93 results on '"Richard C. Condit"'
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2. Whence Feral Vaccinia?
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Richard C. Condit
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Smallpox ,vaccinia ,poxvirus ,viruses ,commentary ,Brazil ,Medicine ,Infectious and parasitic diseases ,RC109-216 - Published
- 2010
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3. Pearls collections: What we can learn about infectious disease and cancer.
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Laura J Knoll, Deborah A Hogan, John M Leong, Joseph Heitman, and Richard C Condit
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Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Published
- 2018
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4. The Brighton Collaboration standardized template for collection of key information for benefit-risk assessment of nucleic acid (RNA and DNA) vaccines
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Jonathan M. Smith, Richard C. Condit, Thomas P. Monath, Sonali Kochhar, Emily R. Smith, Robert T. Chen, James S. Robertson, Jean-Louis Excler, Denny Kim, Marc Gurwith, George N. Pavlakis, Patricia E. Fast, and David Wood
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Benefit-risk ,COVID-19 Vaccines ,Coronavirus disease 2019 (COVID-19) ,Computer science ,030231 tropical medicine ,Computational biology ,Risk Assessment ,Article ,Viral vector ,DNA vaccination ,03 medical and health sciences ,Nucleic Acid Vaccines ,0302 clinical medicine ,CEPI ,Vaccines, DNA ,Humans ,030212 general & internal medicine ,General Veterinary ,General Immunology and Microbiology ,Template ,Public Health, Environmental and Occupational Health ,COVID-19 ,RNA ,Viral Vaccines ,DNA ,Brighton Collaboration ,Infectious Diseases ,Nucleic acid ,Public Opinion ,Key (cryptography) ,Molecular Medicine ,Benefit risk assessment ,Safety ,Coronavirus Infections ,Vaccine - 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.
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- 2020
5. Data Repository: Leaf turgor loss point shapes local and regional distributions of evergreen but not deciduous tropical trees
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Norbert Kunert, Joseph Zailaa, Valentine Herrmann, Helene C. Muller-Landau, S. Joseph Wright, Rolando Pérez, Sean M. McMahon, Richard C. Condit, Steven P. Hubbell, Lawren Sack, Stuart J. Davies, and Kristina J. Anderson-Teixeira
- Abstract
In this repository, the trait data used for the analysis in the article "Leaf turgor loss point shapes local and regional distributions of evergreen but not deciduous tropical trees" is made available. 
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- 2021
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6. The Brighton Collaboration standardized templates for collection of key information for benefit-risk assessment of vaccines by technology (BRAVATO; formerly V3SWG)
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Richard C. Condit, Robert T. Chen, and Sonali Kochhar
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2019-20 coronavirus outbreak ,Benefit-risk ,Technology ,Coronavirus disease 2019 (COVID-19) ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,MEDLINE ,Risk Assessment ,CEPI ,Medicine ,Vaccines ,General Veterinary ,General Immunology and Microbiology ,business.industry ,Viral Vaccine ,Public Health, Environmental and Occupational Health ,COVID-19 ,Viral Vaccines ,medicine.disease ,Brighton Collaboration ,Infectious Diseases ,Key (cryptography) ,Commentary ,Molecular Medicine ,Benefit risk assessment ,Medical emergency ,Safety ,Risk assessment ,business ,Vaccine - Published
- 2020
7. The Brighton Collaboration standardized template for collection of key information for risk/benefit assessment of a Modified Vaccinia Ankara (MVA) vaccine platform
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Heinz Weidenthaler, Richard C. Condit, James S. Robertson, Jean-Louis Excler, Ariane Volkmann, Emily R. Smith, Eric Evans, Robert T. Chen, Anna-Lise Williamson, Thomas Meyer, and Denny Kim
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Vaccine safety ,Modified vaccinia Ankara ,Canada ,viruses ,030231 tropical medicine ,Smallpox vaccine ,Risk/benefit assessment ,Vaccinia virus ,Review ,complex mixtures ,03 medical and health sciences ,Monkeypox ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Viral vector ,Modified Vaccinia Ankara ,medicine ,Vaccinia ,Smallpox ,media_common.cataloged_instance ,Animals ,030212 general & internal medicine ,European union ,Ebola Vaccines ,media_common ,Vaccines ,General Veterinary ,General Immunology and Microbiology ,Ebola vaccine ,business.industry ,Public Health, Environmental and Occupational Health ,medicine.disease ,Brighton Collaboration ,Virology ,Vaccination ,Europe ,Africa, Western ,Infectious Diseases ,chemistry ,Molecular Medicine ,business - 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.
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- 2020
8. 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
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Mike Whelan, Richard C. Condit, Robert T. Chen, Sonali Kochhar, Marc Gurwith, Bettina Klug, James S. Robertson, Jean-Louis Excler, Stephen Drew, Patricia E. Fast, David Wood, Denny Kim, Tamala Mallett Moore, Emily R. Smith, and Najwa Khuri-Bulos
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Societies, Scientific ,2019-20 coronavirus outbreak ,Benefit-risk ,Knowledge management ,COVID-19 Vaccines ,Coronavirus disease 2019 (COVID-19) ,030231 tropical medicine ,Drug Evaluation, Preclinical ,Review ,Vaccines, Attenuated ,Risk Assessment ,Viral vector ,03 medical and health sciences ,0302 clinical medicine ,CEPI ,Medicine ,Humans ,030212 general & internal medicine ,Viral ,Public acceptance ,General Veterinary ,General Immunology and Microbiology ,Live ,business.industry ,Viral Vaccine ,Template ,Public Health, Environmental and Occupational Health ,COVID-19 ,Viral Vaccines ,Brighton Collaboration ,Infectious Diseases ,Attenuated ,Key (cryptography) ,Benefit risk assessment ,Molecular Medicine ,Safety ,business ,Risk assessment ,Vaccine - 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.
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- 2020
9. Vaccines based on replication incompetent Ad26 viral vectors: Standardized template with key considerations for a risk/benefit assessment
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Marc Gurwith, Eric Evans, Frank Tomaka, Hanneke Schuitemaker, Denny Kim, Maarten Leyssen, Richard C. Condit, James S. Robertson, Dirk Heerwegh, Jerome Custers, Macaya Douoguih, Esther Heijnen, Roy van Heesbeen, Georgi Shukarev, Robert T. Chen, and Emily R. Smith
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COVID-19 Vaccines ,030231 tropical medicine ,Genetic Vectors ,medicine.disease_cause ,Risk Assessment ,Article ,Viral vector ,03 medical and health sciences ,0302 clinical medicine ,Immunity ,medicine ,Animals ,Humans ,030212 general & internal medicine ,Vector (molecular biology) ,Neutralizing antibody ,Ebola virus ,General Veterinary ,General Immunology and Microbiology ,biology ,business.industry ,SARS-CoV-2 ,Immunogenicity ,Viral Vaccine ,Public Health, Environmental and Occupational Health ,COVID-19 ,Viral Vaccines ,Replication-incompetent Ad26 ,Ebolavirus ,Virology ,Clinical trial ,Infectious Diseases ,biology.protein ,Molecular Medicine ,Safety ,Benefit/risk ,business ,Vaccine - Abstract
Replication-incompetent adenoviral vectors have been under investigation as a platform to carry a variety of transgenes, and express various antigens as a basis for preventive or therapeutic vaccine development. A replication incompetent adenoviral vector based on human adenovirus type 26 (Ad26) has been evaluated in several clinical trials. The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) was formed to evaluate the safety and features of recombinant viral vector vaccines. This paper reviews the biological features of the Ad26 vectors, including tabulation of safety and risk assessment characteristics of Ad26 vector-based vaccines. Substantial information on immunogenicity, clinical safety, biological characteristics and manufacturing are reported. In the Ad26 vector, deletion of the E1 gene, rendering the vector replication incompetent and providing space for transgene insertion, is combined with additional genetic engineering for vaccine manufacturability and transgene expression optimization. These vaccines are manufactured using the E1-complementing PER.C6® cell line, a continuous, human cell-line that can be cultured in serum-free medium in a suspension to high cell densities, providing an effective and flexible system for high-yield manufacturing. Ad26 vector vaccines have favorable thermostability profiles, compatible with vaccine supply chains. Safety data are compiled in the Ad26 vaccine safety database version 4.0, with unblinded data from 23 ongoing and completed clinical studies for a total of 3912 participants in Ebola, HIV, Malaria, RSV and Filovirus Ad26-based vaccine programs. Overall, all Ad26-based vaccines have been well tolerated, with no significant safety issues identified from the available data in the current Ad26 vaccine safety database. Evaluation of Ad26-based vaccines to further characterize the safety profile is continuing, with more than 90,000 participants vaccinated as of 1st July 2020 (cut-off date). Extensive evaluation of immunogenicity in humans shows strong and durable humoral and cellular immune responses. Clinical trials have not shown meaningful impact of pre-existing immunity to Ad26 on vaccine immunogenicity, even in the presence of Ad26 neutralizing antibody titers or Ad26-targeting T cell responses at baseline. The first vaccine, against Ebola virus, that makes use of the Ad26 vector, received marketing authorization from EC on 1st July 2020, as part of the Ad26.ZEBOV, MVA BN Filo vaccine regimen. New developments based on the Ad26 vector are underway, including a COVID-19 vaccine, which is currently in clinical evaluation.
