT
- Subjects
- Betacoronavirus drug effects, Betacoronavirus immunology, Betacoronavirus pathogenicity, COVID-19, COVID-19 Vaccines, Civil Defense, Coronavirus Infections epidemiology, Coronavirus Infections immunology, Coronavirus Infections virology, Government Regulation, Humans, Immunogenicity, Vaccine, International Cooperation, Patient Safety, Pneumonia, Viral epidemiology, Pneumonia, Viral immunology, Pneumonia, Viral virology, SARS-CoV-2, Vaccines, Inactivated, Viral Vaccines administration & dosage, Viral Vaccines biosynthesis, Coronavirus Infections prevention & control, Drug Approval legislation & jurisprudence, Pandemics prevention & control, Pneumonia, Viral prevention & control, Risk Assessment, Viral Vaccines standards
- Abstract
Inactivated viral vaccines have long been used in humans for diseases of global health threat and are now among the vaccines for COVID-19 under development. The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) has prepared a standardized template to describe the key considerations for the benefit-risk assessment of inactivated viral vaccines. This will help key stakeholders to assess potential safety issues and understand the benefit-risk of the vaccine platform. The standardized and structured assessment provided by the template would also help to contribute to improved communication and support public acceptance of licensed inactivated viral vaccines., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2020
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7. The Brighton Collaboration standardized template for collection of key information for benefit-risk assessment of protein vaccines.
- Author
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Kochhar S, Kim D, Excler JL, Condit RC, Robertson JS, Drew S, Whelan M, Wood D, Fast PE, Gurwith M, Klug B, Khuri-Bulos N, Smith ER, and Chen RT
- Subjects
- Antigens, Viral administration & dosage, Antigens, Viral adverse effects, Antigens, Viral immunology, COVID-19 Vaccines, Coronavirus Infections immunology, Coronavirus Infections prevention & control, Humans, Patient Safety, Risk Assessment, Vaccines, Synthetic administration & dosage, Vaccines, Synthetic adverse effects, Vaccines, Synthetic immunology, Viral Proteins administration & dosage, Viral Proteins adverse effects, Viral Proteins immunology, Viral Vaccines administration & dosage, Viral Vaccines adverse effects, Viral Vaccines immunology
- Abstract
Several protein vaccine candidates are among the COVID-19 vaccines in development. The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) has prepared a standardized template to describe the key considerations for the benefit-risk assessment of protein vaccines. This will help key stakeholders to assess potential safety issues and understand the benefit-risk of such a vaccine platform. The structured and standardized assessment provided by the template would also help contribute to improved public acceptance and communication of licensed protein vaccines., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2020
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8. The Brighton Collaboration standardized template for collection of key information for benefit-risk assessment of nucleic acid (RNA and DNA) vaccines.
- Author
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Kim D, Robertson JS, Excler JL, Condit RC, Fast PE, Gurwith M, Pavlakis G, Monath TP, Smith J, Wood D, Smith ER, Chen RT, and Kochhar S
- Subjects
- COVID-19 Vaccines, Coronavirus Infections genetics, Coronavirus Infections prevention & control, Humans, Public Opinion, Risk Assessment standards, Vaccines, DNA genetics, Viral Vaccines adverse effects, Risk Assessment methods, Vaccines, DNA adverse effects, Vaccines, DNA standards, Viral Vaccines genetics, Viral Vaccines standards
- Abstract
Nucleic acid (DNA and RNA) vaccines are among the most advanced vaccines for COVID-19 under development. The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) has prepared a standardized template to describe the key considerations for the benefit-risk assessment of nucleic acid vaccines. This will facilitate the assessment by key stakeholders of potential safety issues and understanding of overall benefit-risk. The structured assessment provided by the template can also help improve communication and public acceptance of licensed nucleic acid vaccines., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2020
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9. Pearls collections: What we can learn about infectious disease and cancer.
- Author
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Knoll LJ, Hogan DA, Leong JM, Heitman J, and Condit RC
- Subjects
- Humans, Communicable Diseases complications, Neoplasms etiology, Neoplasms prevention & control
- Published
- 2018
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10. High Initial Sputter Rate Found for Vaccinia Virions Using Isotopic Labeling, NanoSIMS, and AFM.
- Author
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Gates SD, Condit RC, Moussatche N, Stewart BJ, Malkin AJ, and Weber PK
- Abstract
High-lateral-resolution secondary ion mass spectrometry (SIMS) has the potential to provide functional and depth resolved information from small biological structures, such as viral particles (virions) and phage, but sputter rate and sensitivity are not characterized at shallow depths relevant to these structures. Here we combine stable isotope labeling of the DNA of vaccinia virions with correlated SIMS imaging depth profiling and atomic force microscopy (AFM) to develop a nonlinear, nonequilibrium sputter rate model for the virions and validate the model on the basis of reconstructing the location of the DNA within individual virions. Our experiments with a Cs
+ beam show an unexpectedly high initial sputter rate (∼100 um2 ·nm·pA-1 ·s-1 ) with a rapid decline to an asymptotic rate of 0.7 um2 ·nm·pA-1 ·s-1 at an approximate depth of 70 nm. Correlated experiments were also conducted with glutaraldehyde-fixed virions, as well as O- and Ga+ beams, yielding similar results. Based on our Cs+ sputter rate model, the labeled DNA in the virion was between 50 and 90 nm depth in the virion core, consistent with expectations, supporting our conclusions. Virion densification was found to be a secondary effect. Accurate isotopic ratios were obtained from the initiation of sputtering, suggesting that isotopic tracers could be successfully used for smaller virions and phage.- Published
- 2018
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11. Editorial introduction to "Ranaviruses and other members of the family Iridoviridae: Their place in the virosphere," a special emphasis section of Virology.
- Author
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Condit RC and Gregory Chinchar V
- Subjects
- Animals, Humans, Iridoviridae classification, Iridoviridae genetics, Ranavirus classification, Ranavirus genetics, DNA Virus Infections virology, Iridoviridae isolation & purification, Ranavirus isolation & purification
- Published
- 2017
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12. Adventitious agents and live viral vectored vaccines: Considerations for archiving samples of biological materials for retrospective analysis.
- Author
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Klug B, Robertson JS, Condit RC, Seligman SJ, Laderoute MP, Sheets R, Williamson AL, Gurwith M, Kochhar S, Chapman L, Carbery B, Mac LM, and Chen RT
- Subjects
- Animals, Humans, Vaccines, Attenuated isolation & purification, Drug Contamination, Preservation, Biological, Technology, Pharmaceutical, Viral Vaccines isolation & purification, Virus Cultivation
- Abstract
Vaccines are one of the most effective public health medicinal products with an excellent safety record. As vaccines are produced using biological materials, there is a need to safeguard against potential contamination with adventitious agents. Adventitious agents could be inadvertently introduced into a vaccine through starting materials used for production. Therefore, extensive testing has been recommended at specific stages of vaccine manufacture to demonstrate the absence of adventitious agents. Additionally, the incorporation of viral clearance steps in the manufacturing process can aid in reducing the risk of adventitious agent contamination. However, for live viral vaccines, aside from possible purification of the virus or vector, extensive adventitious agent clearance may not be feasible. In the event that an adventitious agent is detected in a vaccine, it is important to determine its origin, evaluate its potential for human infection and pathology, and discern which batches of vaccine may have been affected in order to take risk mitigation action. To achieve this, it is necessary to have archived samples of the vaccine and ancillary components, ideally from developmental through to current batches, as well as samples of the biological materials used in the manufacture of the vaccine, since these are the most likely sources of an adventitious agent. The need for formal guidance on such vaccine sample archiving has been recognized but not fulfilled. We summarize in this paper several prior major cases of vaccine contamination with adventitious agents and provide points for consideration on sample archiving of live recombinant viral vector vaccines for use in humans., (Copyright © 2016. Published by Elsevier Ltd.)
- Published
- 2016
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13. Unique safety issues associated with virus-vectored vaccines: Potential for and theoretical consequences of recombination with wild type virus strains.
- Author
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Condit RC, Williamson AL, Sheets R, Seligman SJ, Monath TP, Excler JL, Gurwith M, Bok K, Robertson JS, Kim D, Michael Hendry R, Singh V, Mac LM, and Chen RT
- Subjects
- Animals, Humans, Vaccines, Attenuated adverse effects, Vaccines, Attenuated genetics, Virulence, Viruses, Drug Carriers, Genetic Vectors, Recombination, Genetic, Viral Vaccines adverse effects, Viral Vaccines genetics
- Abstract
In 2003 and 2013, the World Health Organization convened informal consultations on characterization and quality aspects of vaccines based on live virus vectors. In the resulting reports, one of several issues raised for future study was the potential for recombination of virus-vectored vaccines with wild type pathogenic virus strains. This paper presents an assessment of this issue formulated by the Brighton Collaboration. To provide an appropriate context for understanding the potential for recombination of virus-vectored vaccines, we review briefly the current status of virus-vectored vaccines, mechanisms of recombination between viruses, experience with recombination involving live attenuated vaccines in the field, and concerns raised previously in the literature regarding recombination of virus-vectored vaccines with wild type virus strains. We then present a discussion of the major variables that could influence recombination between a virus-vectored vaccine and circulating wild type virus and the consequences of such recombination, including intrinsic recombination properties of the parent virus used as a vector; sequence relatedness of vector and wild virus; virus host range, pathogenesis and transmission; replication competency of vector in target host; mechanism of vector attenuation; additional factors potentially affecting virulence; and circulation of multiple recombinant vectors in the same target population. Finally, we present some guiding principles for vector design and testing intended to anticipate and mitigate the potential for and consequences of recombination of virus-vectored vaccines with wild type pathogenic virus strains., (Copyright © 2016. Published by Elsevier Ltd.)
