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Walsh HA. Preterminal protein, the achilles heel of adenoviridae: Implications for adenoviral infections. World J Pharmacol 2024; 13:97723. [DOI: 10.5497/wjp.v13.i2.97723] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/13/2024] [Revised: 10/03/2024] [Accepted: 11/12/2024] [Indexed: 11/29/2024] Open
Abstract
BACKGROUND Adenoviruses pose a serious health risk particularly in the absence of any clinically approved treatment. As adenoviral infections are quite frequent and recent outbreaks manifest more virulent variant strains, the need to develop an effective treatment remains a priority. The adenoviral protein, preterminal protein (pTP), is one of the key common products of the viral lifecycle as it is necessary to initiate viral replication and hence the infection process. This makes pTP a potential chemotherapeutic target in the search for and development of an effective treatment for adenoviral induced infections. Here we report, for the first time, that glycosylation of pTP in situ prevents binding to ssDNA in vitro.
AIM To explore whether specific structural tailoring of the adenoviral protein pTP, imparts the potential to scupper the viral replication process.
METHODS All chemicals used were of reagent grade. Overexpression of pTP was achieved using the ‘BAC to BAC’ expression system. The presence and relative concentration of the protein was determined throughout the incubation period by the Bradford assay. The pTP was identified by MALDI-TOFF and sodium dodecyl sulphate polyacrylamide gel electrophoresis. For the removal of the aminosugar, a deglycosylase enzyme kit from PROZYME was used. Purification of cloned pTP (6xHis) was done with a ssDNA cellulose column followed by a Ni-NTA column. His-tags were excised with the Tobacco etch virus protease. Protein fractionation was performed with a fraction collector coupled to a UV detector (280 nm) from Pharmacia.
RESULTS The pTP overexpressed in insect cells (Spodoptera frugiperda) (> 96 hours), is unable to bind to ssDNA in vitro. Treatment of this unbound protein with a deglycosidase enzyme that is specific for the removal of truncated unsubstituted O-linked Galβ(1-3)GalNAc-α1 disaccharides bound to Thr or Ser in a glycoprotein, restores binding to ssDNA. Data is presented as a linegraph for both the glycosylated and the deglycosylated proteins. Each point represents the mean of triplicate experiments (from different batches). Means and standard deviation were calculated and plotted on a line graph (with error bars).
CONCLUSION The finding that glycosylation of cloned pTP in situ prevents binding to ssDNA in vitro could aid in the development of an effective treatment of adenoviral infections and/or as an adjunct to complement other anti-adenoviral chemotherapeutic strategies.
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Affiliation(s)
- Harold A Walsh
- Faculty of Pharmacy, Division of Pharmacology, Rhodes University, Grahamstown 6139, Eastern Cape, South Africa
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Gella P, Salas M, Mencía M. Improved artificial origins for phage Φ29 terminal protein-primed replication. Insights into early replication events. Nucleic Acids Res 2014; 42:9792-806. [PMID: 25081208 PMCID: PMC4150772 DOI: 10.1093/nar/gku660] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
The replication machinery of bacteriophage Φ29 is a paradigm for protein-primed replication and it holds great potential for applied purposes. To better understand the early replication events and to find improved origins for DNA amplification based on the Φ29 system, we have studied the end-structure of a double-stranded DNA replication origin. We have observed that the strength of the origin is determined by a combination of factors. The strongest origin (30-fold respect to wt) has the sequence CCC at the 3' end of the template strand, AAA at the 5' end of the non-template strand and 6 nucleotides as optimal unpairing at the end of the origin. We also show that the presence of a correctly positioned displaced strand is important because origins with 5' or 3' ssDNA regions have very low activity. Most of the effect of the improved origins takes place at the passage between the terminal protein-primed and the DNA-primed modes of replication by the DNA polymerase suggesting the existence of a thermodynamic barrier at that point. We suggest that the template and non-template strands of the origin and the TP/DNA polymerase complex form series of interactions that control the critical start of terminal protein-primed replication.
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Affiliation(s)
- Pablo Gella
- Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas - Universidad Autónoma de Madrid), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain
| | - Margarita Salas
- Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas - Universidad Autónoma de Madrid), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain
| | - Mario Mencía
- Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas - Universidad Autónoma de Madrid), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain
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Mi Z, Butt AM, An X, Jiang T, Liu W, Qin C, Cao WC, Tong Y. Genomic analysis of HAdV-B14 isolate from the outbreak of febrile respiratory infection in China. Genomics 2013; 102:448-55. [PMID: 24055951 PMCID: PMC7126778 DOI: 10.1016/j.ygeno.2013.09.001] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2013] [Revised: 09/07/2013] [Accepted: 09/10/2013] [Indexed: 12/14/2022]
Abstract
Human adenovirus type 14 (HAdV-B14) was first reported in 1955 from the Netherlands and since then had been associated with outbreaks of febrile respiratory illness (FRI). In China, sporadic HAdV-B14 infections were first identified in 2010, in Guangzhou and Beijing. In 2012, an outbreak of FRI occurred in Beijing and the etiological agent was determined to be HAdV-B14. We present a complete HAdV-B14 genome sequence isolated from this recent FRI outbreak. Virus in 30 throat swab samples was detected using polymerase chain reaction assays, and confirmed by sequencing of the fiber, hexon and penton genes. Comparative genomics and phylogenetic analysis showed that the newly isolated HAdV-B14 (HAdV-B14 CHN) shared highest sequence homology with a 2006 isolate from the United States and clustered closely with other HAdV-B14 strains. It is expected that data from the present study will help in devising better protocols for virus surveillance, and in developing preventative measures.
Isolation of HAdV-B14 was performed from the outbreak of febrile respiratory illness. Full genome sequence of HAdV-B14 CHN strain has been reported. Several nucleotide substitutions were reported in the HAdV-B14 CHN genome.
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Affiliation(s)
- Zhiqiang Mi
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, People's Republic of China
| | - Azeem Mehmood Butt
- Centre of Excellence in Molecular Biology (CEMB), University of the Punjab, Lahore 53700, Pakistan
| | - Xiaoping An
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, People's Republic of China
| | - Tao Jiang
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, People's Republic of China
| | - Wei Liu
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, People's Republic of China
| | - Chengfeng Qin
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, People's Republic of China
| | - Wu-Chun Cao
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, People's Republic of China.
| | - Yigang Tong
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, People's Republic of China.