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- 2020
10. The Brighton Collaboration standardized template for collection of key information for benefit-risk assessment of viral vector vaccines
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Richard C Condit, Denny Kim, James S. Robertson, Jean-Louis Excler, Marc Gurwith, Thomas P. Monath, George Pavlakis, Patricia E. Fast, Jonathan Smith, Emily R. Smith, Robert T. Chen, and Sonali Kochhar
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Benefit-risk ,Internet ,General Veterinary ,General Immunology and Microbiology ,Template ,Genetic Vectors ,Public Health, Environmental and Occupational Health ,Drug Evaluation, Preclinical ,COVID-19 ,Viral Vaccines ,Brighton Collaboration ,Vaccines, Attenuated ,Risk Assessment ,Article ,Virus ,Infectious Diseases ,CEPI ,Molecular Medicine ,Animals ,Humans ,Viral ,Vector ,Safety ,Vaccine - 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.
- Published
- 2020
11. The Brighton Collaboration standardized template for collection of key information for benefit-risk assessment of protein vaccines
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Sonali Kochhar, Denny Kim, Jean-Louis Excler, Richard C. Condit, James S. Robertson, Stephen Drew, Mike Whelan, David Wood, Patricia E. Fast, Marc Gurwith, Bettina Klug, Najwa Khuri-Bulos, Emily R. Smith, and Robert T Chen
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Benefit-risk ,COVID-19 Vaccines ,030231 tropical medicine ,Risk Assessment ,Article ,03 medical and health sciences ,Viral Proteins ,0302 clinical medicine ,CEPI ,Humans ,030212 general & internal medicine ,Antigens, Viral ,Vaccines, Synthetic ,Recombinant ,General Veterinary ,General Immunology and Microbiology ,Protein ,Template ,Public Health, Environmental and Occupational Health ,COVID-19 ,Viral Vaccines ,Brighton Collaboration ,Infectious Diseases ,Peptide ,Molecular Medicine ,Patient Safety ,Safety ,Coronavirus Infections ,Vaccine - 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.
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- 2020
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12. Innate immune response of human plasmacytoid dendritic cells to poxvirus infection is subverted by vaccinia E3 via its Z-DNA/RNA binding domain.
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Hua Cao, Peihong Dai, Weiyi Wang, Hao Li, Jianda Yuan, Fangjin Wang, Chee-Mun Fang, Paula M Pitha, Jia Liu, Richard C Condit, Grant McFadden, Taha Merghoub, Alan N Houghton, James W Young, Stewart Shuman, and Liang Deng
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Medicine ,Science - 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.
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- 2012
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13. Biochemical analysis of the multifunctional vaccinia mRNA capping enzyme encoded by a temperature sensitive virus mutant
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Paul Gollnick, Amber N. Shatzer, Richard C. Condit, Susan M. D'Costa, Baron McFadden, Jessica Tate, Nicholas M. Lewandowski, and Rachel L. Boldt
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0301 basic medicine ,Guanylyltransferase ,Mutant ,Vaccinia virus ,Biology ,Article ,Cell Line ,Viral Proteins ,03 medical and health sciences ,Multienzyme Complexes ,Transcription (biology) ,Capping enzyme ,Virology ,Chlorocebus aethiops ,Animals ,Humans ,RNA, Messenger ,Gene ,Transcription Initiation, Genetic ,Nucleoside-triphosphatase ,Messenger RNA ,Transcription termination ,Transcription initiation ,Methyltransferases ,Nucleoside-Triphosphatase ,Nucleotidyltransferases ,Molecular biology ,Phosphoric Monoester Hydrolases ,mRNA capping ,Enzyme assay ,3. Good health ,030104 developmental biology ,Transcription Termination, Genetic ,biology.protein ,RNA, Viral ,HeLa Cells - 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.
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- 2016
14. The vaccinia virus E6 protein influences virion protein localization during virus assembly
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Richard C. Condit and Nissin Moussatche
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viruses ,Mutant ,Assembly ,Vaccinia virus ,Biology ,Article ,Virus ,chemistry.chemical_compound ,Virology ,Maturation ,Vaccinia ,Viroplasm ,Microscopy, Immunoelectron ,Microscopy, Confocal ,Viral Core Proteins ,Virus Assembly ,Virion ,Virion membrane ,Membrane ,Structure ,Immunogold labelling ,Protein subcellular localization prediction ,3. Good health ,Cell biology ,Microscopy, Fluorescence ,Membrane protein ,chemistry ,Poxvirus ,Localization ,Mutant Proteins ,Protein Multimerization - 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.
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- 2015
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15. Vaccinia virus protein A3 is required for the production of normal immature virions and for the encapsidation of the nucleocapsid protein L4
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Richard C. Condit, Desyree Murta Jesus, Casey Paulasue Nielsen, Susan M. D'Costa, Nissin Moussatche, and Baron McFadden
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viruses ,Mutant ,Vaccinia virus ,Plasma protein binding ,Biology ,Article ,Cell Line ,chemistry.chemical_compound ,Virology ,Vaccinia ,Animals ,Humans ,Viroplasm ,Nucleocapsid ,Gene ,A3 ,Virus Protein ,Viral Core Proteins ,Virus Assembly ,Virion ,chemistry ,Cell culture ,Core wall ,Protein Binding - 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.
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- 2015
16. Vaccinia Virus Mutations in the L4R Gene Encoding a Virion Structural Protein Produce Abnormal Mature Particles Lacking a Nucleocapsid
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Richard C. Condit, Nissin Moussatche, and Desyree Murta Jesus
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viruses ,Immunology ,Mutant ,Vaccinia virus ,Biology ,Microbiology ,Virus ,law.invention ,chemistry.chemical_compound ,Transcription (biology) ,law ,Virology ,Nucleocapsid ,Gene ,Viral Structural Proteins ,chemistry.chemical_classification ,Structure and Assembly ,Virus Assembly ,Cryoelectron Microscopy ,Virion ,Cell biology ,Enzyme ,chemistry ,Insect Science ,Mutant Proteins ,Vaccinia ,Electron microscope ,DNA - Abstract
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.