- Published
- 2016
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14. An improved high pressure freezing and freeze substitution method to preserve the labile vaccinia virus nucleocapsid.
- Author
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Jesus DM, Moussatche N, and Condit RC
- Subjects
- Animals, Cell Line, Chlorocebus aethiops, Fixatives, Freeze Substitution, Freezing, Virus Assembly, Cryopreservation methods, Microscopy, Electron methods, Nucleocapsid chemistry, Vaccinia virus ultrastructure
- Abstract
In recent years, high pressure freezing and freeze substitution have been widely used for electron microscopy to reveal viral and cellular structures that are difficult to preserve. Vaccinia virus, a member of the Poxviridae family, presents one of the most complex viral structures. The classical view of vaccinia virus structure consists of an envelope surrounding a biconcave core, with a lateral body in each concavity of the core. This classical view was challenged by Peters and Muller (1963), who demonstrated the presence of a folded tubular structure inside the virus core and stated the difficulty in visualizing this structure, possibly because it is labile and cannot be preserved by conventional sample preparation. Therefore, this tubular structure, now called the nucleocapsid, has been mostly neglected over the years. Earlier studies were able to preserve the nucleocapsid, but with low efficiency. In this study, we report the protocol (and troubleshooting) that resulted in preservation of the highest numbers of nucleocapsids in several independent preparations. Using this protocol, we were able to demonstrate an interdependence between the formation of the virus core wall and the nucleocapsid, leading to the hypothesis that an interaction exists between the major protein constituents of these compartments, A3 (core wall) and L4 (nucleocapsid). Our results show that high pressure freezing and freeze substitution can be used in more in-depth studies concerning the nucleocapsid structure and function., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
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15. Biochemical analysis of the multifunctional vaccinia mRNA capping enzyme encoded by a temperature sensitive virus mutant.
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Tate J, Boldt RL, McFadden BD, D'Costa SM, Lewandowski NM, Shatzer AN, Gollnick P, and Condit RC
- Subjects
- Animals, Cell Line, Chlorocebus aethiops, HeLa Cells, Humans, Methyltransferases metabolism, Nucleoside-Triphosphatase metabolism, Nucleotidyltransferases metabolism, RNA, Viral genetics, Vaccinia virus metabolism, Viral Proteins, Methyltransferases genetics, Multienzyme Complexes genetics, Nucleotidyltransferases genetics, Phosphoric Monoester Hydrolases genetics, RNA, Messenger metabolism, Transcription Initiation, Genetic physiology, Transcription Termination, Genetic physiology, Vaccinia virus genetics
- Abstract
Prior biochemical analysis of the heterodimeric vaccinia virus mRNA capping enzyme suggests roles not only in mRNA capping but also in early viral gene transcription termination and intermediate viral gene transcription initiation. Prior phenotypic characterization of Dts36, a temperature sensitive virus mutant affecting the large subunit of the capping enzyme was consistent with the multifunctional roles of the capping enzyme in vivo. We report a biochemical analysis of the capping enzyme encoded by Dts36. Of the three enzymatic activities required for mRNA capping, the guanylyltransferase and methyltransferase activities are compromised while the triphosphatase activity and the D12 subunit interaction are unaffected. The mutant enzyme is also defective in stimulating early gene transcription termination and intermediate gene transcription initiation in vitro. These results confirm that the vaccinia virus mRNA capping enzyme functions not only in mRNA capping but also early gene transcription termination and intermediate gene transcription initiation in vivo., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2016
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16. The vaccinia virus E6 protein influences virion protein localization during virus assembly.
- Author
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Condit RC and Moussatche N
- Subjects
- Microscopy, Confocal, Microscopy, Fluorescence, Microscopy, Immunoelectron, Mutant Proteins genetics, Mutant Proteins metabolism, Viral Core Proteins genetics, Protein Multimerization, Vaccinia virus physiology, Viral Core Proteins metabolism, Virion metabolism, Virus Assembly
- Abstract
Vaccinia virus mutants in which expression of the virion core protein gene E6R is repressed are defective in virion morphogenesis. E6 deficient infections fail to properly package viroplasm into viral membranes, resulting in an accumulation of empty immature virions and large aggregates of viroplasm. We have used immunogold electron microscopy and immunofluorescence confocal microscopy to assess the intracellular localization of several virion structural proteins and enzymes during E6R mutant infections. We find that during E6R mutant infections virion membrane proteins and virion transcription enzymes maintain a normal localization within viral factories while several major core and lateral body proteins accumulate in aggregated virosomes. The results support a model in which vaccinia virions are assembled from at least three substructures, the membrane, the viroplasm and a "pre-nucleocapsid", and that the E6 protein is essential for maintaining proper localization of the seven-protein complex and the viroplasm during assembly., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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17. Vaccinia virus protein A3 is required for the production of normal immature virions and for the encapsidation of the nucleocapsid protein L4.
- Author
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Jesus DM, Moussatche N, McFadden BB, Nielsen CP, D'Costa SM, and Condit RC
- Subjects
- Animals, Cell Line, Humans, Nucleocapsid genetics, Protein Binding, Vaccinia virology, Vaccinia virus genetics, Vaccinia virus physiology, Viral Core Proteins genetics, Virion genetics, Nucleocapsid metabolism, Viral Core Proteins metabolism, Virion physiology, Virus Assembly
- Abstract
Maturation of the vaccinia virion is an intricate process that results in the organization of the viroplasm contained in immature virions into the lateral bodies, core wall and nucleocapsid observed in the mature particles. It is unclear how this organization takes place and studies with mutants are indispensable in understanding this process. By characterizing an inducible mutant in the A3L gene, we revealed that A3, an inner core wall protein, is important for formation of normal immature viruses and also for the correct localization of L4, a nucleocapsid protein. L4 did not accumulate in the viral factories in the absence of A3 and was not encapsidated in the particles that do not contain A3. These data strengthen our previously suggested hypothesis that A3 and L4 interact and that this interaction is critical for proper formation of the core wall and nucleocapsid., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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18. Fine structure of the vaccinia virion determined by controlled degradation and immunolocalization.
- Author
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Moussatche N and Condit RC
- Subjects
- Gene Expression Regulation, Viral, Immunohistochemistry, Mutation, Peptide Hydrolases, Proteolysis, Staining and Labeling, Vaccinia virus chemistry, Vaccinia virus ultrastructure, Virion chemistry, Virion ultrastructure, Virus Assembly, Vaccinia virus physiology, Viral Envelope Proteins physiology, Virion physiology
- Abstract
The vaccinia virion is a membraned, slightly flattened, barrel-shaped particle, with a complex internal structure featuring a biconcave core flanked by lateral bodies. Although the architecture of the purified mature virion has been intensely characterized by electron microscopy, the distribution of the proteins within the virion has been examined primarily using biochemical procedures. Thus, it has been shown that non-ionic and ionic detergents combined or not with a sulfhydryl reagent can be used to disrupt virions and, to a limited degree, separate the constituent proteins in different fractions. Applying a controlled degradation technique to virions adsorbed on EM grids, we were able to immuno-localize viral proteins within the virion particle. Our results show after NP40 and DTT treatment, membrane proteins are removed from the virion surface revealing proteins that are associated with the lateral bodies and the outer layer of the core wall. Combined treatment using high salt and high DTT removed lateral body proteins and exposed proteins of the internal core wall. Cores treated with proteases could be disrupted and the internal components were exposed. Cts8, a mutant in the A3 protein, produces aberrant virus that, when treated with NP-40 and DTT, releases to the exterior the virus DNA associated with other internal core proteins. With these results, we are able to propose a model for the structure the vaccinia virion., (Published by Elsevier Inc.)
- Published
- 2015
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19. Live virus vaccines based on a yellow fever vaccine backbone: standardized template with key considerations for a risk/benefit assessment.