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Walsh MP, Seto J, Tirado D, Chodosh J, Schnurr D, Seto D, Jones MS. Computational analysis of human adenovirus serotype 18. Virology 2010; 404:284-92. [PMID: 20542532 PMCID: PMC2902685 DOI: 10.1016/j.virol.2010.05.013] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2010] [Revised: 03/01/2010] [Accepted: 05/13/2010] [Indexed: 11/26/2022]
Abstract
The genome of the sole remaining unsequenced member of species A, human adenovirus type 18 (HAdV-A18), has been sequenced and analyzed. Members of species A are implicated as gastrointestinal pathogens and were shown to be tumorigenic in rodents. These whole genome and in silico proteome data are important as references for reexamining and integrating earlier work and observations based on lower resolution techniques, such as restriction enzyme digestion patterns, particularly for hypotheses based on pre-genomics data. Additionally, the genome of HAdV-A18 will also serve as reference for current studies examining the molecular evolution and origins of human and simian adenoviruses, particularly genome recombination studies. Applications of this virus as a potential vector for gene delivery protocols may be practical as data accumulate on this and other adenovirus genomes.
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Affiliation(s)
- Michael P. Walsh
- Department of Bioinformatics and Computational Biology, George Mason University. 10900 University Blvd., MSN 5B3, Manassas, VA 20110, USA
| | - Jason Seto
- Department of Bioinformatics and Computational Biology, George Mason University. 10900 University Blvd., MSN 5B3, Manassas, VA 20110, USA
| | - Damaris Tirado
- Clinical Investigation Facility, David Grant USAF Medical Center. Travis, CA 94535, USA
| | - James Chodosh
- Department of Ophthalmology, Howe Laboratory, Massachusetts Eye and Ear Infirmary, Harvard Medical School. Boston, MA 02114. USA
| | - David Schnurr
- Viral and Rickettsial Disease Laboratory, California Department of Public Health, Richmond, California, United States of America
| | - Donald Seto
- Department of Bioinformatics and Computational Biology, George Mason University. 10900 University Blvd., MSN 5B3, Manassas, VA 20110, USA
| | - Morris S. Jones
- Clinical Investigation Facility, David Grant USAF Medical Center. Travis, CA 94535, USA
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Abstract
Whole-genome sequencing of human adenovirus type 11 (HAdV-11) strain QS, isolated in China, was conducted, and its sequence was compared with the sequences of strains within the species of HAdVs. The HAdV-11 QS genome contains 34,755 nucleotides. Similar to the other HAdV subgenus B sequences, the HAdV-11 QS genome coded 37 functional proteins and could be divided into four early, two intermediate, and five late transcription regions. The amino acid sequences of the fiber and the hypervariable regions (HVRs) within the hexon gene of HAdV-11 QS were identical to the corresponding sequences of the HAdV-11a strain; further analyses that compared those amino acid sequences with the amino acid sequences of the HAdV species subgenus B:2 strains revealed that the highest degree of homology (>99.2%) existed between HAdV-11 QS and the prototypical HAdV-14 strain, except for a few coding sequences of HVRs within the hexon gene, DNA polymerase, pVI, and pre-terminal protein. This indicate that HAdV-11 strain QS, isolated in China, is a recombinant adenovirus of HAdV-14, and the recombination analyses also confirmed this finding. It is difficult to clarify the time and manner of the recombination, and further investigations are required to determine whether the emergence of recombination between HAdV-11a and HAdV-14 might increase virulence, thereby posing a new global challenge with regard to acute respiratory diseases in the near future.
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Seto J, Walsh MP, Mahadevan P, Purkayastha A, Clark JM, Tibbetts C, Seto D. Genomic and bioinformatics analyses of HAdV-14p, reference strain of a re-emerging respiratory pathogen and analysis of B1/B2. Virus Res 2009; 143:94-105. [PMID: 19463726 DOI: 10.1016/j.virusres.2009.03.011] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2008] [Revised: 03/22/2009] [Accepted: 03/23/2009] [Indexed: 11/25/2022]
Abstract
Unlike other human adenovirus (HAdV) species, B is divided into subspecies B1 and B2. Originally this was partly based on restriction enzyme (RE) analysis. B1 members, except HAdV-50, are commonly associated with respiratory diseases while B2 members are rarely associated with reported respiratory diseases. Recently two members of B2 have been identified in outbreaks of acute respiratory disease (ARD). One, HAdV-14, has re-emerged after an apparent 52-year absence. Genomic analysis and bioinformatics data are reported for HAdV-14 prototype for use as a reference and to understand and counter its re-emergence. The data complement and extend the original criteria for subspecies designation, unique amongst the adenoviruses, and highlight differences between B1 and B2, representing the first comprehensive analysis of this division. These data also provide finer granularity into the pathoepidemiology of the HAdVs. Whole genome analysis uncovers heterogeneous identity structures of the hexon and fiber genes amongst the HAdV-14 and the B1/B2 subspecies, which may be important in prescient vaccine development. Analysis of cell surface proteins provides insight into HAdV-14 tropism, accounting for its role as a respiratory pathogen. This HAdV-14 prototype genome is also a reference for applications of B2 adenoviruses as vectors for vaccine development and gene therapy.
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Affiliation(s)
- Jason Seto
- Department of Bioinformatics and Computational Biology, George Mason University, 10900 University Blvd., MSN 5B3, Manassas, VA 20110, USA.
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Robinson CM, Shariati F, Gillaspy AF, Dyer DW, Chodosh J. Genomic and bioinformatics analysis of human adenovirus type 37: new insights into corneal tropism. BMC Genomics 2008; 9:213. [PMID: 18471294 PMCID: PMC2397415 DOI: 10.1186/1471-2164-9-213] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2007] [Accepted: 05/09/2008] [Indexed: 01/22/2023] Open
Abstract
Background Human adenovirus type 37 (HAdV-37) is a major etiologic agent of epidemic keratoconjunctivitis, a common and severe eye infection associated with long-term visual morbidity due to persistent corneal inflammation. While HAdV-37 has been known for over 20 years as an important cause, the complete genome sequence of this serotype has yet to be reported. A detailed bioinformatics analysis of the genome sequence of HAdV-37 is extremely important to understanding its unique pathogenicity in the eye. Results We sequenced and annotated the complete genome of HAdV-37, and performed genomic and bioinformatics comparisons with other HAdVs to identify differences that might underlie the unique corneal tropism of HAdV-37. Global pairwise genome alignment with HAdV-9, a human species D adenovirus not associated with corneal infection, revealed areas of non-conserved sequence principally in genes for the virus fiber (site of host cell binding), penton (host cell internalization signal), hexon (principal viral capsid structural protein), and E3 (site of several genes that mediate evasion of the host immune system). Phylogenetic analysis revealed close similarities between predicted proteins from HAdV-37 of species D and HAdVs from species B and E. However, virtual 2D gel analyses of predicted viral proteins uncovered unexpected differences in pI and/or size of specific proteins thought to be highly similar by phylogenetics. Conclusion This genomic and bioinformatics analysis of the HAdV-37 genome provides a valuable tool for understanding the corneal tropism of this clinically important virus. Although disparities between HAdV-37 and other HAdV within species D in genes encoding structural and host receptor-binding proteins were to some extent expected, differences in the E3 region suggest as yet unknown roles for this area of the genome. The whole genome comparisons and virtual 2D gel analyses reported herein suggest potent areas for future studies.