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- 2014
17. Editorial introduction to 'Ranaviruses and other members of the family Iridoviridae: Their place in the virosphere,' a special emphasis section of Virology
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Richard C. Condit and V. Gregory Chinchar
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0106 biological sciences ,0301 basic medicine ,Family Iridoviridae ,Section (typography) ,Ranavirus ,Library science ,Biology ,01 natural sciences ,DNA Virus Infections ,Iridoviridae ,010602 entomology ,03 medical and health sciences ,030104 developmental biology ,Virology ,Animals ,Humans ,Emphasis (typography) ,Introductory Journal Article - Published
- 2017
18. The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG)
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Baevin Carbery, Michael Hendry, Robert T. Chen, Marc Gurwith, Lisa Mac, Richard C. Condit, Stephen J. Seligman, Jean-Louis Excler, Arifa S. Khan, Kenneth I. Berns, Rebecca L. Sheets, Louisa E. Chapman, Anna-Lise Williamson, Najwa Khuri-Bulos, Bettina Klug, and James S. Robertson
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Drug-Related Side Effects and Adverse Reactions ,International Cooperation ,viruses ,Genetic Vectors ,Article ,Viral vector ,Immunology and Microbiology(all) ,Viral Vector ,Humans ,Medicine ,Public acceptance ,Vaccines ,Clinical Trials as Topic ,Drug Carriers ,Vaccines, Synthetic ,General Veterinary ,General Immunology and Microbiology ,business.industry ,Viral Vaccine ,Public Health, Environmental and Occupational Health ,Viral Vaccines ,Virology ,veterinary(all) ,Clinical trial ,Infectious Diseases ,Immunization ,Molecular Medicine ,Safety ,business - 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.
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- 2015
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19. Biochemical and Biophysical Properties of a Putative Hub Protein Expressed by Vaccinia Virus
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Robert McKenna, Travis W. Bainbridge, Susan M. D'Costa, Balasubramanian Venkatakrishnan, Nicole E. Kay, Richard C. Condit, Michael R. Bubb, and Reuben E. Judd
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DNA Replication ,Polyadenylation ,viruses ,Vaccinia virus ,RNA-binding protein ,macromolecular substances ,Biology ,Virus Replication ,Microbiology ,Biochemistry ,DNA-binding protein ,Virus ,Viral Proteins ,chemistry.chemical_compound ,Protein structure ,Endoribonucleases ,Vaccinia ,Humans ,Phosphorylation ,Molecular Biology ,musculoskeletal, neural, and ocular physiology ,DNA replication ,Cell Biology ,Molecular biology ,Protein Structure, Tertiary ,Cell biology ,nervous system ,chemistry ,Viral replication ,Transcription Termination, Genetic ,DNA, Viral ,HeLa Cells - 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
20. Vaccinia virions deficient in transcription enzymes lack a nucleocapsid
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Richard C. Condit, Karen Kelley, Byung-Ho Kang, Nissin Moussatche, and Baron D.H. McFadden
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Transcription, Genetic ,viruses ,Mutant ,Vaccinia virus ,Biology ,Virus ,Article ,Cell Line ,chemistry.chemical_compound ,Viral Proteins ,Transcription (biology) ,Virology ,Vaccinia ,Animals ,Humans ,Nucleocapsid ,Cryoelectron Microscopy ,Virion ,RNA ,Virus structure ,chemistry ,Cell culture ,Poxvirus ,Ultrastructure ,DNA - 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.
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- 2012
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21. Unique Safety Issues Associated with Virus Vectored Vaccines: Potential for and Theoretical Consequences of Recombination with Wild Type Virus Strains
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Richard C. Condit, James S. Robertson, Thomas P. Monath, Vidisha Singh, Marc Gurwith, Rebecca L. Sheets, Robert T. Chen, Denny Kim, Jean-Louis Excler, Lisa M. Mac, Stephen J. Seligman, R. Michael Hendry, Anna-Lise Williamson, and Karin Bok
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0301 basic medicine ,viruses ,Genetic Vectors ,Virulence ,Context (language use) ,Biology ,Vaccines, Attenuated ,Virus ,Article ,03 medical and health sciences ,Animals ,Humans ,Vector (molecular biology) ,Recombination, Genetic ,Drug Carriers ,Attenuated vaccine ,General Immunology and Microbiology ,General Veterinary ,Transmission (medicine) ,Viral Vaccine ,Wild type ,Public Health, Environmental and Occupational Health ,Viral Vaccines ,Virology ,030104 developmental biology ,Infectious Diseases ,Viruses ,Molecular Medicine - 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.
- Published
- 2016
22. An improved high pressure freezing and freeze substitution method to preserve the labile vaccinia virus nucleocapsid
- Author
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Desyree Murta Jesus, Richard C. Condit, and Nissin Moussatche
- Subjects
0301 basic medicine ,Freeze Substitution ,viruses ,Vaccinia virus ,Biology ,Virus ,Viral Structure ,Cell Line ,03 medical and health sciences ,chemistry.chemical_compound ,Fixatives ,Structural Biology ,Chlorocebus aethiops ,Freezing ,Animals ,Poxviridae ,Virus Structure ,Nucleocapsid ,Cryopreservation ,030102 biochemistry & molecular biology ,Virus Assembly ,Virus core ,biology.organism_classification ,Virology ,Microscopy, Electron ,030104 developmental biology ,Freeze substitution ,chemistry ,Biophysics ,High pressure freezing ,Vaccinia - 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.
- Published
- 2016
23. Biological Characterization and Next-Generation Genome Sequencing of the Unclassified Cotia Virus SPAn232 (Poxviridae)
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Rolf Renne, Yajie Yang, Márcia Attias, Laila C. Schnellrath, Richard C. Condit, Nissin Moussatche, Jianhong Hu, Priscila P. Afonso, Clarissa R. Damaso, Desyree Murta Jesus, and Patrícia M. Silva
- Subjects
Genes, Viral ,Swine ,viruses ,Molecular Sequence Data ,Immunology ,Myxoma virus ,Chick Embryo ,Genome, Viral ,Cross Reactions ,Virus Replication ,Suipoxvirus ,Microbiology ,Virus ,Capripoxvirus ,Mice ,Cytopathogenic Effect, Viral ,Neutralization Tests ,Virology ,Chlorocebus aethiops ,Animals ,Humans ,Poxviridae ,Amino Acid Sequence ,Orthopoxvirus ,Phylogeny ,Yatapoxvirus ,Genetics ,biology ,High-Throughput Nucleotide Sequencing ,biology.organism_classification ,Macaca mulatta ,Rats ,Viral Tropism ,Genetic Diversity and Evolution ,Insect Science ,Rabbits ,Sequence Alignment ,Leporipoxvirus - 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
24. Myxoma Virus M064 Is a Novel Member of the Poxvirus C7L Superfamily of Host Range Factors That Controls the Kinetics of Myxomatosis in European Rabbits
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Grant McFadden, Nissin Moussatche, Mary K. Reinhard, Richard C. Condit, Jia Liu, and Sonia Wennier
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Gene Expression Regulation, Viral ,Immunology ,Virulence ,Myxoma virus ,Biology ,Microbiology ,Virulence factor ,Cell Line ,Pathogenesis ,Gene Knockout Techniques ,Viral Proteins ,Myxomatosis, Infectious ,Virology ,Gene Order ,medicine ,Animals ,Gene ,Regulation of gene expression ,Myxomatosis ,Virus Assembly ,medicine.disease ,biology.organism_classification ,Kinetics ,Viral Tropism ,Insect Science ,Tissue tropism ,Pathogenesis and Immunity ,Rabbits - Abstract
The myxoma virus (MYXV) carries three tandem C7L-like host range genes (M062R, M063R, and M064R). However, despite the fact that the sequences of these three genes are similar, they possess very distinctive functions in vivo . The role of M064 in MYXV pathogenesis was investigated and compared to the roles of M062 and M063. We report that M064 is a virulence factor that contributes to MYXV pathogenesis but lacks the host range properties associated with M062 and M063.