- Author
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Monath TP, Seligman SJ, Robertson JS, Guy B, Hayes EB, Condit RC, Excler JL, Mac LM, Carbery B, and Chen RT
- Subjects
- Humans, Randomized Controlled Trials as Topic, Risk Assessment, Vaccines, Attenuated adverse effects, Vaccines, Attenuated genetics, Vaccines, Synthetic adverse effects, Vaccines, Synthetic genetics, Drug Carriers, Genetic Vectors, Viral Vaccines adverse effects, Viral Vaccines genetics, Yellow fever virus genetics
- Abstract
The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) was formed to evaluate the safety of live, recombinant viral vaccines incorporating genes from heterologous viruses inserted into the backbone of another virus (so-called "chimeric virus vaccines"). Many viral vector vaccines are in advanced clinical trials. The first such vaccine to be approved for marketing (to date in Australia, Thailand, Malaysia, and the Philippines) is a vaccine against the flavivirus, Japanese encephalitis (JE), which employs a licensed vaccine (yellow fever 17D) as a vector. In this vaccine, two envelope proteins (prM-E) of YF 17D virus were exchanged for the corresponding genes of JE virus, with additional attenuating mutations incorporated into the JE gene inserts. Similar vaccines have been constructed by inserting prM-E genes of dengue and West Nile into YF 17D virus and are in late stage clinical studies. The dengue vaccine is, however, more complex in that it requires a mixture of four live vectors each expressing one of the four dengue serotypes. This vaccine has been evaluated in multiple clinical trials. No significant safety concerns have been found. The Phase 3 trials met their endpoints in terms of overall reduction of confirmed dengue fever, and, most importantly a significant reduction in severe dengue and hospitalization due to dengue. However, based on results that have been published so far, efficacy in preventing serotype 2 infection is less than that for the other three serotypes. In the development of these chimeric vaccines, an important series of comparative studies of safety and efficacy were made using the parental YF 17D vaccine virus as a benchmark. In this paper, we use a standardized template describing the key characteristics of the novel flavivirus vaccine vectors, in comparison to the parental YF 17D vaccine. The template facilitates scientific discourse among key stakeholders by increasing the transparency and comparability of information. The Brighton Collaboration V3SWG template may also be useful as a guide to the evaluation of other recombinant viral vector vaccines., (Copyright © 2014. Published by Elsevier Ltd.)
- Published
- 2015
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20. The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG).
- Author
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Chen RT, Carbery B, Mac L, Berns KI, Chapman L, Condit RC, Excler JL, Gurwith M, Hendry M, Khan AS, Khuri-Bulos N, Klug B, Robertson JS, Seligman SJ, Sheets R, and Williamson AL
- Subjects
- Clinical Trials as Topic, Humans, Vaccines, Synthetic adverse effects, Vaccines, Synthetic genetics, Vaccines, Synthetic immunology, Viral Vaccines genetics, Viral Vaccines immunology, Drug Carriers adverse effects, Drug-Related Side Effects and Adverse Reactions prevention & control, Genetic Vectors, International Cooperation, Viral Vaccines adverse effects
- Abstract
Recombinant viral vectors provide an effective means for heterologous antigen expression in vivo and thus represent promising platforms for developing novel vaccines against human pathogens from Ebola to tuberculosis. An increasing number of candidate viral vector vaccines are entering human clinical trials. The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) was formed to improve our ability to anticipate potential safety issues and meaningfully assess or interpret safety data, thereby facilitating greater public acceptance when licensed., (Published by Elsevier Ltd.)
- Published
- 2015
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21. Vaccinia virus mutations in the L4R gene encoding a virion structural protein produce abnormal mature particles lacking a nucleocapsid.
- Author
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Jesus DM, Moussatche N, and Condit RC
- Subjects
- Cryoelectron Microscopy, Mutant Proteins genetics, Mutant Proteins metabolism, Nucleocapsid ultrastructure, Vaccinia virus genetics, Vaccinia virus ultrastructure, Viral Structural Proteins genetics, Virion ultrastructure, Nucleocapsid metabolism, Vaccinia virus physiology, Viral Structural Proteins metabolism, Virion metabolism, Virus Assembly
- Abstract
Unlabelled: Electron micrographs from the 1960s revealed the presence of an S-shaped tubular structure in the center of the vaccinia virion core. Recently, we showed that packaging of virus transcription enzymes is necessary for the formation of the tubular structure, suggesting that the structure is equivalent to a nucleocapsid. Based on this study and on what is known about nucleocapsids of other viruses, we hypothesized that in addition to transcription enzymes, the tubular structure also contains the viral DNA and a structural protein as a scaffold. The vaccinia virion structural protein L4 stands out as the best candidate for the role of a nucleocapsid structural protein because it is abundant, it is localized in the center of the virion core, and it binds DNA. In order to gain more insight into the structure and relevance of the nucleocapsid, we analyzed thermosensitive and inducible mutants in the L4R gene. Using a cryo-fixation method for electron microscopy (high-pressure freezing followed by freeze-substitution) to preserve labile structures like the nucleocapsid, we were able to demonstrate that in the absence of functional L4, mature particles with defective internal structures are produced under nonpermissive conditions. These particles do not contain a nucleocapsid. In addition, the core wall of these virions is abnormal. This suggests that the nucleocapsid interacts with the core wall and that the nucleocapsid structure might be more complex than originally assumed., Importance: The vaccinia virus nucleocapsid has been neglected since the 1960s due to a lack of electron microscopy techniques to preserve this labile structure. With the advent of cryo-fixation techniques, like high-pressure freezing/freeze-substitution, we are now able to consistently preserve and visualize the nucleocapsid. Because vaccinia virus early transcription is coupled to the viral core structure, detailing the structure of the nucleocapsid is indispensable for determining the mechanisms of vaccinia virus core-directed transcription. The present study represents our second attempt to understand the structure and biological significance of the nucleocapsid. We demonstrate the importance of the protein L4 for the formation of the nucleocapsid and reveal in addition that the nucleocapsid and the core wall may be associated, suggesting a higher level of complexity of the nucleocapsid than predicted. In addition, we prove the utility of high-pressure freezing in preserving the vaccinia virus nucleocapsid., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)
- Published
- 2014
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22. Editorial introduction to "Giant Viruses" special issue of Virology.
- Author
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Fischer MG and Condit RC
- Subjects
- DNA Viruses classification, DNA Viruses genetics, Mimiviridae genetics, DNA Viruses physiology, Genome Size, Genome, Viral genetics
- Published
- 2014
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- View/download PDF
23. Biochemical and biophysical properties of a putative hub protein expressed by vaccinia virus.
- Author
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Kay NE, Bainbridge TW, Condit RC, Bubb MR, Judd RE, Venkatakrishnan B, McKenna R, and D'Costa SM
- Subjects
- DNA Replication physiology, DNA, Viral biosynthesis, DNA, Viral genetics, Endoribonucleases genetics, HeLa Cells, Humans, Phosphorylation physiology, Protein Structure, Tertiary, Vaccinia genetics, Vaccinia metabolism, Viral Proteins genetics, Endoribonucleases metabolism, Transcription Termination, Genetic physiology, Vaccinia virus physiology, Viral Proteins metabolism, Virus Replication physiology
- Abstract
H5 is a constitutively expressed, phosphorylated vaccinia virus protein that has been implicated in viral DNA replication, post-replicative gene expression, and virus assembly. For the purpose of understanding the role of H5 in vaccinia biology, we have characterized its biochemical and biophysical properties. Previously, we have demonstrated that H5 is associated with an endoribonucleolytic activity. In this study, we have shown that this cleavage results in a 3'-OH end suitable for polyadenylation of the nascent transcript, corroborating a role for H5 in vaccinia transcription termination. Furthermore, we have shown that H5 is intrinsically disordered, with an elongated rod-shaped structure that preferentially binds double-stranded nucleic acids in a sequence nonspecific manner. The dynamic phosphorylation status of H5 influences this structure and has implications for the role of H5 in multiple processes during virus replication.
- Published
- 2013
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24. Vaccinia virions deficient in transcription enzymes lack a nucleocapsid.
- Author
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McFadden BD, Moussatche N, Kelley K, Kang BH, and Condit RC
- Subjects
- Animals, Cell Line, Cryoelectron Microscopy, Humans, Vaccinia virus genetics, Vaccinia virus ultrastructure, Viral Proteins genetics, Virion genetics, Virion ultrastructure, Nucleocapsid ultrastructure, Transcription, Genetic, Vaccinia virus physiology, Viral Proteins metabolism, Virion physiology
- Abstract
The poxvirus virion contains an inner tubular nucleocapsid structure. The nucleocapsid is apparently labile to conventional electron microscopy fixation procedures and has therefore been largely ignored for decades. Advancements in electron microscopy sample preparation, notably high pressure freezing, better preserve the nucleocapsid structure. Using high pressure freezing and electron microscopy, we have compared the virion structures of wt virus and mutant viruses known to be deficient in packaging of viral transcription enzymes. We show that the mutant viruses lack a defined nucleocapsid. These results support the hypothesis that the nucleocapsid contains the viral DNA genome complexed with viral transcription enzymes and structural proteins. The studies open the door to further investigation of the composition and ultrastructure of the poxvirus nucleocapsid., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
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25. Biological characterization and next-generation genome sequencing of the unclassified Cotia virus SPAn232 (Poxviridae).