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Affiliation(s)
- Christopher M Robinson
- Molecular Pathogenesis of Eye Infection Research Center, Dean A. McGee Eye Institute, 608 Stanton L, Young Blvd., Oklahoma City, OK 73104, USA.
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Hindley CE, Davidson AD, Matthews DA. Relationship between adenovirus DNA replication proteins and nucleolar proteins B23.1 and B23.2. J Gen Virol 2008; 88:3244-3248. [PMID: 18024892 PMCID: PMC2884980 DOI: 10.1099/vir.0.83196-0] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Adenovirus infection subverts nucleolar structure and function. B23 is a nucleolar protein present in two isoforms (B23.1 and B23.2) and both isoforms have been identified as stimulatory factors for adenovirus DNA replication. Here, it is demonstrated that the two isoforms of B23, B23.1 and B23.2, interact and co-localize differently with viral DNA replication proteins pTP and DBP in adenovirus-infected cells. Thus, the mechanism by which the two proteins stimulate viral DNA replication is likely to differ. These data also demonstrate the importance of testing both isoforms of B23 for interactions with viral proteins and nucleic acids.
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Affiliation(s)
- Clemence E Hindley
- Department of Cellular and Molecular Medicine, University Walk, University of Bristol, Bristol BS8 1TD, UK
| | - Andrew D Davidson
- Department of Cellular and Molecular Medicine, University Walk, University of Bristol, Bristol BS8 1TD, UK
| | - David A Matthews
- Department of Cellular and Molecular Medicine, University Walk, University of Bristol, Bristol BS8 1TD, UK
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Netherton C, Moffat K, Brooks E, Wileman T. A guide to viral inclusions, membrane rearrangements, factories, and viroplasm produced during virus replication. Adv Virus Res 2007; 70:101-82. [PMID: 17765705 PMCID: PMC7112299 DOI: 10.1016/s0065-3527(07)70004-0] [Citation(s) in RCA: 164] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Virus replication can cause extensive rearrangement of host cell cytoskeletal and membrane compartments leading to the “cytopathic effect” that has been the hallmark of virus infection in tissue culture for many years. Recent studies are beginning to redefine these signs of viral infection in terms of specific effects of viruses on cellular processes. In this chapter, these concepts have been illustrated by describing the replication sites produced by many different viruses. In many cases, the cellular rearrangements caused during virus infection lead to the construction of sophisticated platforms in the cell that concentrate replicase proteins, virus genomes, and host proteins required for replication, and thereby increase the efficiency of replication. Interestingly, these same structures, called virus factories, virus inclusions, or virosomes, can recruit host components that are associated with cellular defences against infection and cell stress. It is possible that cellular defence pathways can be subverted by viruses to generate sites of replication. The recruitment of cellular membranes and cytoskeleton to generate virus replication sites can also benefit viruses in other ways. Disruption of cellular membranes can, for example, slow the transport of immunomodulatory proteins to the surface of infected cells and protect against innate and acquired immune responses, and rearrangements to cytoskeleton can facilitate virus release.
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Affiliation(s)
- Christopher Netherton
- Vaccinology Group, Pirbright Laboratories, Institute for Animal Health, Surrey, United Kingdom
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Stock R, Harste G, Madisch I, Heim A. A rapid quantitative PCR-based assay for testing antiviral agents against human adenoviruses demonstrates type specific differences in ribavirin activity. Antiviral Res 2006; 72:34-41. [PMID: 16650905 DOI: 10.1016/j.antiviral.2006.03.009] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2006] [Accepted: 03/20/2006] [Indexed: 11/19/2022]
Abstract
Human adenovirus (HAdV) infections are increasingly frequent and potentially fatal as a disseminated disease in highly immunocompromised patients. Determining the in vitro sensitivity of HAdV to antiviral agents is not an easy task because HAdV CPE reduction assays are difficult to interpret and may take more than 1 week. We developed a phenotypic assay for testing the antiviral activity during the first round of replication using HAdV DNA concentration as an objective readout within 30 h. After evaluating the assay with cidofovir, we focused on determining the antiviral of ribavirin against different HAdV serotypes because clinical response of HAdV infections towards ribavirin treatment varied considerably. Several HAdV prototypes (1, 2, 5, 11, 31, 34, 48) associated with disseminated infections and clinical isolates were tested. Predominating HAdV of species C were more sensitive to ribavirin (HAdV-2 and -5: EC(50)<10 microM, EC(99) 111 and 104 microM, respectively) than HAdV of other species, for example HAdV-31 (EC(50) 56 microM, EC(99)>500 microM). Differential ribavirin sensitivity of HAdV types may contribute to the variable outcome of ribavirin therapy. Rapid screening of antiviral agents with the rapid qPCR-based assay against a multitude of HAdV serotypes may also facilitate development of future antiviral agents.
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Affiliation(s)
- Rüdiger Stock
- Institute for Virology, German National Reference Laboratory for Adenoviruses, Hannover Medical School, Germany
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Zhang Q, Su X, Gong S, Zeng Q, Zhu B, Wu Z, Peng T, Zhang C, Zhou R. Comparative genomic analysis of two strains of human adenovirus type 3 isolated from children with acute respiratory infection in southern China. J Gen Virol 2006; 87:1531-1541. [PMID: 16690917 DOI: 10.1099/vir.0.81515-0] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Human adenovirus type 3 (HAdV-3) is a causative agent of acute respiratory disease, which is prevalent throughout the world, especially in Asia. Here, the complete genome sequences of two field strains of HAdV-3 (strains GZ1 and GZ2) isolated from children with acute respiratory infection in southern China are reported (GenBank accession nos DQ099432 and DQ105654, respectively). The genomes were 35,273 bp (GZ1) and 35,269 bp (GZ2) and both had a G+C content of 51 mol%. They shared 99% nucleotide identity and the four early and five late regions that are characteristic of human adenoviruses. Thirty-nine protein- and two RNA-coding sequences were identified in the genome sequences of both strains. Protein pX had a predicted molecular mass of 8.3 kDa in strain GZ1; this was lower (7.6 kDa) in strain GZ2. Both strains contained 10 short inverted repeats, in addition to their inverted terminal repeats (111 bp). Comparative whole-genome analysis revealed 93 mismatches and four insertions/deletions between the two strains. Strain GZ1 infection produced a typical cytopathic effect, whereas strain GZ2 did not; non-synonymous substitutions in proteins of GZ2 may be responsible for this difference.