- Published
- 2012
25. Temperature-sensitive mutant in the vaccinia virus E6 protein produce virions that are transcriptionally inactive
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Audra L. Strahl, Carson Rodeffer, Olga Boyd, Nissin Moussatche, and Richard C. Condit
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Viral Plaque Assay ,Temperature-sensitive mutant ,Virus genetics ,DNA, Viral/biosynthesis/genetics ,Hot Temperature ,Transcription, Genetic ,viruses ,Mutant ,Virus Transcription ,Virus Attachment ,Vaccinia virus ,Biology ,Biochemistry, biophysics & molecular biology [F05] [Life sciences] ,Virus Replication ,Article ,Cell Line ,Late protein ,03 medical and health sciences ,Virology ,Gene expression ,Chlorocebus aethiops ,Animals ,Virion/genetics/metabolism/physiology ,Biochimie, biophysique & biologie moléculaire [F05] [Sciences du vivant] ,Viral Core Proteins/genetics/metabolism ,030304 developmental biology ,0303 health sciences ,Viral Core Proteins ,030302 biochemistry & molecular biology ,DNA replication ,Virion ,Virus Internalization ,Molecular biology ,Virus assembly ,Viral replication ,Poxvirus ,DNA, Viral ,Mutation ,Vaccinia virus/genetics/metabolism/physiology - 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
- 2010
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26. Phenotypic analysis of a temperature sensitive mutant in the large subunit of the vaccinia virus mRNA capping enzyme
- Author
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Sayuri E.M. Kato, Amber N. Shatzer, and Richard C. Condit
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Hot Temperature ,Genes, Viral ,Protein subunit ,Mutant ,Mutation, Missense ,Vaccinia virus ,Viral Plaque Assay ,Biology ,Article ,Cell Line ,Viral Proteins ,03 medical and health sciences ,Transcription (biology) ,Virology ,Chlorocebus aethiops ,Gene expression ,Animals ,RNA, Messenger ,Temperature sensitive mutant ,Gene ,030304 developmental biology ,0303 health sciences ,Messenger RNA ,Genes, Essential ,030302 biochemistry & molecular biology ,DNA replication ,Temperature-sensitive mutant ,Nucleotidyltransferases ,Molecular biology ,mRNA capping ,Protein Subunits ,Amino Acid Substitution ,RNA, Viral ,Transcription - 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
27. 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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Steven G. Cresawn and Richard C. Condit
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Gene Expression Regulation, Viral ,Isatin ,Genes, Viral ,Transcription, Genetic ,Vaccinia virus ,Biology ,Isatin-β-thiosemicarbazone ,DNA-binding protein ,Article ,Virus ,Viral Proteins ,03 medical and health sciences ,chemistry.chemical_compound ,Transcription (biology) ,Virology ,RNA polymerase ,Drug Resistance, Viral ,Genetics ,Animals ,Humans ,Point Mutation ,Gene ,Alleles ,Polymerase ,030304 developmental biology ,0303 health sciences ,030302 biochemistry & molecular biology ,DNA-Directed RNA Polymerases ,Marker rescue ,DNA-Binding Proteins ,Elongation factor ,Protein Subunits ,chemistry ,Drug resistance ,biology.protein ,Transcriptional Elongation Factors ,Vaccinia ,Transcription - Abstract
Treatment of wild-type vaccinia virus infected cells with the anti-poxviral drug isatin-β-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
28. Parapoxviruses of seals and sea lions make up a distinct subclade within the genus Parapoxvirus
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Elliott R. Jacobson, Richard C. Condit, Michael Walsh, Hendrik H. Nollens, Paul A. Klein, Jorge A. Hernandez, and Frances M. D. Gulland
- Subjects
Seals, Earless ,Molecular Sequence Data ,Zoology ,Genome, Viral ,Poxviridae Infections ,DNA sequencing ,Monophyly ,Phylogenetics ,Virology ,Animals ,Pinniped ,Sea lion ,Phylogeny ,Seal ,Parapoxvirus ,Base Sequence ,Phylogenetic tree ,biology ,Strain (biology) ,Genetic Variation ,Subclade ,Sequence Analysis, DNA ,biology.organism_classification ,Sea Lions ,Poxvirus ,DNA, Viral ,Host range ,Sequence Alignment - 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
29. 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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Robert T. Chen, Jean-Louis Excler, Lisa Marie Mac, Richard C. Condit, Baevin Carbery, Stephen J. Seligman, Thomas P. Monath, James S. Robertson, Edward B. Hayes, and Bruno Guy
- Subjects
Vaccine safety ,viruses ,Genetic Vectors ,Risk/benefit assessment ,Medicina tropical ,Yellow fever vaccine ,Vaccines, Attenuated ,Risk Assessment ,Article ,Malalties víriques ,Viral vector ,Dengue fever ,Tropical medicine ,Vacunes antivíriques ,Immunology and Microbiology(all) ,Humans ,Medicine ,Dengue vaccine ,Randomized Controlled Trials as Topic ,Duck embryo vaccine ,Drug Carriers ,Vaccines, Synthetic ,Vaccines ,Vectors genètics ,General Veterinary ,General Immunology and Microbiology ,biology ,business.industry ,Viral vaccines ,Viral Vaccine ,Yellow fever ,Public Health, Environmental and Occupational Health ,Viral Vaccines ,Brighton Collaboration ,biology.organism_classification ,medicine.disease ,veterinary(all) ,Virology ,Genetic vectors ,Flavivirus ,Infectious Diseases ,Immunology ,Molecular Medicine ,Yellow fever virus ,business ,medicine.drug ,Virus diseases - 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.
- Published
- 2014
30. Complementation Analysis of the Dales Collection of Vaccinia Virus Temperature-Sensitive Mutants
- Author
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Richard C. Condit, Susan M. D'Costa, Charles Buck, and Cari A. Lackner
- Subjects
DNA Replication ,Mutant ,Reversion ,Vaccinia virus ,Biology ,Protein Serine-Threonine Kinases ,Genetic analysis ,Virus ,Cell Line ,DNA Glycosylases ,chemistry.chemical_compound ,Virology ,Chlorocebus aethiops ,Animals ,genetics ,Uracil-DNA Glycosidase ,vaccinia ,N-Glycosyl Hydrolases ,Genetics ,Strain (chemistry) ,Genetic Complementation Test ,Temperature ,DNA-Directed RNA Polymerases ,Molecular biology ,Complementation ,Protein Subunits ,IHD-W ,chemistry ,temperature-sensitive ,complementation ,Mutation ,Temperature sensitive ,Vaccinia - 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
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31. 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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Paul D. Gershon, Donald R. Latner, Richard C. Condit, Carina Storrs, and Joseph M. Thompson
- Subjects
termination ,Methyltransferase ,Transcription, Genetic ,Mutant ,Vaccinia virus ,elongation ,E1 ,03 medical and health sciences ,Structure-Activity Relationship ,VP39 ,Transcription (biology) ,Virology ,RNA, Messenger ,vaccinia ,Polymerase ,030304 developmental biology ,0303 health sciences ,biology ,MRNA modification ,030302 biochemistry & molecular biology ,poly(A) polymerase ,RNA ,Polynucleotide Adenylyltransferase ,Processivity ,Methyltransferases ,O-methyltransferase ,Molecular biology ,poxvirus ,biology.protein ,Mutagenesis, Site-Directed ,J3 ,RNA, Viral ,methyltransferase ,transcription ,Transcription Factors - 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
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32. Editorial introduction to 'Giant Viruses' special issue of Virology
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Richard C. Condit and Matthias G. Fischer
- Subjects
biology ,Genome Size ,Virology ,DNA Viruses ,Mimiviridae ,Giant Virus ,Computational biology ,Genome, Viral ,biology.organism_classification ,Genome size - Published
- 2014
33. Interaction between the J3R Subunit of Vaccinia Virus Poly(A) Polymerase and the H4L Subunit of the Viral RNA Polymerase
- Author
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Richard C. Condit, Edward G. Niles, Donald R. Latner, and Mohamed R. Mohamed
- Subjects
Termination factor ,RNA-dependent RNA polymerase ,Vaccinia virus ,RNA polymerase II ,Cell Line ,Viral Proteins ,chemistry.chemical_compound ,Virology ,RNA polymerase ,Chlorocebus aethiops ,Animals ,Polymerase ,biology ,poly(A) polymerase ,Polynucleotide Adenylyltransferase ,DNA-Directed RNA Polymerases ,Molecular biology ,capping enzyme ,Terminator (genetics) ,poxvirus ,chemistry ,biology.protein ,Transcription factor II D ,Transcription factor II B ,Transcription Factors - Abstract
J3R, the 39-kDa subunit of vaccinia virus poly(A) polymerase, is a multifunctional protein that catalyzes (nucleoside-2′-O-)-methyltransferase activity, serves as a poly(A) polymerase stimulatory factor, and acts as a postreplicative positive transcription elongation factor. Prior results support an association between poly(A) polymerase and the virion RNA polymerase. A possible direct interaction between J3R and H4L subunit of virion RNA polymerase was evaluated. J3R was shown to specifically bind to H4L amino acids 235–256, C terminal to NPH I binding site on H4L. H4L binds to the C-terminal region of J3R between amino acids 169 and 333. The presence of a J3R binding site near to the NPH I binding region on H4L led us to evaluate a physical interaction between NPH I and J3R. The NPH I binding site was located on J3R between amino acids 169 and 249, and J3R was shown to bind to NPH I between amino acids 457 and 524. To evaluate a role for J3R in early gene mRNA synthesis, transcription termination, and/or release, a transcription-competent extract prepared from cells infected with mutant virus lacking J3R, J3-7. Analysis of transcription activity demonstrated that J3R is not required for early mRNA synthesis and is not an essential factor in early gene transcription termination or transcript release in vitro. J3R interaction with NPH I and H4L may serve as a docking site for J3R on the virion RNA polymerase, linking transcription to mRNA cap formation and poly(A) addition.