- Author
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Afonso PP, Silva PM, Schnellrath LC, Jesus DM, Hu J, Yang Y, Renne R, Attias M, Condit RC, Moussatché N, and Damaso CR
- Subjects
- Amino Acid Sequence, Animals, Chick Embryo, Chlorocebus aethiops, Cross Reactions immunology, Cytopathogenic Effect, Viral, Genes, Viral, Humans, Macaca mulatta, Mice, Molecular Sequence Data, Neutralization Tests, Phylogeny, Poxviridae physiology, Rabbits, Rats, Sequence Alignment, Swine, Viral Tropism, Virus Replication physiology, Genome, Viral, High-Throughput Nucleotide Sequencing, Poxviridae classification, Poxviridae genetics
- Abstract
Cotia virus (COTV) SPAn232 was isolated in 1961 from sentinel mice at Cotia field station, São Paulo, Brazil. Attempts to classify COTV within a recognized genus of the Poxviridae have generated contradictory findings. Studies by different researchers suggested some similarity to myxoma virus and swinepox virus, whereas another investigation characterized COTV SPAn232 as a vaccinia virus strain. Because of the lack of consensus, we have conducted an independent biological and molecular characterization of COTV. Virus growth curves reached maximum yields at approximately 24 to 48 h and were accompanied by virus DNA replication and a characteristic early/late pattern of viral protein synthesis. Interestingly, COTV did not induce detectable cytopathic effects in BSC-40 cells until 4 days postinfection and generated viral plaques only after 8 days. We determined the complete genomic sequence of COTV by using a combination of the next-generation DNA sequencing technologies 454 and Illumina. A unique contiguous sequence of 185,139 bp containing 185 genes, including the 90 genes conserved in all chordopoxviruses, was obtained. COTV has an interesting panel of open reading frames (ORFs) related to the evasion of host defense, including two novel genes encoding C-C chemokine-like proteins, each present in duplicate copies. Phylogenetic analysis revealed the highest amino acid identity scores with Cervidpoxvirus, Capripoxvirus, Suipoxvirus, Leporipoxvirus, and Yatapoxvirus. However, COTV grouped as an independent branch within this clade, which clearly excluded its classification as an Orthopoxvirus. Therefore, our data suggest that COTV could represent a new poxvirus genus.
- Published
- 2012
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26. Innate immune response of human plasmacytoid dendritic cells to poxvirus infection is subverted by vaccinia E3 via its Z-DNA/RNA binding domain.
- Author
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Cao H, Dai P, Wang W, Li H, Yuan J, Wang F, Fang CM, Pitha PM, Liu J, Condit RC, McFadden G, Merghoub T, Houghton AN, Young JW, Shuman S, and Deng L
- Subjects
- Animals, Chloroquine pharmacology, Dendritic Cells drug effects, Dendritic Cells metabolism, Down-Regulation, Humans, Interferon-alpha biosynthesis, Membrane Glycoproteins metabolism, Mice, Myeloid Differentiation Factor 88 metabolism, Myxoma virus genetics, Myxoma virus immunology, Myxoma virus pathogenicity, Phosphatidylinositol 3-Kinases metabolism, Phosphoinositide-3 Kinase Inhibitors, Protein Structure, Tertiary, Proto-Oncogene Proteins c-akt antagonists & inhibitors, Proto-Oncogene Proteins c-akt metabolism, RNA-Binding Proteins chemistry, RNA-Binding Proteins genetics, Rabbits, Toll-Like Receptor 7 metabolism, Tumor Necrosis Factor-alpha biosynthesis, Vaccinia virus genetics, Vaccinia virus pathogenicity, Viral Proteins chemistry, Viral Proteins genetics, Dendritic Cells immunology, Dendritic Cells virology, Immunity, Innate, RNA-Binding Proteins immunology, Vaccinia virus immunology, Viral Proteins immunology
- Abstract
Plasmacytoid dendritic cells (pDCs) play important roles in antiviral innate immunity by producing type I interferon (IFN). In this study, we assess the immune responses of primary human pDCs to two poxviruses, vaccinia and myxoma virus. Vaccinia, an orthopoxvirus, was used for immunization against smallpox, a contagious human disease with high mortality. Myxoma virus, a Leporipoxvirus, causes lethal disease in rabbits, but is non-pathogenic in humans. We report that myxoma virus infection of human pDCs induces IFN-α and TNF production, whereas vaccinia infection does not. Co-infection of pDCs with myxoma virus plus vaccinia blocks myxoma induction effects. We find that heat-inactivated vaccinia (Heat-VAC; by incubating the virus at 55°C for 1 h) gains the ability to induce IFN-α and TNF in primary human pDCs. Induction of IFN-α in pDCs by myxoma virus or Heat-VAC is blocked by chloroquine, which inhibits endosomal acidification required for TLR7/9 signaling, and by inhibitors of cellular kinases PI3K and Akt. Using purified pDCs from genetic knockout mice, we demonstrate that Heat-VAC-induced type I IFN production in pDCs requires the endosomal RNA sensor TLR7 and its adaptor MyD88, transcription factor IRF7 and the type I IFN feedback loop mediated by IFNAR1. These results indicate that (i) vaccinia virus, but not myxoma virus, expresses inhibitor(s) of the poxvirus sensing pathway(s) in pDCs; and (ii) Heat-VAC infection fails to produce inhibitor(s) but rather produces novel activator(s), likely viral RNA transcripts that are sensed by the TLR7/MyD88 pathway. Using vaccinia gene deletion mutants, we show that the Z-DNA/RNA binding domain at the N-terminus of the vaccinia immunomodulatory E3 protein is an antagonist of the innate immune response of human pDCs to poxvirus infection and TLR agonists. The myxoma virus ortholog of vaccinia E3 (M029) lacks the N-terminal Z-DNA/RNA binding domain, which might contribute to the immunostimulating properties of myxoma virus.
- Published
- 2012
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27. Whence feral vaccinia?
- Author
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Condit RC
- Subjects
- Animals, Brazil epidemiology, Disease Reservoirs virology, Humans, Vaccinia virology, Animals, Wild virology, Vaccinia veterinary, Vaccinia virus isolation & purification
- Published
- 2010
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28. Vaccinia H5 is a multifunctional protein involved in viral DNA replication, postreplicative gene transcription, and virion morphogenesis.
- Author
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D'Costa SM, Bainbridge TW, Kato SE, Prins C, Kelley K, and Condit RC
- Subjects
- DNA Replication, DNA, Viral biosynthesis, Humans, Transcription, Genetic, Virion physiology, Virus Replication, DNA-Binding Proteins physiology, Vaccinia virus physiology, Viral Proteins physiology
- Abstract
The vaccinia H5 protein has been implicated in several steps of virus replication including DNA synthesis, postreplicative gene transcription, and virion morphogenesis. Our recent mapping of mutants in the consolidated Condit-Dales collection identified a temperature-sensitive vaccinia mutant in the H5R gene (Dts57). We demonstrate here that Dts57 has a DNA negative phenotype, strongly suggesting a direct role for H5 in DNA replication. We used a temperature shift protocol to determine the impact of H5 temperature sensitivity on postreplicative gene expression and observed changes in the pattern of postreplicative viral mRNA metabolism consistent with a role of H5 in postreplicative transcription. Finally, using a rifampicin release temperature shift protocol, we show that H5 is involved in multiple steps of virion morphogenesis. These data demonstrate directly that H5 plays roles in DNA replication, transcription and morphogenesis in vivo.
- Published
- 2010
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29. Surf and turf: mechanism of enhanced virus spread during poxvirus infection.
- Author
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Condit RC
- Abstract
Commentary on Doceul, V.; Hollinshead, M.; van der Linden, L.; Smith, G.L. Repulsion of superinfecting virions: a mechanism for rapid virus spread. Science2010, 327, 873-876.
- Published
- 2010
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- View/download PDF
30. The E6 protein from vaccinia virus is required for the formation of immature virions.
- Author
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Boyd O, Turner PC, Moyer RW, Condit RC, and Moussatche N
- Subjects
- Animals, Cell Line, Chlorocebus aethiops, DNA, Viral biosynthesis, DNA, Viral genetics, Isopropyl Thiogalactoside, Mutation, Vaccinia virus genetics, Vaccinia virus metabolism, Viral Core Proteins genetics, Viral Plaque Assay, Virion physiology, Vaccinia virus physiology, Viral Core Proteins metabolism, Virus Replication
- Abstract
An IPTG-inducible mutant in the E6R gene of vaccinia virus was used to study the role of the E6 virion core protein in viral replication. In the absence of the inducer, the mutant exhibited a normal pattern DNA replication, concatemer resolution and late gene expression, but it showed an inhibition of virion structural protein processing it failed to produce infectious particles. Electron microscopic analysis showed that in the absence of IPTG viral morphogenesis was arrested before IV formation: crescents, aberrant or empty IV-like structures, and large aggregated virosomes were observed throughout the cytoplasm. The addition of IPTG to release a 12-h block showed that virus infectious particles could be formed in the absence of de novo DNA synthesis. Our observations show that in the absence of E6 the association of viroplasm with viral membrane crescents is impaired., (Published by Elsevier Inc.)