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Affiliation(s)
- Qiwei Zhang
- Central Laboratory, Guangzhou Children's Hospital, Guangzhou 510120, China
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China
| | - Xiaobo Su
- South China Sea Institute of Oceanology, LED, Chinese Academy of Sciences, Guangzhou 510301, China
| | - Sitang Gong
- Central Laboratory, Guangzhou Children's Hospital, Guangzhou 510120, China
| | - Qiyi Zeng
- Central Laboratory, Guangzhou Children's Hospital, Guangzhou 510120, China
| | - Bing Zhu
- Central Laboratory, Guangzhou Children's Hospital, Guangzhou 510120, China
| | - Zaohe Wu
- South China Sea Institute of Oceanology, LED, Chinese Academy of Sciences, Guangzhou 510301, China
| | - Tao Peng
- Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510663, China
| | - Chuyu Zhang
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China
| | - Rong Zhou
- South China Sea Institute of Oceanology, LED, Chinese Academy of Sciences, Guangzhou 510301, China
- Central Laboratory, Guangzhou Children's Hospital, Guangzhou 510120, China
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Abou El Hassan MAI, Braam SR, Kruyt FAE. A real-time RT-PCR assay for the quantitative determination of adenoviral gene expression in tumor cells. J Virol Methods 2005; 133:53-61. [PMID: 16300837 DOI: 10.1016/j.jviromet.2005.10.019] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2005] [Revised: 10/06/2005] [Accepted: 10/12/2005] [Indexed: 11/21/2022]
Abstract
Oncolytic adenoviruses are exploited as possible anticancer agents in clinical trails. To monitor adenoviral gene expression, a real-time RT-PCR method with a LightCycler was developed that allows the rapid and easy quantification of a number of early and late adenoviral genes in infected tumor cells. Primers were designed that can amplify the spliced forms of the genes encoding E1A13S, DNA polymerase (Pol), pre-terminal protein (pTP), adenoviral death protein (ADP), Hexon (Hex) and Penton (Pent) genes. Standard curves were generated using two-fold serial dilutions of cDNAs derived from non-small cell lung cancer (NSCLC) H460 cells infected for 24h with wild-type adenovirus serotype 5. For all genes correlation coefficients of the standard curves of 0.984 or higher were obtained. The dynamic range of the assay was sufficient to allow the quantitative determination of adenoviral gene expression during a lytic cycle. This RT-PCR assay could be used as a research tool to study the effect of host-cell factors or exogenous treatments on adenoviral gene expression. As example, it is shown that the procedure is suitable to detect changes in adenoviral gene expression in infected H460 cells treated with paclitaxel that is known to enhance the antitumor effect of oncolytic adenoviruses.
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Purkayastha A, Su J, McGraw J, Ditty SE, Hadfield TL, Seto J, Russell KL, Tibbetts C, Seto D. Genomic and bioinformatics analyses of HAdV-4vac and HAdV-7vac, two human adenovirus (HAdV) strains that constituted original prophylaxis against HAdV-related acute respiratory disease, a reemerging epidemic disease. J Clin Microbiol 2005; 43:3083-94. [PMID: 16000418 PMCID: PMC1169186 DOI: 10.1128/jcm.43.7.3083-3094.2005] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/25/2004] [Revised: 03/21/2005] [Accepted: 04/06/2005] [Indexed: 11/20/2022] Open
Abstract
Vaccine strains of human adenovirus serotypes 4 and 7 (HAdV-4vac and HAdV-7vac) have been used successfully to prevent adenovirus-related acute respiratory disease outbreaks. The genomes of these two vaccine strains have been sequenced, annotated, and compared with their prototype equivalents with the goals of understanding their genomes for molecular diagnostics applications, vaccine redevelopment, and HAdV pathoepidemiology. These reference genomes are archived in GenBank as HAdV-4vac (35,994 bp; AY594254) and HAdV-7vac (35,240 bp; AY594256). Bioinformatics and comparative whole-genome analyses with their recently reported and archived prototype genomes reveal six mismatches and four insertions-deletions (indels) between the HAdV-4 prototype and vaccine strains, in contrast to the 611 mismatches and 130 indels between the HAdV-7 prototype and vaccine strains. Annotation reveals that the HAdV-4vac and HAdV-7vac genomes contain 51 and 50 coding units, respectively. Neither vaccine strain appears to be attenuated for virulence based on bioinformatics analyses. There is evidence of genome recombination, as the inverted terminal repeat of HAdV-4vac is initially identical to that of species C whereas the prototype is identical to species B1. These vaccine reference sequences yield unique genome signatures for molecular diagnostics. As a molecular forensics application, these references identify the circulating and problematic 1950s era field strains as the original HAdV-4 prototype and the Greider prototype, from which the vaccines are derived. Thus, they are useful for genomic comparisons to current epidemic and reemerging field strains, as well as leading to an understanding of pathoepidemiology among the human adenoviruses.
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Affiliation(s)
- Anjan Purkayastha
- Bioinformatics and Computational Biology Program, School of Computational Sciences, George Mason University, 10900 University Boulevard, MSN 5B3, Manassas, Virginia 20110, USA
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Purkayastha A, Su J, Carlisle S, Tibbetts C, Seto D. Genomic and bioinformatics analysis of HAdV-7, a human adenovirus of species B1 that causes acute respiratory disease: implications for vector development in human gene therapy. Virology 2005; 332:114-29. [PMID: 15661145 DOI: 10.1016/j.virol.2004.10.041] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2004] [Revised: 07/25/2004] [Accepted: 10/26/2004] [Indexed: 01/11/2023]
Abstract
Human adenovirus serotype 7 (HAdV-7) is a reemerging pathogen identified in acute respiratory disease (ARD), particularly in epidemics affecting basic military trainee populations of otherwise healthy young adults. The genome has been sequenced and annotated (GenBank accession no. ). Comparative genomics and bioinformatics analyses of the HAdV-7 genome sequence provide insight into its natural history and phylogenetic relationships. A putative origin of HAdV-7 from a chimpanzee host is observed. This has implications within the current biotechnological interest of using chimpanzee adenoviruses as vectors for human gene therapy and DNA vaccine delivery. Rapid genome sequencing and analyses of this species B1 member provide an example of exploiting accurate low-pass DNA sequencing technology in pathogen characterization and epidemic outbreak surveillance through the identification, validation, and application of unique pathogen genome signatures.