- Published
- 2001
34. An Emergent Poxvirus from Humans and Cattle in Rio de Janeiro State: Cantagalo Virus May Derive from Brazilian Smallpox Vaccine
- Author
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Nissin Moussatche, Richard C. Condit, Joseph J. Esposito, and Clarissa R. Damaso
- Subjects
viruses ,Molecular Sequence Data ,Cattle Diseases ,Hemagglutinins, Viral ,Poxviridae Infections ,Biology ,Polymerase Chain Reaction ,Virus ,law.invention ,Microbiology ,Emergent virus ,Disease Outbreaks ,chemistry.chemical_compound ,law ,Virology ,Chlorocebus aethiops ,Animals ,Humans ,Orthopoxvirus ,Amino Acid Sequence ,Smallpox vaccine ,Buffalopox ,Cantagalo virus ,Vero Cells ,Polymerase chain reaction ,Phylogeny ,smallpox vaccine ,Cowpox virus ,Poxviridae ,virus diseases ,emergent virus ,Exanthema ,biology.organism_classification ,vaccinia virus ,chemistry ,Cattle ,Female ,Vaccinia ,Sequence Alignment ,Brazil - Abstract
The biological properties of poxvirus isolates from skin lesions on dairy cows and milkers during recent exanthem episodes in Cantagalo County, Rio de Janeiro State, Brazil, were more like vaccinia virus (VV) than cowpox virus. PCR amplification of the hemagglutinin (HA) gene substantiated the isolate classification as an Old World orthopoxvirus, and alignment of the HA sequences with those of other orthopoxviruses indicated that all the isolates represented a single strain of VV, which we have designated Cantagalo virus (CTGV). HA sequences of the Brazilian smallpox vaccine strain (VV-IOC), used over 20 years ago, and CTGV showed 98.2% identity; phylogeny inference of CTGV, VV-IOC, and 12 VV strains placed VV-IOC and CTGV together in a distinct clade. Viral DNA restriction patterns and protein profiles showed a few differences between VV-IOC and CTGV. Together, the data suggested that CTGV may have derived from VV-IOC by persisting in an indigenous animal(s), accumulating polymorphisms, and now emerging in cattle and milkers as CTGV. CTGV may represent the first case of long-term persistence of vaccinia in the New World.
- Published
- 2000
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35. The Vaccinia Virus Bifunctional Gene J3 (Nucleoside-2′-O-)-methyltransferase and Poly(A) Polymerase Stimulatory Factor Is Implicated as a Positive Transcription Elongation Factor by Two Genetic Approaches
- Author
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Richard C. Condit, Ying Xiang, Jackie I. Lewis, Jeremy Condit, and Donald R. Latner
- Subjects
Genetic Markers ,Genes, Viral ,Blotting, Western ,Mutant ,Vaccinia virus ,Biology ,Virus ,chemistry.chemical_compound ,Suppression, Genetic ,Transcription (biology) ,Virology ,Chlorocebus aethiops ,Animals ,Point Mutation ,Vero Cells ,Gene ,Polymerase ,Genetics ,Point mutation ,Wild type ,Polynucleotide Adenylyltransferase ,Sequence Analysis, DNA ,Peptide Elongation Factors ,Molecular biology ,chemistry ,Mutagenesis ,biology.protein ,DNA - Abstract
Vaccinia virus genes A18 and G2 affect the elongation and termination of postreplicative viral gene transcription in opposite ways. Viruses with mutations in gene A18 produce abnormally long transcripts, indicating that A18 is a negative transcription elongation factor. Viruses containing mutations in gene G2 produce transcripts that are abnormally short, truncated specifically from their 3′ ends, indicating that G2 is a positive transcription elongation factor. Despite the fact that both A18 and G2 are essential genes, A18-G2 double-mutant viruses are viable, presumably because the effects of the mutations are mutually compensatory. In addition, the anti-poxviral drug isatin-β-thiosemicarbazone (IBT) seems to enhance elongation during a vaccinia infection: IBT treatment of a wildtype vaccinia infection induces a phenotype identical to an A18 mutant infection, and G2 mutant viruses are dependent on IBT for growth, presumably because IBT restores the G2 mutant truncated transcripts to a normal length. These observations inspire two independent genetic selections that have now been used to identify an additional vaccinia gene, J3, that regulates postreplicative transcription elongation. In the first selection, a single virus that contains an extragenic suppressor of the A18 temperature-sensitive mutant, Cts23, was isolated. In the second selection, several spontaneous IBT-dependent (IBTd) mutant viruses were isolated and characterized genetically. Marker rescue mapping and DNA sequence analysis show that the extragenic suppressor of Cts23 contains a point mutation in the J3 gene, while each of seven new IBTd mutants contains null mutations in the J3 gene. The J3 protein has previously been identified as a (nucleoside-2′-O-)-methyltransferase and as a processivity subunit for the heterodimeric viral poly(A) polymerase. The nature of the two independent selections used to isolate the J3 mutants strongly suggests that the J3 protein serves as a positive postreplicative transcription elongation factor during a normal virus infection.