- Published
- 2010
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- View/download PDF
31. Temperature-sensitive mutant in the vaccinia virus E6 protein produce virions that are transcriptionally inactive.
- Author
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Boyd O, Strahl AL, Rodeffer C, Condit RC, and Moussatche N
- Subjects
- Animals, Cell Line, Chlorocebus aethiops, DNA, Viral biosynthesis, DNA, Viral genetics, Mutation, Vaccinia virus genetics, Vaccinia virus metabolism, Viral Core Proteins genetics, Viral Plaque Assay, Virion genetics, Virion metabolism, Virion physiology, Virus Attachment, Virus Internalization, Hot Temperature, Transcription, Genetic, Vaccinia virus physiology, Viral Core Proteins metabolism, Virus Replication
- Abstract
The vaccinia virus E6R gene encodes a late protein that is packaged into virion cores. A temperature-sensitive mutant was used to study the role of this protein in viral replicative cycle. Cts52 has a P226L missense mutation in the E6R gene, shows a two-log reduction in plaque formation, but displays normal patterns of gene expression, late protein processing and DNA replication during infection. Mutant virions produced at 40 degrees C were similar in their morphology to wt virions grown at 40 degrees C. The particle to infectivity ratio was 50 times higher in purified Cts52 grown at 40 degrees C when compared to the mutant grown at permissive temperature. In vitro characterization of Cts-52 particles grown at 40 degrees C revealed no differences in protein composition or in DNA content and the mutant virions could bind and enter cells. However, core particles prepared from Cts52 grown at 40 degrees C failed to transcribe in vitro. Our results show that E6 in the virion has either a direct or an indirect role in viral transcription., (Published by Elsevier Inc.)
- Published
- 2010
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- View/download PDF
32. In vitro susceptibility of sea lion poxvirus to cidofovir.
- Author
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Nollens HH, Gulland FM, Jacobson ER, Hernandez JA, Klein PA, Walsh MT, and Condit RC
- Subjects
- Animals, Cells, Cultured, Cidofovir, Cytopathogenic Effect, Viral drug effects, Cytosine pharmacology, Kidney cytology, Kidney virology, Microbial Sensitivity Tests, Parapoxvirus classification, Parapoxvirus physiology, Poxviridae Infections virology, Antiviral Agents pharmacology, Cytosine analogs & derivatives, Organophosphonates pharmacology, Parapoxvirus drug effects, Poxviridae Infections veterinary, Sea Lions virology
- Abstract
Parapoxviruses of seals and sea lions are commonly encountered pathogens with zoonotic potential. The antiviral activity of the antiviral compounds isatin-beta-thiosemicarbazone, rifampicin, acyclovir, cidofovir and phosphonoacetic acid against a parapoxvirus (SLPV-1) isolated from a Californian sea lions (Zalophus californianus) was evaluated. Cidofovir was able to reduce virus-induced cytopathic effect of SLPV-1 in confluent monolayers when used in concentrations greater than 2microg/ml. A decreasing virus yield was observed in the presence of increasing concentrations of cidofovir, which confirmed the ability of cidofovir to inhibit SLPV-1 replication. The in vitro efficacy of cidofovir against SLPV-1 indicates the therapeutic potential of cidofovir for the treatment of infections of humans and pinnipeds with parapoxviruses of seals and sea lions. This study confirms the previously proposed therapeutic potential of cidofovir for the treatment of parapoxvirus infections.
- Published
- 2008
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33. Phenotypic analysis of a temperature sensitive mutant in the large subunit of the vaccinia virus mRNA capping enzyme.
- Author
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Shatzer AN, Kato SE, and Condit RC
- Subjects
- Amino Acid Substitution genetics, Animals, Cell Line, Chlorocebus aethiops, Genes, Viral, Hot Temperature, Protein Subunits genetics, Protein Subunits metabolism, RNA, Messenger biosynthesis, RNA, Viral biosynthesis, Viral Plaque Assay, Viral Proteins biosynthesis, Genes, Essential, Mutation, Missense, Nucleotidyltransferases genetics, Nucleotidyltransferases metabolism, Vaccinia virus genetics
- Abstract
The heterodimeric vaccinia virus mRNA capping enzyme is a multifunctional enzyme, encoded by genes D1R and D12L. Published biochemical experiments demonstrate that, in addition to mRNA capping, the enzyme is involved in early viral gene transcription termination and intermediate viral gene transcription initiation. This paper presents the phenotypic characterization of Dts36, a temperature sensitive mutant in the large subunit of the mRNA capping enzyme (G705D), encoded by gene D1R. At the non-permissive temperature, Dts36 displays decreased steady state levels of some early RNAs, suggesting a defect in mRNA capping. Mutant infections also show decreased steady state levels of some early proteins, while DNA replication and post-replicative gene expression are absent. Under non-permissive conditions, the mutant directs synthesis of longer-than-normal early mRNAs from some genes, demonstrating that early gene transcription termination is defective. If mutant infections are initiated at the permissive temperature and shifted to the non-permissive temperature late during infection, steady state levels of intermediate gene transcripts decrease while the levels of late gene transcripts remain constant, consistent with a defect in intermediate gene transcription initiation. In addition to its previously described role in mRNA capping, the results presented in this study provide the first in vivo evidence that the vaccinia virus mRNA capping enzyme plays a role in early gene transcription termination and intermediate gene transcription.
- Published
- 2008
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34. Marker rescue mapping of the combined Condit/Dales collection of temperature-sensitive vaccinia virus mutants.
- Author
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Kato SE, Moussatche N, D'Costa SM, Bainbridge TW, Prins C, Strahl AL, Shatzer AN, Brinker AJ, Kay NE, and Condit RC
- Subjects
- Animals, Cell Line, Chlorocebus aethiops, Genes, Viral, Genetic Complementation Test, Hot Temperature, Sequence Analysis, DNA, Viral Plaque Assay, Chromosome Mapping, Mutation, Vaccinia virus genetics
- Abstract
Complementation analysis of the combined Condit/Dales collection of vaccinia virus temperature-sensitive mutants has been reported (Lackner, C.A., D'Costa, S.M., Buck, C., Condit, R.C., 2003. Complementation analysis of the Dales collection of vaccinia virus temperature-sensitive mutants. Virology 305, 240-259), however not all complementation groups have previously been assigned to single genes on the viral genome. We have used marker rescue to map at least one representative of each complementation group to a unique viral gene. The final combined collection contains 124 temperature-sensitive mutants affecting 38 viral genes, plus five double mutants.
- Published
- 2008
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- View/download PDF
35. Purification and properties of the vaccinia virus mRNA processing factor.
- Author
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D'Costa SM, Bainbridge TW, and Condit RC
- Subjects
- HeLa Cells, Humans, Inclusion Bodies, Viral genetics, Inclusion Bodies, Viral metabolism, Phosphorylation, Protein Processing, Post-Translational physiology, RNA, Messenger genetics, RNA, Messenger metabolism, RNA, Viral genetics, RNA, Viral metabolism, RNA-Binding Proteins genetics, Transcription, Genetic physiology, Vaccinia virus genetics, Viral Proteins genetics, Virus Replication physiology, RNA Processing, Post-Transcriptional physiology, RNA-Binding Proteins isolation & purification, RNA-Binding Proteins metabolism, Vaccinia virus metabolism, Viral Proteins isolation & purification, Viral Proteins metabolism
- Abstract
The mRNAs encoding the vaccinia virus F17 protein and the cowpox A-type inclusion protein are known to possess sequence-homogeneous 3' ends, generated by a post-transcriptional cleavage event. By using partially purified extracts, we have previously shown that the same factor probably cleaves both the F17 and A-type inclusion protein transcripts and that the cleavage factor is either virus-coded or virus-induced during the post-replicative phase of virus replication. In this study, we have purified the cleavage factor from vaccinia-infected HeLa cells using column chromatography and gel filtration. The factor eluted from the gel filtration column with an apparent molecular mass of approximately 440 kDa. Mass spectrometric analyses of the proteins present in the peak active fractions revealed the presence of at least one vaccinia protein with a high degree of certainty, the H5R gene product. To extend this finding, extracts were prepared from HeLa cells infected with vaccinia virus overexpressing His-tagged H5, chromatographed on a nickel affinity column, and eluted using an imidazole gradient. Cleavage activity eluted with the peak of His-tagged H5. Gel filtration of the affinity-purified material further demonstrated that cleavage activity and His-tagged H5 co-chromatographed with an apparent molecular mass of 463 kDa. We therefore conclude that H5 is specifically associated with post-transcriptional cleavage of F17R transcripts. In addition, we show that dephosphorylation of a cleavage competent extract with a nonspecific phosphatase abolishes cleavage activity implying a role for phosphorylation in cleavage activity.