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Affiliation(s)
- Anjan Purkayastha
- Bioinformatics and Computational Biology, School of Computational Sciences, George Mason University, 10900 University Boulevard, MSN 5B3, Manassas, VA 20110, USA
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15
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Purkayastha A, Ditty SE, Su J, McGraw J, Hadfield TL, Tibbetts C, Seto D. Genomic and bioinformatics analysis of HAdV-4, a human adenovirus causing acute respiratory disease: implications for gene therapy and vaccine vector development. J Virol 2005; 79:2559-72. [PMID: 15681456 PMCID: PMC546560 DOI: 10.1128/jvi.79.4.2559-2572.2005] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2004] [Accepted: 10/13/2004] [Indexed: 11/20/2022] Open
Abstract
Human adenovirus serotype 4 (HAdV-4) is a reemerging viral pathogenic agent implicated in epidemic outbreaks of acute respiratory disease (ARD). This report presents a genomic and bioinformatics analysis of the prototype 35,990-nucleotide genome (GenBank accession no. AY594253). Intriguingly, the genome analysis suggests a closer phylogenetic relationship with the chimpanzee adenoviruses (simian adenoviruses) rather than with other human adenoviruses, suggesting a recent origin of HAdV-4, and therefore species E, through a zoonotic event from chimpanzees to humans. Bioinformatics analysis also suggests a pre-zoonotic recombination event, as well, between species B-like and species C-like simian adenoviruses. These observations may have implications for the current interest in using chimpanzee adenoviruses in the development of vectors for human gene therapy and for DNA-based vaccines. Also, the reemergence, surveillance, and treatment of HAdV-4 as an ARD pathogen is an opportunity to demonstrate the use of genome determination as a tool for viral infectious disease characterization and epidemic outbreak surveillance: for example, rapid and accurate low-pass sequencing and analysis of the genome. In particular, this approach allows the rapid identification and development of unique probes for the differentiation of family, species, serotype, and strain (e.g., pathogen genome signatures) for monitoring epidemic outbreaks of ARD.
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MESH Headings
- Adenovirus Infections, Human/epidemiology
- Adenovirus Infections, Human/prevention & control
- Adenovirus Infections, Human/therapy
- Adenoviruses, Human/classification
- Adenoviruses, Human/genetics
- Adenoviruses, Human/pathogenicity
- Cell Line, Tumor
- Computational Biology
- DNA, Viral/chemistry
- DNA, Viral/genetics
- Genetic Therapy
- Genome, Viral
- Humans
- Molecular Sequence Data
- Phylogeny
- Respiratory Tract Infections/epidemiology
- Respiratory Tract Infections/prevention & control
- Respiratory Tract Infections/transmission
- Respiratory Tract Infections/virology
- Viral Vaccines/administration & dosage
- Viral Vaccines/genetics
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Affiliation(s)
- Anjan Purkayastha
- School of Bioinformatics and Computational Biology, School of Computational Sciences, George Mason University, 10900 University Blvd., Manassas, VA 20110, USA
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16
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Lauer KP, Llorente I, Blair E, Seto J, Krasnov V, Purkayastha A, Ditty SE, Hadfield TL, Buck C, Tibbetts C, Seto D. Natural variation among human adenoviruses: genome sequence and annotation of human adenovirus serotype 1. J Gen Virol 2004; 85:2615-2625. [PMID: 15302955 DOI: 10.1099/vir.0.80118-0] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
The 36,001 base pair DNA sequence of human adenovirus serotype 1 (HAdV-1) has been determined, using a 'leveraged primer sequencing strategy' to generate high quality sequences economically. This annotated genome (GenBank AF534906) confirms anticipated similarity to closely related species C (formerly subgroup), human adenoviruses HAdV-2 and -5, and near identity with earlier reports of sequences representing parts of the HAdV-1 genome. A first round of HAdV-1 sequence data acquisition used PCR amplification and sequencing primers from sequences common to the genomes of HAdV-2 and -5. The subsequent rounds of sequencing used primers derived from the newly generated data. Corroborative re-sequencing with primers selected from this HAdV-1 dataset generated sparsely tiled arrays of high quality sequencing ladders spanning both complementary strands of the HAdV-1 genome. These strategies allow for rapid and accurate low-pass sequencing of genomes. Such rapid genome determinations facilitate the development of specific probes for differentiation of family, serotype, subtype and strain (e.g. pathogen genome signatures). These will be used to monitor epidemic outbreaks of acute respiratory disease in a defined test bed by the Epidemic Outbreak Surveillance (EOS) project.
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Affiliation(s)
- Kim P Lauer
- Bioinformatics and Computational Biology, School of Computational Sciences, George Mason University, 10900 University Boulevard, MSN 5B3, Manassas, VA 20110, USA
| | - Isabel Llorente
- Bioinformatics and Computational Biology, School of Computational Sciences, George Mason University, 10900 University Boulevard, MSN 5B3, Manassas, VA 20110, USA
| | - Eric Blair
- Bioinformatics and Computational Biology, School of Computational Sciences, George Mason University, 10900 University Boulevard, MSN 5B3, Manassas, VA 20110, USA
| | - Jason Seto
- Bioinformatics and Computational Biology, School of Computational Sciences, George Mason University, 10900 University Boulevard, MSN 5B3, Manassas, VA 20110, USA
| | - Vladimir Krasnov
- Bioinformatics and Computational Biology, School of Computational Sciences, George Mason University, 10900 University Boulevard, MSN 5B3, Manassas, VA 20110, USA
| | - Anjan Purkayastha
- Epidemic Outbreak Surveillance (EOS) Consortium, 5201 Leesburg Pike, Suite 1401, Falls Church, VA 22041, USA
- HQ USAF Surgeon General Office, Directorate of Modernization (SGR), 5201 Leesburg Pike, Suite 1401, Falls Church, VA 22041, USA
- Bioinformatics and Computational Biology, School of Computational Sciences, George Mason University, 10900 University Boulevard, MSN 5B3, Manassas, VA 20110, USA
| | - Susan E Ditty
- Epidemic Outbreak Surveillance (EOS) Consortium, 5201 Leesburg Pike, Suite 1401, Falls Church, VA 22041, USA
- Division of Microbiology, Department of Infectious and Parasitic Diseases Pathology, Armed Forces Institute of Pathology, 5300 Georgia Avenue NW, Washington, DC 20306, USA
| | - Ted L Hadfield
- Epidemic Outbreak Surveillance (EOS) Consortium, 5201 Leesburg Pike, Suite 1401, Falls Church, VA 22041, USA
- Division of Microbiology, Department of Infectious and Parasitic Diseases Pathology, Armed Forces Institute of Pathology, 5300 Georgia Avenue NW, Washington, DC 20306, USA
| | - Charles Buck
- Department of Virology, American Type Culture Collection (ATCC), Manassas, VA 20108, USA
| | - Clark Tibbetts
- Epidemic Outbreak Surveillance (EOS) Consortium, 5201 Leesburg Pike, Suite 1401, Falls Church, VA 22041, USA
- HQ USAF Surgeon General Office, Directorate of Modernization (SGR), 5201 Leesburg Pike, Suite 1401, Falls Church, VA 22041, USA
| | - Donald Seto
- Epidemic Outbreak Surveillance (EOS) Consortium, 5201 Leesburg Pike, Suite 1401, Falls Church, VA 22041, USA
- HQ USAF Surgeon General Office, Directorate of Modernization (SGR), 5201 Leesburg Pike, Suite 1401, Falls Church, VA 22041, USA
- Bioinformatics and Computational Biology, School of Computational Sciences, George Mason University, 10900 University Boulevard, MSN 5B3, Manassas, VA 20110, USA
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17
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Mysiak ME, Bleijenberg MH, Wyman C, Holthuizen PE, van der Vliet PC. Bending of adenovirus origin DNA by nuclear factor I as shown by scanning force microscopy is required for optimal DNA replication. J Virol 2004; 78:1928-35. [PMID: 14747557 PMCID: PMC369512 DOI: 10.1128/jvi.78.4.1928-1935.2004] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Nuclear factor I (NFI) is a transcription factor that binds to the adenovirus type 5 (Ad5) origin of replication and recruits the adenovirus DNA polymerase, thereby stimulating initiation of DNA replication in vitro. Using scanning force microscopy, we demonstrate that NFI induces a 60 degrees bend upon binding to the origin. The A/T-rich region preceding the core recognition sequence of NFI influences the DNA bend angle, since substitution of A/T base pairs by G/C base pairs severely decreases bending. Mutations in the A/T-rich region do not affect binding of NFI to DNA. However, mutations that reduce the protein-induced bend lead to a loss of NFI-stimulated replication, indicating that DNA bending is functionally important. In contrast, basal initiation or DNA binding of the polymerase is not impaired by these origin mutations. We conclude that binding of NFI to the Ad5 origin causes structural changes in DNA that are essential for the stimulatory function of NFI in replication. We propose that NFI-induced origin bending facilitates the assembly of a functional initiation complex.