- Published
- 2000
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36. Analysis of a Temperature-Sensitive Vaccinia Virus Mutant in the Viral mRNA Capping Enzyme Isolated by Clustered Charge-to-Alanine Mutagenesis and Transient Dominant Selection
- Author
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Xuekun Xing, Richard C. Condit, Daniel E Hassett, Jackie I. Lewis, and Luke Delange
- Subjects
Guanylyltransferase ,Viral protein ,viruses ,Molecular Sequence Data ,Mutant ,Mutagenesis (molecular biology technique) ,Vaccinia virus ,Viral Plaque Assay ,Biology ,medicine.disease_cause ,Virus ,Cell Line ,Viral Proteins ,Multienzyme Complexes ,Capping enzyme ,Virology ,Morphogenesis ,medicine ,Amino Acid Sequence ,RNA, Messenger ,Virus quantification ,Mutation ,Alanine ,Temperature ,Virion ,Methyltransferases ,Telomere ,Nucleotidyltransferases ,Molecular biology ,Phosphoric Monoester Hydrolases ,Phenotype ,Mutagenesis, Site-Directed ,RNA, Viral ,Protein Processing, Post-Translational - Abstract
We have previously reported the successful development of a targeted genetic method for the creation of temperature-sensitive vaccinia virus mutants [D. E. Hassett and R. C. Condit (1994)Proc. Natl. Acad. Sci. USA91, 4554–4558]. This method has now been applied to the large subunit of the multifunctional vaccinia virus capping enzyme, encoded by gene D1R. Ten clustered charge-to-alanine mutations were created in a cloned copy of D1R. Four of these mutations were successfully transferred into the viral genome using transient dominant selection, and each of these four mutations yielded viruses with plaque phenotypes different from that of wild-type virus. Two of the mutant viruses, 516 and 793, were temperature sensitive in a plaque assay. Mutant 793 was also temperature sensitive in a one-step growth experiment. Phenotypic characterization of the 793 virus under both permissive and nonpermissive conditions revealed nearly normal patterns of viral protein and mRNA synthesis. Under nonpermissive conditions the 793 virus was defective in telomere resolution and blocked at an intermediate stage of viral morphogenesis.In vitroassays of various capping enzyme activities revealed that in permeabilized virions, enzyme guanylylate intermediate formation was reduced and methyltransferase activity was thermolabile, while in solubilized virion extracts enzyme guanylylate activity was reduced and both guanylyltransferase and methyltransferase activities were absent. Thus, the 793 mutation affects at least two separate enzymatic activities of the capping enzyme, guanylyltransferase and methyltransferase, and when incorporated into the virus genome, the mutation yields a virus that is temperature sensitive for growth, telomere resolution, and virion morphogenesis.
- Published
- 1997
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37. Mutation of Vaccinia Virus Gene G2R Causes Suppression of Gene A18R ts Mutants: Implications for Control of Transcription
- Author
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Ying Xiang, Jackie I. Lewis, and Richard C. Condit
- Subjects
Gene Expression Regulation, Viral ,Transcription, Genetic ,Mutant ,DNA Mutational Analysis ,Pair-rule gene ,Vaccinia virus ,Biology ,Cell Line ,03 medical and health sciences ,Viral Proteins ,Suppression, Genetic ,Transcription (biology) ,Virology ,Chlorocebus aethiops ,Animals ,Late viral transcription ,Gene ,030304 developmental biology ,Suppressor mutation ,Genetics ,Adenosine Triphosphatases ,0303 health sciences ,030302 biochemistry & molecular biology ,DNA Helicases ,Resistance mutation ,Phenotype ,Molecular biology ,Repressor Proteins - Abstract
This report provides genetic evidence that two vaccinia virus genes, A18R and G2R, both of which affect the fidelity of viral transcription in vivo, interact with each other or act on a common biochemical pathway. Previous experiments with the antipoxviral drug isatin-β-thiosemicarbazone suggest that lethal mutation of gene G2R would compensate for mutations in gene A18R. We therefore tested the hypothesis that gene G2R is an extragenic suppressor of A18R mutations. First, we constructed a recombinant which contains both a G2R deletion mutation and an A18R temperature-sensitive mutation and found that this recombinant was viable. Second, we isolated both cold-sensitive and temperature-insensitive phenotypic revertants of A18R temperature-sensitive mutants and found in both cases that the revertants contained G2R mutations. In the case of the cold-sensitive revertants, we were able to prove that the cold-sensitive phenotype mapped to the G2R gene. Combined with the biochemical data on A18R and G2R, these results imply that the A18R and G2R genes interact with each other either directly or indirectly in a fashion which affects the fidelity of intermediate and late viral transcription.
- Published
- 1996
- Full Text
- View/download PDF
38. Use of Lysolecithin-Permeabilized Infected-Cell Extracts to Investigate thein VitroBiochemical Phenotypes of Poxvirus ts Mutations Altered in Viral Transcription Activity
- Author
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Michelle Quinn, Jackie I. Lewis, Linda Christen, Richard C. Condit, and Edward G. Niles
- Subjects
Gene Expression Regulation, Viral ,Cell Membrane Permeability ,Base Sequence ,Transcription, Genetic ,General transcription factor ,biology ,Termination factor ,Molecular Sequence Data ,Response element ,Lysophosphatidylcholines ,Vaccinia virus ,E-box ,Promoter ,RNA polymerase II ,Nucleotidyltransferases ,Molecular biology ,Phenotype ,Sp3 transcription factor ,Virology ,Mutation ,TAF2 ,biology.protein ,Humans ,Promoter Regions, Genetic - Abstract
Lysolecithin permeabilization of vaccinia virus-infected cells was employed to prepare extracts that support faithful transcription initiationin vitroon plasmids possessing early, intermediate, and late viral gene promoters. Conditions which optimize transcription from each promoter were defined. Thein vitrosystem was used to investigate the multifunctional viral mRNA capping enzyme, which also functions as the viral early gene transcription termination factor (VTF) and a viral intermediate gene transcription initiation factor. A low level of signal-dependent termination of early gene transcription was observedin vitrowhich could be elevated by the addition of pure mRNA capping enzyme. VTF-dependent transcription termination was found to be restricted to templates that possessed an early promoter. This restriction mimics that observedin vivoand demonstrates that transcription termination is limited to RNA polymerase molecules that recognize early rather than intermediate or late gene promoters. Extracts prepared from cells infected at the nonpermissive temperature with a virus containing a ts mutation in gene D12L, which encodes the small subunit of VTF, are incapable of supporting both early gene transcription termination and intermediate gene transcription initiation. Both activities are restored upon addition of the purified wild-type mRNA capping enzyme.
- Published
- 1996
- Full Text
- View/download PDF
39. The Vaccinia Virus A18R Gene Product Is a DNA-dependent ATPase
- Author
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Richard C. Condit and Christopher D. Bayliss
- Subjects
Genes, Viral ,viruses ,Restriction Mapping ,Vaccinia virus ,RNA polymerase II ,Biology ,Early viral transcription ,Biochemistry ,Cell Line ,Gene product ,chemistry.chemical_compound ,Transcription (biology) ,Animals ,Drosophila Proteins ,Humans ,Cloning, Molecular ,Molecular Biology ,Gene ,Adenosine Triphosphatases ,DNA Helicases ,Cell Biology ,Virology ,Molecular biology ,RNA Helicase A ,Recombinant Proteins ,DNA-Binding Proteins ,Kinetics ,chemistry ,Mutagenesis, Site-Directed ,biology.protein ,DNA ,Nucleotide excision repair - Abstract
The predicted amino acid sequence of the vaccinia virus gene A18R shows significant homology to the human ERCC3 gene product, which is a member of the DEXH subfamily of the DNA and RNA helicase superfamily II and which plays a role in both RNA polymerase II transcription and nucleotide excision repair of DNA. The vaccinia virus A18R gene product is expressed throughout infection and is encapsidated in virions. Vaccinia virions containing mutant A18R gene product are defective in early viral transcription in vitro, and infection with A18R mutant virus results in aberrant viral transcription late during infection. Thus we hypothesize that the vaccinia virus A18R gene product is a helicase that plays a role in viral transcription and possibly DNA repair. As a first test of this hypothesis, we have affinity purified an amino-terminal polyhistidine-tagged A18R protein and shown that it has DNA-dependent ATPase activity. The A18R ATPase activity is stimulated by both single-stranded and double-stranded DNA and by RNA.DNA hybrids, but not by either single-stranded or double-stranded RNA.