- Published
- 2008
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36. The vaccinia virus E8R gene product is required for formation of transcriptionally active virions.
- Author
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Kato SE, Condit RC, and Moussatché N
- Subjects
- Gene Expression Regulation, Viral, Mutation, Temperature, Tumor Cells, Cultured, Vaccinia virus genetics, Viral Proteins physiology, Virion physiology, Membrane Proteins physiology, Transcription, Genetic physiology, Vaccinia virus chemistry, Viral Proteins analysis, Virion genetics
- Abstract
Two vaccinia virus temperature-sensitive mutants were mapped to the E8R gene and subjected to phenotypic characterization. Dts23 contains a missense mutation in the coding region of E8R (L81F), and in Cts19 the initiating methionine codon is changed from ATG to ATA (M1I). The two ts mutants display normal patterns of gene expression and DNA replication during infection. The E8 protein is synthesized exclusively late during infection and packaged into virion cores Western blot analysis revealed that E8 synthesis is reduced in Dts23 infected cells at permissive (31 degrees C) and non-permissive temperature (39.7 degrees C) and absent in Cts19 infection under both conditions. Dts23 virions produced at 39.7 degrees C were indistinguishable in appearance from wt virions. Cts19 fails to produce identifiable viral structures when incubated at 39.7 degrees C. Purified Dts23 virions produced at 39.7 degrees C contain reduced amounts of E8 and have a high particle to infectivity ratio; purified Cts19 virions grown at 31 degrees C also show reduced infectivity and do not contain detectable E8. Dts23 grown at 39.7 degrees C could enter cells but failed to synthesize early mRNA or produce CPE. Soluble extracts from mutant virions were active in a promoter dependent in vitro transcription assay, however intact mutant cores were defective in transcription. We suggest that E8 plays a subtle role in virion core structure that impacts directly or indirectly on core transcription.
- Published
- 2007
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37. Vaccinia, Inc.--probing the functional substructure of poxviral replication factories.
- Author
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Condit RC
- Subjects
- Animals, Cytoplasm metabolism, Cytoplasm virology, Humans, Viral Proteins metabolism, Virus Replication, Poxviridae physiology
- Abstract
Poxviruses replicate in viral factories in the host cell cytoplasm. In this issue of Cell Host & Microbe, Katsafanas and Moss identify discrete poxviral factory subdomains that contain locally synthesized virus proteins and specifically imported host proteins, and within which viral transcription and translation take place simultaneously. These factories thus comprise a highly structured virus organelle that isolates and coordinates late viral replication events, facilitating competition for cellular resources.
- Published
- 2007
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38. Vaccinia virus temperature-sensitive mutants in the A28 gene produce non-infectious virions that bind to cells but are defective in entry.
- Author
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Turner PC, Dilling BP, Prins C, Cresawn SG, Moyer RW, and Condit RC
- Subjects
- 3' Untranslated Regions, Animals, Cell Fusion, Cell Line, Chlorocebus aethiops, DNA Viruses genetics, DNA Viruses pathogenicity, DNA Viruses physiology, Genome, Viral, Haplorhini, Recombination, Genetic, Temperature, Vaccinia virus immunology, Vaccinia virus physiology, Viral Vaccines, Virion genetics, Virion physiology, Genes, Viral, Mutation, Vaccinia virus genetics, Vaccinia virus pathogenicity
- Abstract
The vaccinia virus temperature-sensitive mutations Cts6 and Cts9 were mapped by marker rescue and DNA sequencing to the A28 gene. Cts6 and Cts9 contain an identical 2-bp deletion truncating the A28 protein and removing the fourth conserved cysteine near the C-terminus. Cts9 mutant virions produced at 40 degrees C were non-infectious and unable to cause cytopathic effect. However, the mutant A28 protein localized to purified mature virions (MV) at 31 degrees C and 40 degrees C. MV of Cts9 produced at 40 degrees C bound to cells but did not enter cells. Low pH treatment of Cts9-infected cells at 18 h p.i. failed to produce fusion from within at 40 degrees C, but gave fusion at 31 degrees C. Adsorption of Cts9 mutant virions to cells followed by low pH treatment showed a defect in fusion from without. The Cts9 phenotype suggests that the A28 protein is involved in both virus entry and cell-cell fusion, and supports the linkage between the two processes.
- Published
- 2007
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- View/download PDF
39. A targeted approach to identification of vaccinia virus postreplicative transcription elongation factors: genetic evidence for a role of the H5R gene in vaccinia transcription.
- Author
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Cresawn SG and Condit RC
- Subjects
- Alleles, Animals, DNA-Directed RNA Polymerases chemistry, DNA-Directed RNA Polymerases genetics, Drug Resistance, Viral, Genes, Viral, Humans, Isatin analogs & derivatives, Isatin pharmacology, Point Mutation, Protein Subunits genetics, Transcription, Genetic, Vaccinia virus drug effects, Viral Proteins physiology, DNA-Binding Proteins physiology, Gene Expression Regulation, Viral, Transcriptional Elongation Factors genetics, Vaccinia virus genetics, Viral Proteins genetics
- Abstract
Treatment of wild-type vaccinia virus infected cells with the anti-poxviral drug isatin-beta-thiosemicarbazone (IBT) induces the viral postreplicative transcription apparatus to synthesize longer-than-normal mRNAs through an unknown mechanism. Prior studies have shown that virus mutants resistant to or dependent on IBT affect proteins involved in control of viral postreplicative transcription elongation, including G2, J3, and the viral RNA polymerase. Prior studies also suggest that there exist additional unidentified vaccinia genes that influence transcription elongation. The present study was undertaken to target candidate transcription elongation factor genes in an error-prone mutagenesis protocol to determine whether IBT-resistant or -dependent alleles could be isolated in those candidate genes. Mutagenesis of genes in which IBT resistance alleles have previously been isolated, namely A24R (encoding the second largest RNA polymerase subunit, rpo132) and G2R (encoding a positive transcription elongation factor), resulted in isolation of novel IBT resistance and dependence alleles therefore providing proof of principle of the targeted mutagenesis technique. The vaccinia H5 protein has been implicated previously in transcription elongation by virtue of its association with the positive elongation factor G2. Mutagenesis of the vaccinia H5R gene resulted in a novel H5R IBT resistance allele, strongly suggesting that H5 is a positive transcription elongation factor.
- Published
- 2007
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40. Mapping and phenotypic analysis of spontaneous isatin-beta-thiosemicarbazone resistant mutants of vaccinia virus.
- Author
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Cresawn SG, Prins C, Latner DR, and Condit RC
- Subjects
- Animals, Catalytic Domain genetics, Cell Line, DNA-Directed RNA Polymerases chemistry, Drug Resistance, Viral, Gene Expression Profiling, Humans, Isatin analogs & derivatives, Isatin pharmacology, Models, Chemical, Mutation, Peptide Elongation Factors, Phenotype, RNA, Messenger metabolism, RNA, Viral metabolism, Transcriptional Elongation Factors genetics, Vaccinia virus drug effects, Viral Proteins chemistry, DNA-Directed RNA Polymerases genetics, Gene Expression Regulation, Viral, Vaccinia virus genetics, Viral Proteins genetics
- Abstract
Treatment of wild type vaccinia virus infected cells with the anti-poxviral drug isatin-beta-thiosemicarbazone (IBT) induces the viral postreplicative transcription apparatus to synthesize longer-than-normal mRNAs through an unknown mechanism. Previous studies have shown that virus mutants resistant to or dependent on IBT affect genes involved in control of viral postreplicative transcription elongation. This study was initiated in order to identify additional viral genes involved in control of vaccinia postreplicative transcription elongation. Eight independent, spontaneous IBT resistant mutants of vaccinia virus were isolated. Marker rescue experiments mapped two mutants to gene G2R, which encodes a previously characterized postreplicative gene positive transcription elongation factor. Three mutants mapped to the largest subunit of the viral RNA polymerase, rpo147, the product of gene J6R. One mutant contained missense mutations in both G2R and A24R (rpo132, the second largest subunit of the RNA polymerase). Two mutants could not be mapped, however sequence analysis demonstrated that neither of these mutants contained mutations in previously identified IBT resistance or dependence genes. Phenotypic and biochemical analysis of the mutants suggests that they possess defects in transcription elongation that compensate for the elongation enhancing effects of IBT. The results implicate the largest subunit of the RNA polymerase (rpo147) in the control of elongation, and suggest that there exist additional gene products which mediate intermediate and late transcription elongation in vaccinia virus.
- Published
- 2007
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41. Parapoxviruses of seals and sea lions make up a distinct subclade within the genus Parapoxvirus.