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Affiliation(s)
- Monika E Mysiak
- Department of Physiological Chemistry, University Medical Center Utrecht, and Centre for Biomedical Genetics, 3584 CG Utrecht, The Netherlands
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18
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Abstract
Replication of the adenovirus genome is catalysed by adenovirus DNA polymerase in which the adenovirus preterminal protein acts as a protein primer. DNA polymerase and preterminal protein form a heterodimer which, in the presence of the cellular transcription factors NFI/CTFI and NFIII/Oct-1, binds to the origin of DNA replication. DNA replication is initiated by DNA polymerase mediated transfer of dCMP onto preterminal protein. Further DNA synthesis is catalysed by DNA polymerase in a strand displacement mechanism which also requires adenovirus DNA binding protein. Here, we discuss the role of individual proteins in this process as revealed by biochemical analysis, mutagenesis and molecular modelling.
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Affiliation(s)
- H Liu
- Centre for Biomolecular Science, Biomolecular Science Building, The University of St. Andrews, North Haugh, St. Andrews, KY16 9ST, UK
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19
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Hösel M, Webb D, Schröer J, Doerfler W. The abortive infection of Syrian hamster cells with human adenovirus type 12. Curr Top Microbiol Immunol 2003; 272:415-40. [PMID: 12747558 DOI: 10.1007/978-3-662-05597-7_14] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/02/2023]
Abstract
Human adenovirus type 12 (Ad12) induces undifferentiated tumors in newborn Syrian hamsters, and this tumor model has been investigated in detail in our laboratory. One of the characteristics of the Ad12-hamster cell system is a strictly abortive infection cycle. In this chapter, we summarize previous and more recent results of studies on the interaction of Ad12 with the nonpermissive BHK21 hamster cell line. The block of Ad12 replication lies before viral DNA replication and late gene transcription which cannot be detected with the most sensitive techniques. Ad12 adsorption, cellular uptake and transport of the viral DNA to the nucleus are less efficient in the nonpermissive hamster cells than in permissive human cells. However, most of the early functions of the Ad12 genome are expressed in BHK21 cells, though at a low level. In the downstream region, the first exon, of the major late promoter (MLP) of Ad12 DNA, a mitigator element of 33 nucleotide pairs in length has been identified which contributes to the inactivity of the MLP in hamster cells and its markedly decreased activity in human cells. The E1 functions of Ad2 or Ad5 are capable of partly complementing the Ad12 deficiencies in hamster cells in that Ad12 viral DNA replication and late gene transcription can proceed, e.g. in a BHK hamster cell line, BHK297-C131,which carries in an integrated form and constitutively expresses the E1 region of Ad5 DNA. Nevertheless, the late Ad12 mRNAs, which are synthesized in this system with the authentic nucleotide sequence, fail to be translated to structural viral proteins. Hence, infectious virions are not produced in the partly complementing system. Probably there is also a translational block for late Ad12 mRNAs in hamster cells. We have recently shown that the overexpression of the Ad12 preterminal protein (pTP) gene or of the E1A gene facilitates the synthesis of full-length, authentic Ad12 DNA in BHK21 cells infected with Ad12. Apparently the pTP has a hitherto unknown function in eliciting full cycles of Ad12 DNA replication even in nonpermissive BHK21 cells when sufficient levels of Ad12 pTP are produced. We pursue the possibility that the completely abortive infection cycle of Ad12 in hamster cells ensures the survival of Ad12-induced hamster tumor cells which all carry, integrated in their genomes, multiple copies of Ad12 DNA. In this way, the viral genomes are immortalized and expanded in a huge number of tumor cells.
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Affiliation(s)
- M Hösel
- Institut für Genetik, Universität zu Köln, 50931 Köln, Germany.
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20
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de Jong RN, van der Vliet PC, Brenkman AB. Adenovirus DNA replication: protein priming, jumping back and the role of the DNA binding protein DBP. Curr Top Microbiol Immunol 2003; 272:187-211. [PMID: 12747551 DOI: 10.1007/978-3-662-05597-7_7] [Citation(s) in RCA: 42] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/11/2023]
Abstract
The adenovirus (Ad) genome is a linear double-stranded (ds) molecule containing about 36 kilobase pairs. At each end of the genome an approximately 100 base pair (bp) inverted terminal repeat (ITR) is found, the exact length depending on the serotype. To the 5'-end of each ITR, a 55-kDa terminal protein (TP) is covalently coupled. The Ad DNA replication system was one of the first replication systems that could be reconstituted in vitro (Challberg and Kelly 1979). The system requires three virally encoded proteins: precursor TP (pTP), DNA polymerase (Pol) and the DNA binding protein (DBP). In addition, three stimulating human cellular proteins have been identified. These are the transcription factors NFI (Nagata et al. 1982) and Oct-1 (Pruijn et al. 1986) and the type I topoisomerase NFII (Nagata et al. 1983). Ad DNA replication uses a protein primer for replication initiation. The transition from initiation to elongation is marked by a jumping back mechanism (King and van der Vliet 1994), followed by elongation. In order to elongate DBP is required. In this review we discuss the roles of DBP during initiation and elongation and we relate biochemical data on the jumping back mechanism used by Ad Pol to the recently solved crystal structure of a Pol alpha-like replication complex (Franklin et al. 2001). We comment on the conditions and possible functions of jumping back and propose a model to describe the jumping back mechanism.