- Published
- 1995
40. 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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Hao Li, Liang Deng, Hua Cao, Richard C. Condit, Jianda Yuan, Chee-Mun Fang, Alan N. Houghton, Paula M. Pitha, Taha Merghoub, Fangjin Wang, Stewart Shuman, Weiyi Wang, James W. Young, Grant McFadden, Jia Liu, and Peihong Dai
- Subjects
Anatomy and Physiology ,viruses ,lcsh:Medicine ,chemistry.chemical_compound ,Mice ,Phosphatidylinositol 3-Kinases ,0302 clinical medicine ,Interferon ,Viral classification ,Immune Physiology ,Orthopoxvirus ,lcsh:Science ,Phosphoinositide-3 Kinase Inhibitors ,0303 health sciences ,Multidisciplinary ,Membrane Glycoproteins ,virus diseases ,RNA-Binding Proteins ,hemic and immune systems ,Chloroquine ,Innate Immunity ,3. Good health ,030220 oncology & carcinogenesis ,Cytokines ,Medicine ,Rabbits ,medicine.drug ,Research Article ,Immunology ,Down-Regulation ,Myxoma virus ,Vaccinia virus ,Biology ,Microbiology ,Virus ,03 medical and health sciences ,Viral Proteins ,Immune system ,Virology ,medicine ,Animals ,Humans ,030304 developmental biology ,Innate immune system ,Tumor Necrosis Factor-alpha ,lcsh:R ,Immunity ,Interferon-alpha ,Viral Vaccines ,TLR7 ,Dendritic Cells ,biology.organism_classification ,Immunity, Innate ,Protein Structure, Tertiary ,chemistry ,Toll-Like Receptor 7 ,Immune System ,Myeloid Differentiation Factor 88 ,lcsh:Q ,Clinical Immunology ,Vaccinia ,DNA viruses ,Proto-Oncogene Proteins c-akt - 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
41. The vaccinia virus A18R protein plays a role in viral transcription during both the early and the late phases of infection
- Author
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D A Simpson and Richard C. Condit
- Subjects
Genes, Viral ,Transcription, Genetic ,viruses ,Immunology ,Vaccinia virus ,Early viral transcription ,Microbiology ,Virus ,Cell Line ,Viral Proteins ,chemistry.chemical_compound ,VP40 ,Viral entry ,Virology ,Vaccinia ,Viral structural protein ,Humans ,Poxviridae ,biology ,Temperature ,biology.organism_classification ,Viral replication ,chemistry ,Insect Science ,Mutation ,Research Article - Abstract
The vaccinia virus gene A18R is essential for virus infection. The loss of A18R protein function results in unregulated transcription late during virus infection from regions of the viral genome which are normally transcriptionally quiescent. We have characterized A18R protein expression in cells infected with wild-type virus and the A18R temperature-sensitive mutant Cts23. The A18R protein is expressed during early and late phases of infection. The A18R protein expressed by Cts23 virus at permissive and nonpermissive temperatures is significantly less stable than the wild-type A18R protein. The A18R protein was identified as a virion component and localized by detergent extraction to the virion core. Virions purified from cells infected with the A18R temperature-sensitive mutants Cts4, Cts22, and Cts23 are defective in early viral transcription in vitro. The mutant transcription defect is not attributable to gross defects in virion structure or virion DNA-dependent RNA polymerase activity. We conclude that the A18R protein plays a role in viral transcription during the early phase of infection as well as during the late phase.
- Published
- 1994
42. Temperature-Sensitive Mutants in the Vaccinia Virus A18R Gene Increase Double-Stranded RNA Synthesis as a Result of Aberrant Viral Transcription
- Author
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C.D. Bayliss and Richard C. Condit
- Subjects
Gene Expression Regulation, Viral ,Isatin ,Genes, Viral ,Transcription, Genetic ,Mutant ,Vaccinia virus ,Biology ,Virus ,Transcription (biology) ,Virology ,Endoribonucleases ,2',5'-Oligoadenylate Synthetase ,Late viral transcription ,Orthopoxvirus ,Gene ,RNA, Double-Stranded ,Regulation of gene expression ,RNA ,biology.organism_classification ,Molecular biology ,Enzyme Activation ,RNA, Ribosomal ,Mutation ,Dactinomycin ,RNA, Viral - Abstract
Mutations in the vaccinia gene A18R cause activation of the cellular ribonucleolytic 2-5A pathway. To determine the mechanism of 2-5A pathway activation, mutant infections were analyzed for synthesis of double-stranded RNA and for transcription of individual virus genes. At late times postinfection, A18R mutant-infected cells contained an increased amount of complementary RNA and a higher steady state level of RNA from regions of the genome transcribed normally only early in the infection. The phenotype of A18R ts mutants is indistinguishable from that of wild-type infections done in the presence of isatin-beta-thiosemicarbazone (IBT). Actinomycin D is a potent inhibitor of activation of the 2-5A pathway in IBT-treated wt infections. Based on these observations, we conclude that the phenotype induced by A18R mutants or by IBT treatment of wt infections is caused by a loss of control of late viral transcription.
- Published
- 1993
43. Poxvirus Replication
- Author
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Richard C. Condit and Richard W. Moyer
- Published
- 2010
44. The E6 protein from vaccinia virus is required for the formation of immature virions
- Author
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Richard C. Condit, Nissin Moussatche, Peter C. Turner, Richard W. Moyer, and Olga Boyd
- Subjects
Isopropyl Thiogalactoside ,DNA, Viral/biosynthesis/genetics ,Concatemer ,viruses ,Mutant ,Vaccinia virus ,Viral Plaque Assay ,Biology ,Biochemistry, biophysics & molecular biology [F05] [Life sciences] ,Virus Replication ,Virion/physiology ,Virus ,Article ,Cell Line ,chemistry.chemical_compound ,Virology ,Chlorocebus aethiops ,Viroplasm ,Animals ,Biochimie, biophysique & biologie moléculaire [F05] [Sciences du vivant] ,Viral Core Proteins/genetics/metabolism ,Viral Core Proteins ,DNA replication ,Virion ,Viral membrane ,Molecular biology ,chemistry ,Viral replication ,DNA, Viral ,Mutation ,Vaccinia ,Vaccinia virus/genetics/metabolism/physiology - 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 twelve-hour 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
- 2010
45. Vaccinia H5 is a multifunctional protein involved in viral DNA replication, postreplicative gene transcription, and virion morphogenesis
- Author
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Karen Kelley, Travis W. Bainbridge, Susan M. D'Costa, Sayuri E.M. Kato, Richard C. Condit, and Cindy Prins
- Subjects
DNA Replication ,Virion morphogenesis ,Transcription, Genetic ,Mutant ,Vaccinia virus ,Biology ,Virus Replication ,ts ,Article ,Scaffold ,chemistry.chemical_compound ,Viral Proteins ,Transcription (biology) ,Virology ,Gene expression ,Vaccinia ,Humans ,Gene ,Genetics ,DNA synthesis ,DNA replication ,Virion ,DNA-Binding Proteins ,H5 ,Viral replication ,chemistry ,Poxvirus ,DNA, Viral ,Postreplicative gene transcription ,DNA - 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
- 2009
46. A Vaccinia virus isatin-β-thiosemicarbazone resistance mutation maps in the viral gene encoding the 132-kDa subunit of RNA polymerase
- Author
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Ronald J. Meis, Richard C. Condit, Radiya F. Pacha, Zahra Fathi, and Rachel Easterly
- Subjects
Isatin ,Genes, Viral ,viruses ,Mutant ,Drug Resistance ,Vaccinia virus ,Virus ,chemistry.chemical_compound ,Plasmid ,Virology ,Poxviridae ,Orthopoxvirus ,Cloning, Molecular ,Southern blot ,biology ,DNA-Directed RNA Polymerases ,Cosmids ,biology.organism_classification ,Resistance mutation ,Molecular biology ,Blotting, Southern ,chemistry ,Mutation ,Vaccinia ,DNA Probes ,Plasmids - Abstract
The mutation in a vaccinia virus mutant resistant to inhibition by isatin-β-thiosemicarbazone was mapped by marker rescue. DNA from the resistant mutant was cloned into cosmid and plasmid vectors and tested for its ability to convert wild-type vaccinia virus to IBT resistant virus in a helper-mediated marker rescue protocol. Resistance was mapped in this way to a 0.9-kb DNA fragment derived from the Hin dIII A fragment of vaccinia genome. Southern blot hybridization using this DNA as a probe demonstrated that the 0.9-kb fragment is contained within the DNA sequence encoding the second largest subunit of vaccinia RNA polymerase, rpol32. Thus, mutation of rpo132 can cause resistance to IBT in vaccinia virus.