- Author
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Nollens HH, Gulland FM, Jacobson ER, Hernandez JA, Klein PA, Walsh MT, and Condit RC
- Subjects
- Animals, Base Sequence, DNA, Viral chemistry, DNA, Viral genetics, Genetic Variation, Genome, Viral, Molecular Sequence Data, Parapoxvirus genetics, Parapoxvirus isolation & purification, Phylogeny, Poxviridae Infections virology, Sequence Alignment, Sequence Analysis, DNA, Parapoxvirus classification, Poxviridae Infections veterinary, Sea Lions virology, Seals, Earless virology
- Abstract
Poxviruses of seals and sea lions have been tentatively identified as both orthopoxviruses and parapoxviruses, but their exact identity remained unconfirmed. Here, poxviral DNA sequences were generated from 39 clinical cases and compared to sequences from earlier poxvirus isolates from seals (Phocidae) and sea lions (Otariidae). Six genetically distinct poxvirus strains were detected, of which three were previously unrecognized. All detected strains were closely related to the parapoxviruses, confirming their classification as members of the genus Parapoxvirus. A phylogenetic analysis showed that pinniped parapoxviruses form a monophyletic group within the genus Parapoxvirus. Parapoxviruses from Atlantic pinnipeds were phylogenetically distant from those of Pacific pinnipeds. Parapoxviruses from phocids and otariids that inhabit the same geographical region were also phylogenetically distant, suggesting that parapoxviruses are not commonly transmitted between free-ranging phocids and otariids. However, one strain was detected in two otariid species, suggesting that pinniped parapoxviruses are capable of infecting multiple species within a phylogenetic family.
- Published
- 2006
- Full Text
- View/download PDF
42. In a nutshell: structure and assembly of the vaccinia virion.
- Author
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Condit RC, Moussatche N, and Traktman P
- Subjects
- Viral Proteins genetics, Viral Proteins metabolism, Virion genetics, Vaccinia virus genetics, Vaccinia virus metabolism, Vaccinia virus ultrastructure, Virion metabolism, Virion ultrastructure, Virus Assembly
- Abstract
Poxviruses comprise a large family of viruses characterized by a large, linear dsDNA genome, a cytoplasmic site of replication and a complex virion morphology. The most notorious member of the poxvirus family is variola, the causative agent of smallpox. The laboratory prototype virus used for the study of poxviruses is vaccinia, the virus that was used as a live, naturally attenuated vaccine for the eradication of smallpox. Both the morphogenesis and structure of poxvirus virions are unique among viruses. Poxvirus virions apparently lack any of the symmetry features common to other viruses such as helical or icosahedral capsids or nucleocapsids. Instead poxvirus virions appear as "brick shaped" or "ovoid" membrane-bound particles with a complex internal structure featuring a walled, biconcave core flanked by "lateral bodies." The virion assembly pathway involves a remarkable fabrication of membrane-containing crescents and immature virions, which evolve into mature virions in a process that is unparalleled in virology. As a result of significant advances in poxvirus genetics and molecular biology during the past 15 years, we can now positively identify over 70 specific gene products contained in poxvirus virions, and we can describe the effects of mutations in over 50 specific genes on poxvirus assembly. This review summarizes these advances and attempts to assemble them into a comprehensible and thoughtful picture of poxvirus structure and assembly.
- Published
- 2006
- Full Text
- View/download PDF
43. Pathology and preliminary characterization of a parapoxvirus isolated from a California sea lion (Zalophus californianus).
- Author
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Nollens HH, Jacobson ER, Gulland FM, Beusse DO, Bossart GD, Hernandez JA, Klein PA, and Condit RC
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, California epidemiology, DNA, Viral analysis, Disease Outbreaks veterinary, Fatal Outcome, Female, Microscopy, Electron veterinary, Molecular Sequence Data, Parapoxvirus classification, Parapoxvirus genetics, Phylogeny, Polymerase Chain Reaction veterinary, Poxviridae Infections epidemiology, Poxviridae Infections pathology, Poxviridae Infections virology, Sequence Alignment veterinary, Skin pathology, Skin virology, Parapoxvirus isolation & purification, Poxviridae Infections veterinary, Sea Lions virology
- Abstract
Cutaneous pox-like lesions are a common complication in the rehabilitation of pinnipeds. However, the exact identity, taxonomy, and host range of pinniped parapoxviruses remain unknown. During a poxvirus outbreak in May 2003 in California sea lions (Zalophus californianus) at a marine mammal rehabilitation facility, multiple raised, firm, 1-3-cm skin nodules from the head, neck, and thorax of one sea lion weanling pup that spontaneously died were collected. Histologically, the nodules were characterized by inflammation and necrosis of the dermis and epidermis, acanthosis, and ballooning degeneration of the stratum spinosum. Large, coalescing eosinophilic cytoplasmic inclusions were observed in the ballooned cells. A parapoxvirus (sea lion poxvirus 1, SLPV-1) was isolated on early passage California sea lion kidney cells inoculated with a tissue homogenate of a skin nodule. The morphology of the virions on electron microscopy was consistent with that of parapoxviruses. Partial sequencing of the genomic region encoding the putative major virion envelope antigen p42K confirmed the assignment of the sea lion poxvirus to the genus Parapoxvirus. Although SLPV-1 is most closely related to the poxvirus of harbor seals of the European North Sea, it is significantly different from orf virus, bovine papular stomatitis virus, pseudocowpox virus and the parapoxvirus of New Zealand red deer.
- Published
- 2006
- Full Text
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44. Temperature-sensitive mutants in the vaccinia virus 4b virion structural protein assemble malformed, transcriptionally inactive intracellular mature virions.
- Author
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Kato SE, Strahl AL, Moussatche N, and Condit RC
- Subjects
- Animals, Cell Line, Chlorocebus aethiops, DNA, Viral chemistry, DNA, Viral genetics, DNA, Viral isolation & purification, Electrophoresis, Gel, Pulsed-Field, Gene Expression Regulation, Viral, Genetic Markers, Kidney, Mutation, Plasmids genetics, Temperature, Thermodynamics, Transcription, Genetic, Virion physiology, Vaccinia virus genetics, Viral Structural Proteins genetics, Virion genetics
- Abstract
Two noncomplementing vaccinia virus temperature-sensitive mutants, Cts8 and Cts26, were mapped to the A3L gene, which encodes the major virion structural protein, 4b. The two ts mutants display normal patterns of gene expression, DNA replication, telomere resolution, and protein processing during infection. Morphogenesis during mutant infections is normal through formation of immature virions with nucleoids (IVN) but appears to be defective in the transition from IVN to intracellular mature virus (IMV). In mutant infections, aberrant particles that have the appearance of malformed IMV accumulate. The mutant particles are wrapped in Golgi-derived membranes and exported from cells. Purified mutant particles are indistinguishable from wt particles in protein and DNA composition; however, they are defective in a permeabilized-virion-directed transcription reaction despite containing significant (Cts8) or even normal (Cts26) levels of specific transcription enzymes. These results indicate that the 4b protein is required for proper metamorphosis of IMV from IVN and that proper organization of the IMV structure is required to produce a transcriptionally active virion particle.
- Published
- 2004
- Full Text
- View/download PDF
45. An isatin-beta-thiosemicarbazone-resistant vaccinia virus containing a mutation in the second largest subunit of the viral RNA polymerase is defective in transcription elongation.
- Author
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Prins C, Cresawn SG, and Condit RC
- Subjects
- Biotinylation, Cell Line, Tumor, Cytoplasm metabolism, DNA metabolism, DNA-Directed RNA Polymerases metabolism, Dose-Response Relationship, Drug, HeLa Cells, Humans, Models, Genetic, Models, Molecular, Mutation, Plasmids metabolism, Polymerase Chain Reaction, Salts pharmacology, Sarcosine pharmacology, Software, Time Factors, Uridine Triphosphate chemistry, DNA-Directed RNA Polymerases genetics, Drug Resistance, Viral, Isatin analogs & derivatives, Isatin pharmacology, RNA, Viral, Sarcosine analogs & derivatives, Transcription, Genetic, Vaccinia virus metabolism
- Abstract
The vaccinia virus RNA polymerase is a multi-subunit enzyme that contains eight subunits in the postreplicative form. A prior study of a virus called IBT(r90), which contains a mutation in the A24 gene encoding the RPO132 subunit of the RNA polymerase, demonstrated that the mutation results in resistance to the anti-poxvirus drug isatin-beta-thiosemicarbazone (IBT). In this study, we utilized an in vitro transcription elongation assay to determine the effect of this mutation on transcription elongation. Both wild type and IBT(r90) polymerase complexes were studied with regard to their ability to pause during elongation, their stability in a paused state, their ability to release transcripts, and their elongation rate. We have determined that the IBT(r90) complex is specifically defective in elongation compared with the WT complex, pausing longer and more frequently than the WT complex. We have built a homology model of the RPO132 subunit with the yeast pol II rpb2 subunit to propose a structural mechanism for this elongation defect.