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Affiliation(s)
- R N de Jong
- University Medical Center, Department of Physiological Chemistry and Center for Biomedical Genetics, Utrecht, The Netherlands.
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21
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Stone D, Furthmann A, Sandig V, Lieber A. The complete nucleotide sequence, genome organization, and origin of human adenovirus type 11. Virology 2003; 309:152-65. [PMID: 12726735 DOI: 10.1016/s0042-6822(02)00085-5] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
The complete DNA sequence and transcription map of human adenovirus type 11 are reported here. This is the first published sequence for a subgenera B human adenovirus and demonstrates a genome organization highly similar to those of other human adenoviruses. All of the genes from the early, intermediate, and late regions are present in the expected locations of the genome for a human adenovirus. The genome size is 34,794 bp in length and has a GC content of 48.9%. Sequence alignment with genomes of groups A (Ad12), C (Ad5), D (Ad17), E (Simian adenovirus 25), and F (Ad40) revealed homologies of 64, 54, 68, 75, and 52%, respectively. Detailed genomic analysis demonstrated that Ads 11 and 35 are highly conserved in all areas except the hexon hypervariable regions and fiber. Similarly, comparison of Ad11 with subgroup E SAV25 revealed poor homology between fibers but high homology in proteins encoded by all other areas of the genome. We propose an evolutionary model in which functional viruses can be reconstituted following fiber substitution from one serotype to another. According to this model either the Ad11 genome is a derivative of Ad35, from which the fiber was substituted with Ad7, or the Ad35 genome is the product of a fiber substitution from Ad21 into the Ad11 genome. This model also provides a possible explanation for the origin of group E Ads, which are evolutionarily derived from a group C fiber substitution into a group B genome.
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Affiliation(s)
- Daniel Stone
- Division of Medical Genetics, University of Washington, Seattle, WA 98195, USA
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22
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van Breukelen B, Brenkman AB, Holthuizen PE, van der Vliet PC. Adenovirus type 5 DNA binding protein stimulates binding of DNA polymerase to the replication origin. J Virol 2003; 77:915-22. [PMID: 12502807 PMCID: PMC140850 DOI: 10.1128/jvi.77.2.915-922.2003] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The adenovirus (Ad) DNA-binding protein (DBP) is essential for the elongation phase of Ad DNA replication by unwinding the template in an ATP-independent fashion, employing its capacity to form multimers. DBP also enhances the rate of initiation, with the highest levels obtained at low concentrations of Ad DNA polymerase (Pol). Here, we show that stimulation of initiation depends on the template conformation. Maximal stimulation, up to 15-fold, is observed on double-stranded or viral TP-containing origins. The stimulation is reduced on partially single-stranded origins and DBP does not enhance initiation any more once the origin is completely unwound. This suggests a role for DBP in origin unwinding that is comparable to its unwinding capacity during elongation. However, mutant DBP proteins defective in unwinding and elongation can still enhance initiation on ds templates. DBP also stimulates the binding of nuclear factor I (NFI) to the origin and lowers the K(m) for coupling of the first nucleotide to the precursor terminal protein by Pol. Mobility shift experiments reveal that DBP stimulates the binding of Pol on double-stranded origin and nonorigin DNA but not on single-stranded DNA. This effect is specific for DBP and is also seen with other DNA Pols. Our results suggest that, rather than by origin unwinding, DBP enhances initiation by modulating the origin conformation such that DNA Pol can bind more efficiently.
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Affiliation(s)
- Bas van Breukelen
- Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Center Utrecht, The Netherlands
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23
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Brenkman AB, Breure EC, van der Vliet PC. Molecular architecture of adenovirus DNA polymerase and location of the protein primer. J Virol 2002; 76:8200-7. [PMID: 12134025 PMCID: PMC155156 DOI: 10.1128/jvi.76.16.8200-8207.2002] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2002] [Accepted: 05/13/2002] [Indexed: 11/20/2022] Open
Abstract
Adenovirus (Ad) DNA polymerase (pol) belongs to the distinct subclass of the polalpha family of DNA pols that employs the precursor terminal protein (pTP) as primer. Ad pol forms a stable heterodimer with this primer, and together, they bind specifically to the core origin in order to start replication. After initiation of Ad replication, the resulting pTP-trinucleotide intermediate jumps back and pTP starts to dissociate. Compared to free Ad pol, the pTP-pol complex shows reduced polymerase and exonuclease activities, but the reason for this is not understood. Furthermore, the interaction domains between these proteins have not been defined and the contribution of each protein to origin binding is unclear. To address these questions, we used oligonucleotides with a translocation block and show here that pTP binds at the entrance of the primer binding groove of Ad pol, thereby explaining the decreased synthetic activities of the pTP-pol complex and providing insight into how pTP primes Ad replication. Employing an exonuclease-deficient mutant polymerase, we further show that the polymerase and exonuclease active sites of Ad pol are spatially distinct and that the exonuclease activity of Ad pol is located at the N-terminal part of the protein. In addition, by probing the distances between both active sites and the surface of Ad pol, we show that Ad pol binds a DNA region of 14 to 15 nucleotides. Based on these results, a model for binding of the pTP-pol complex at the origin of replication is proposed.
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Affiliation(s)
- Arjan B Brenkman
- University Medical Centre, Department of Physiological Chemistry and Centre for Biomedical Genetics, Utrecht, The Netherlands
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24
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Affiliation(s)
- E Sadowy
- Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
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25
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Brenkman AB, Heideman MR, Truniger V, Salas M, van der Vliet PC. The (I/Y)XGG motif of adenovirus DNA polymerase affects template DNA binding and the transition from initiation to elongation. J Biol Chem 2001; 276:29846-53. [PMID: 11390396 DOI: 10.1074/jbc.m103159200] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Adenovirus DNA polymerase (Ad pol) is a eukaryotic-type DNA polymerase involved in the catalysis of protein-primed initiation as well as DNA polymerization. The functional significance of the (I/Y)XGG motif, highly conserved among eukaryotic-type DNA polymerases, was analyzed in Ad pol by site-directed mutagenesis of four conserved amino acids. All mutant polymerases could bind primer-template DNA efficiently but were impaired in binding duplex DNA. Three mutant polymerases required higher nucleotide concentrations for effective polymerization and showed higher exonuclease activity on double-stranded DNA. These observations suggest a local destabilization of DNA substrate at the polymerase active site. In agreement with this, the mutant polymerases showed reduced initiation activity and increased K(m)(app) for the initiating nucleotide, dCMP. Interestingly, one mutant polymerase, while capable of elongating on the primer-template DNA, failed to elongate after protein priming. Further investigation of this mutant polymerase showed that polymerization activity decreased after each polymerization step and ceased completely after formation of the precursor terminal protein-trinucleotide (pTP-CAT) initiation intermediate. Our results suggest that residues in the conserved motif (I/Y)XGG in Ad pol are involved in binding the template strand in the polymerase active site and play an important role in the transition from initiation to elongation.