- Published
- 1991
47. Genetic and molecular biological characterization of a vaccinia virus gene which renders the virus dependent on isatin-β-thiosemicarbazone (IBT)
- Author
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Ronald J. Meis and Richard C. Condit
- Subjects
Isatin ,Untranslated region ,Genes, Viral ,Transcription, Genetic ,Molecular Sequence Data ,Restriction Mapping ,Mutant ,Vaccinia virus ,Virus Replication ,Cell Line ,Gene product ,Viral Proteins ,Restriction map ,Transcription (biology) ,Virology ,Animals ,Amino Acid Sequence ,RNA, Messenger ,Orthopoxvirus ,Cloning, Molecular ,Gene ,Viral Structural Proteins ,Genetics ,Oligoribonucleotides ,Base Sequence ,biology ,Adenine Nucleotides ,Genetic Complementation Test ,Nucleic acid sequence ,Chromosome Mapping ,Blotting, Northern ,biology.organism_classification ,Molecular biology ,Mutation ,RNA, Viral ,Chromosome Deletion - Abstract
We have sequenced and analyzed the transcription of a gene capable of rendering vaccinia virus (VV) dependent upon isatin-beta-thiosemicarbazone (IBT) for growth. Marker rescue analysis of an IBT-dependent mutant of VV, IBTd-1, and a temperature-sensitive mutant of VV, ts56, both of which require IBT to grow at 40 degrees, showed that both lesions mapped to gene G2R. VV mutants with G2R deletions were constructed and shown to also be dependent upon IBT for growth. The nucleotide sequence changes responsible for IBTd-1, ts56, and the gene G2R deletion mutants were determined, and taken together show that IBT dependence results from inactivation of the orf G2R gene product. Gene G2R, which has the capacity to encode a 26-kDa protein, is transcribed solely early during infection. The 1.3-kb mRNA contains a 5' untranslated region of almost 600 nucleotides, and terminates about 20 nucleotides downstream from an early transcription termination signal. Transcription analyses of three flanking genes, as well as the map positions of the VV mutants ts11 and ts60 are also presented.
- Published
- 1991
48. Phenotypic characterization of a vaccinia virus temperature-sensitive complementation group affecting a virion component
- Author
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Richard C. Condit and Zahra Fathi
- Subjects
Genes, Viral ,biology ,viruses ,Genetic Complementation Test ,Mutant ,Temperature ,Virion ,Vaccinia virus ,biology.organism_classification ,Molecular biology ,Virus ,Cell Line ,chemistry.chemical_compound ,Phenotype ,Chordopoxvirinae ,chemistry ,Virology ,Animals ,Poxviridae ,Orthopoxvirus ,Vaccinia ,Thermolabile ,Gene - Abstract
Genetic and biochemical evidence is presented which shows that the product of the vaccinia virus gene 18R is a virion protein. Western blot analysis of virion proteins using anti-18R serum detects a 78,000-Da protein, localized in the virus core. Of five ts mutants which map to gene 18R, two mutants, ts 10 and ts 44, possess thermolabile virions. Temperature shifts performed during single-step growth of ts 44 suggest that precursors required for virion maturation accumulate during nonpermissive infections with ORF 18R mutants and that protein synthesis is required for recovery from nonpermissive condition.
- Published
- 1991
49. Marker rescue mapping of the combined Condit/Dales collection of temperature sensitive vaccinia virus mutants
- Author
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Nicole E. Kay, Travis W. Bainbridge, Sayuri E.M. Kato, Nissin Moussatche, Alyson J. Brinker, Cindy Prins, Richard C. Condit, Susan M. D'Costa, Audra L. Strahl, and Amber N. Shatzer
- Subjects
Hot Temperature ,Genes, Viral ,viruses ,Mutant ,Vaccinia virus ,Viral Plaque Assay ,Biology ,medicine.disease_cause ,Genome ,Virus ,Article ,Cell Line ,03 medical and health sciences ,chemistry.chemical_compound ,Virology ,Chlorocebus aethiops ,medicine ,Genetics ,Animals ,Gene ,Temperature sensitive mutant ,030304 developmental biology ,0303 health sciences ,Mutation ,030302 biochemistry & molecular biology ,Genetic Complementation Test ,Chromosome Mapping ,Sequence Analysis, DNA ,Temperature-sensitive mutant ,Complementation ,chemistry ,Mapping ,Poxvirus ,Vaccinia - 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
50. Extrachromosomal recombination in vaccinia-infected cells requires a functional DNA polymerase participating at a level other than DNA replication
- Author
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Enzo Paoletti, Robert J. Colinas, and Richard C. Condit
- Subjects
DNA Replication ,Genetic Markers ,Phosphonoacetic Acid ,Cancer Research ,Hot Temperature ,DNA polymerase ,Vaccinia virus ,DNA-Directed DNA Polymerase ,Virus Replication ,chemistry.chemical_compound ,Plasmid ,Sequence Homology, Nucleic Acid ,Virology ,Extrachromosomal DNA ,Animals ,Hydroxyurea ,Gene ,Cells, Cultured ,Recombination, Genetic ,biology ,Plasmid recombination ,Cytarabine ,DNA replication ,beta-Galactosidase ,Molecular biology ,Infectious Diseases ,Lac Operon ,chemistry ,DNA, Viral ,Mutation ,biology.protein ,Homologous recombination ,DNA ,Plasmids - Abstract
Homologous recombination was measured in vaccinia-infected cells cotransfected with two plasmid recombination substrates. One plasmid contains a vaccinia protein lacZ coding region bearing a 1.1 kb 3' terminal deletion while the other plasmid contains a non-promoted lacZ coding region bearing a 1.1 kb 5' terminal deletion. Homologous recombination occurring between the 825 bp of lacZ common to both plasmids regenerates a functional lacZ gene from which B-galactosidase expression was measured. The entire 3 kb lacZ gene was used as a positive control. A panel of thermosensitive mutants was screened in cells either transfected with the positive control plasmid or cotransfected with the recombination substrates. A DNA - mutant, ts42, known to map to the viral DNA polymerase gene was found to be defective in recombination. Significantly, other DNA - mutants, ts17 or ts25, or other DNA polymerase mutants did not exhibit a defect in recombination similar to ts42. Inhibitors of viral DNA synthesis did not uniformly affect recombination. Cytosine arabinoside and aphidicolin inhibited B-galactosidase expression from the recombination substrates but not from the positive control plasmid, whereas hydroxyurea enhanced expression from both. Marker rescue with the cloned wildtype DNA polymerase gene repaired the defect in ts42. Southern and western analyses demonstrated that B-galactosidase activity was consistent with a recombined lacZ gene and unit size 116 kDa protein. Measurement of plasmid and viral DNA replication in cells infected with the different DNA - mutants indicated that recombination was independent of plasmid and viral DNA replication. Together these results suggest that the vaccinia DNA polymerase participates in homologous recombination at a level other than that of DNA replication.
- Published
- 1990
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