- Published
- 2004
- Full Text
- View/download PDF
46. Post-transcription cleavage generates the 3' end of F17R transcripts in vaccinia virus.
- Author
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D'Costa SM, Antczak JB, Pickup DJ, and Condit RC
- Subjects
- Animals, Base Sequence, Cell Line, RNA Processing, Post-Transcriptional, RNA Splice Sites, Single-Strand Specific DNA and RNA Endonucleases metabolism, Vaccinia virus metabolism, Viral Proteins genetics, Viral Proteins metabolism, Gene Expression Regulation, Viral, RNA, Messenger chemistry, RNA, Messenger metabolism, RNA, Viral chemistry, RNA, Viral metabolism, Vaccinia virus genetics
- Abstract
Most vaccinia virus intermediate and late mRNAs possess 3' ends that are extremely heterogeneous in sequence. However, late mRNAs encoding the cowpox A-type inclusion protein (ATI), the second largest subunit of the RNA polymerase, and the late telomeric transcripts possess homogeneous 3' ends. In the case of the ATI mRNA, it has been shown that the homogeneous 3' end is generated by a post-transcriptional endoribonucleolytic cleavage event. We have determined that the F17R gene also produces homogeneous transcripts generated by a post-transcriptional cleavage event. Mapping of in vivo mRNA shows that the major 3' end of the F17R transcript maps 1262 nt downstream of the F17R translational start site. In vitro transcripts spanning the in vivo 3' end are cleaved in an in vitro reaction using extracts from virus infected cells, and the site of cleavage is the same both in vivo and in vitro. Cleavage is not observed using extract from cells infected in the presence of hydroxyurea; therefore, the cleavage factor is either virus-coded or virus-induced during the post-replicative phase of virus replication. The cis-acting sequence responsible for cleavage is orientation specific and the factor responsible for cleavage activity has biochemical properties similar to the factor required for cleavage of ATI transcripts. Partially purified cleavage factor generates cleavage products of expected size when either the ATI or F17R substrates are used in vitro, strongly suggesting that cleavage of both transcripts is mediated by the same factor.
- Published
- 2004
- Full Text
- View/download PDF
47. An alternative genetic method to test essential vaccinia virus early genes.
- Author
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Kato SE, Greco FA, Damaso CR, Condit RC, and Moussatché N
- Subjects
- Blotting, Western, Culture Media, Escherichia coli, Genetic Techniques, Hypoxanthine Phosphoribosyltransferase genetics, Immediate-Early Proteins metabolism, Mutagenesis, Insertional, Recombination, Genetic, Vaccinia virus genetics, Genes, Essential, Immediate-Early Proteins genetics, Selection, Genetic, Vaccinia virus growth & development
- Abstract
The vaccinia virus F11L gene product was identified during search for additional factors involved in the control of post-replicative viral gene transcription elongation. F11L is a 1065 base pairs (354 aminoacids) gene expressed early during infection with no attributed function. The F11L gene is conserved in many but not all poxviruses. The essential presence of the F11L gene was tested using two different genetic methods. F11L gene disruption by insertion of a selectable cassette containing the Escherichia coli guanine phosphoribosyl transferase gene driven by the viral early-late 7.5K transcriptional promoter resulted exclusively in recombinant viruses containing both the wild type and disrupted alleles, indicating that the F11L gene was essential. However, an alternative test, using transient dominant selection to insert nonsense mutations into the F11L gene, proved that the F11L gene was non-essential for growth in culture. These experiments suggest that misleading results can be obtained using gene insertional mutagenesis as a test of essential presence of the gene. The experiments also provide genetic data on the probability of co-insertion of linked mutations in vaccinia virus genome using transient dominant selection.
- Published
- 2004
- Full Text
- View/download PDF
48. Complementation analysis of the dales collection of vaccinia virus temperature-sensitive mutants.
- Author
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Lackner CA, D'Costa SM, Buck C, and Condit RC
- Subjects
- Animals, Cell Line, Chlorocebus aethiops, DNA Replication, DNA-Directed RNA Polymerases genetics, N-Glycosyl Hydrolases genetics, Protein Serine-Threonine Kinases genetics, Protein Subunits, Temperature, Uracil-DNA Glycosidase, DNA Glycosylases, Genetic Complementation Test, Mutation, Vaccinia virus genetics
- Abstract
A collection of randomly generated temperature-sensitive (ts) vaccinia virus (strain IHD-W) mutants were reported by S. Dales et al., (1978, Virology, 84, 403-428) in 1978 and characterized by electron microscopy. We have performed further genetic analysis on the Dales collection of mutants to make the mutants more useful to the scientific community. We obtained the entire Dales collection, 97 mutants, from the American Type Culture Center (ATCC). All 97 mutants were grown and reassessed for temperature sensitivity. Of these, 16 mutants were either very leaky or showed unacceptably high reversion indices even after plaque purification and therefore were not used for further analysis. The remaining 81 ts mutants were used to perform a complete complementation analysis with each other and the existing Condit collection of ts vaccinia virus (strain WR) mutants. Twenty-two of these 81 Dales mutants were dropped during complementation analysis due to erratic or weak behavior in the complementation test. Of the 59 mutants that were fit for further investigation, 30 fall into 13 of Condit's existing complementation groups, 5 comprise 3 previously identified complementation groups independent of the Condit collection, and 24 comprise 18 new complementation groups. The 59 mutants which were successfully characterized by complementation will be accessioned by and made available to the scientific community through the ATCC.
- Published
- 2003
- Full Text
- View/download PDF
49. Identification and expression of immunogenic proteins of a disease-associated marine turtle herpesvirus.
- Author
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Coberley SS, Condit RC, Herbst LH, and Klein PA
- Subjects
- Alphaherpesvirinae chemistry, Alphaherpesvirinae classification, Alphaherpesvirinae genetics, Animals, Base Sequence, Capsid genetics, DNA, Viral, Herpesviridae classification, Herpesviridae genetics, Herpesviridae Infections virology, Humans, Molecular Sequence Data, Serine Endopeptidases genetics, Viral Envelope Proteins genetics, Viral Proteins genetics, Herpesviridae chemistry, Herpesviridae Infections veterinary, Turtles virology, Viral Proteins analysis
- Abstract
Herpesviruses are associated with several diseases of marine turtles, including lung-eye-trachea disease (LETD) and fibropapillomatosis. Two approaches were used to identify immunodominant antigens of LETV, the LETD-associated herpesvirus. The first approach targeted glycoprotein B, which is known to be immunogenic and neutralizing in other species. The second strategy identified LETV proteins recognized on Western blots by antibodies in immune green turtle plasma. A 38-kDa protein was resolved by two-dimensional gel electrophoresis, sequenced, and identified as a scaffolding protein encoded by the overlapping open reading frames of UL26 and UL26.5. Glycoprotein B and the scaffolding protein were cloned and expressed in Escherichia coli. The expressed proteins were recognized on Western blots by antibodies in immune green turtle plasma. Phylogenetic studies based on UL26, DNA polymerase, and glycoprotein B revealed that LETV clusters with the alphaherpesviruses.
- Published
- 2002
- Full Text
- View/download PDF
50. The positive transcription elongation factor activity of the vaccinia virus J3 protein is independent from its (nucleoside-2'-O-) methyltransferase and poly(A) polymerase stimulatory functions.
- Author
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Latner DR, Thompson JM, Gershon PD, Storrs C, and Condit RC
- Subjects
- Mutagenesis, Site-Directed, Polynucleotide Adenylyltransferase chemistry, RNA, Messenger biosynthesis, RNA, Viral biosynthesis, Structure-Activity Relationship, Vaccinia virus growth & development, Methyltransferases physiology, Polynucleotide Adenylyltransferase physiology, Transcription Factors physiology, Transcription, Genetic
- Abstract
Previous genetic and biochemical experiments have shown that the vaccinia virus J3 protein has three different roles in mRNA synthesis and modification. First, J3 is a (nucleoside-2'-O-)methyltransferase which methylates the 2' position of the first transcribed nucleotide, thus converting a cap-0 to a cap-1 structure at the 5' ends of mRNAs. Second, J3 is a processivity factor for the virus coded poly(A) polymerase. Third, J3 has recently been shown to have intermediate and late gene positive transcription elongation factor activity in vivo. Previous experiments have shown that the poly(A) polymerase stimulatory activity and the (nucleoside-2'-O-)methyltransferase activity are two independent functions of the protein that can be genetically separated through site-directed mutagenesis. In this article, the relationship between the J3-mediated transcription elongation activity and the two other functions of the protein was investigated by constructing several site-directed mutant viruses that contain specific defects in either methyltransferase or poly(A) polymerase processivity functions. The results demonstrate that the J3 positive transcription elongation factor activity is a third independent function of the protein that is genetically separable from its two other functions in mRNA modification. The results also show that neither the poly(A) polymerase stimulatory nor the methyltransferase activities of the J3 protein is essential for virus growth in cell culture.
- Published
- 2002
- Full Text
- View/download PDF
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