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Affiliation(s)
- A B Brenkman
- University Medical Center, Department of Physiological Chemistry and Center for Biomedical Genetics, Utrecht, The Netherlands
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26
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Gonzalez-Huici V, Salas M, Hermoso JM. Sequence requirements for protein-primed initiation and elongation of phage O29 DNA replication. J Biol Chem 2000; 275:40547-53. [PMID: 11006291 DOI: 10.1074/jbc.m007170200] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The double-stranded linear DNA of Bacillus subtilis phage O29 is replicated by a mechanism in which a terminal protein (TP) acts as a primer. The second 3'-terminal nucleotide of the template directs the incorporation of the 5'-terminal nucleotide into the TP, giving rise to the initiation complex TP-dAMP. Elongation then proceeds by a sliding-back mechanism in which the dAMP covalently linked to the TP pairs to the 3'-terminal nucleotide of the template strand to recover full-length DNA. We have studied the sequence requirements for efficient initiation of replication using mutated TP-free double-stranded DNA fragments. Efficient initiation only requires the terminal repetition 5'-AA. The 3'-terminal T, although not used as template, increases the affinity of DNA polymerase for the initiator nucleotide; in addition, although to a minor extent, the third 3'-terminal position also directs the formation of the initiation complex and modulates the initiation rate at the second position. Efficient elongation requires a previous sliding-back, demanding again a repetition of two nucleotides at the 3' end; if the sliding-back is prevented, a residual elongation can proceed directly from the second position or after jumping back from the third to the first position.
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Affiliation(s)
- V Gonzalez-Huici
- Centro de Biologia Molecular Severo Ochoa (Consejo Superior de Investigaciones Cientificas-Universidad Autónoma Madrid), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain
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27
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Liu H, Naismith JH, Hay RT. Identification of conserved residues contributing to the activities of adenovirus DNA polymerase. J Virol 2000; 74:11681-9. [PMID: 11090167 PMCID: PMC112450 DOI: 10.1128/jvi.74.24.11681-11689.2000] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Adenovirus codes for a DNA polymerase that is a member of the DNA polymerase alpha family and uses a protein primer for initiation of DNA synthesis. It contains motifs characteristic of a proofreading 3'-5'-exonuclease domain located in the N-terminal region and several polymerase motifs located in the C-terminal region. To determine the role of adenovirus DNA polymerase in DNA replication, 22 site-directed mutations were introduced into the conserved DNA polymerase motifs in the C-terminal region of adenovirus DNA polymerase and the mutant forms were expressed in insect cells using a baculovirus expression system. Each mutant enzyme was tested for DNA binding activity, the ability to interact with pTP, DNA polymerase catalytic activity, and the ability to participate in the initiation of adenovirus DNA replication. The mutant phenotypes identify functional domains within the adenovirus DNA polymerase and allow discrimination between the roles of conserved residues in the various activities carried out by the protein. Using the functional data in this study and the previously published structure of the bacteriophage RB69 DNA polymerase (J. Wang et al., Cell 89:1087-1099, 1997), it is possible to envisage how the conserved domains in the adenovirus DNA polymerase function.
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Affiliation(s)
- H Liu
- Centre for Biomolecular Science, The University of St. Andrews, North Haugh, St. Andrews KY16 9ST, United Kingdom
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28
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Lieber A, Kay MA, Li ZY. Nuclear import of moloney murine leukemia virus DNA mediated by adenovirus preterminal protein is not sufficient for efficient retroviral transduction in nondividing cells. J Virol 2000; 74:721-34. [PMID: 10623734 PMCID: PMC111592 DOI: 10.1128/jvi.74.2.721-734.2000] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/1999] [Accepted: 10/07/1999] [Indexed: 11/20/2022] Open
Abstract
Moloney murine leukemia virus (MoMLV)-derived vectors require cell division for efficient transduction, which may be related to an inability of the viral DNA-protein complex to cross the nuclear membrane. In contrast, adenoviruses (Ad) can efficiently infect nondividing cells. This property may be due to the presence of multiple nuclear translocation signals in a number of Ad proteins, which are associated with the incoming viral genomes. Of particular interest is the Ad preterminal protein (pTP), which binds alone or in complex with the Ad polymerase to specific sequences in the Ad inverted terminal repeat. The goal of this study was to test whether coexpression of pTP with retroviral DNA carrying pTP-binding sites would facilitate nuclear import of the viral preintegration complex and transduction of quiescent cells. In preliminary experiments, we demonstrated that the karyophylic pTP can coimport plasmid DNA into the nuclei of growth-arrested cells. Retroviral transduction studies were performed with G(1)/S-arrested LTA cells or stationary-phase human primary fibroblasts. These studies demonstrated that pTP or pTP-Ad polymerase conferred nuclear import of retroviral DNA upon arrested cells when the retrovirus vector contained the corresponding binding motifs. However, pTP-mediated nuclear translocation of MoMLV DNA in nondividing cells was not sufficient for stable transduction. Additional cellular factors activated during S phase or DNA repair synthesis were required for efficient retroviral integration.
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Affiliation(s)
- A Lieber
- Division of Medical Genetics, University of Washington, Seattle, Washington 98195, USA.
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Botting CH, Hay RT. Characterisation of the adenovirus preterminal protein and its interaction with the POU homeodomain of NFIII (Oct-1). Nucleic Acids Res 1999; 27:2799-805. [PMID: 10373599 PMCID: PMC148491 DOI: 10.1093/nar/27.13.2799] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
Formation of the preinitiation complex for adenovirus DNA replication involves the incoming preterminal protein-adenovirus DNA polymerase heterodimer being positioned at the origin of replication by protein-DNA and protein-protein interactions. Preterminal protein directly binds to the cellular transcription factor nuclear factor III (Oct-1), via the POU homeodomain. Co-precipitation of POU with individual domains of preterminal protein expressed by in vitro translation indicated that POU contacts multiple sites on preterminal protein. Partial proteolysis of preterminal protein in the presence or absence of POU homeodomain demonstrated that many sites accessible to proteases in free preterminal protein were resistant to cleavage in the presence of POU homeodomain. The accessibility of sites in free preterminal protein to cleavage by trypsin was strongly dependent on the ionic strength, suggesting that preterminal protein may undergo a sodium chloride-induced conformational change. It is therefore likely that the POU homeodomain contacts a number of sites on preterminal protein to induce a conformational change which may influence the initiation of adenovirus DNA replication.
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Affiliation(s)
- C H Botting
- School of Biomedical Sciences, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, UK
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30
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Pitcovski J, Mualem M, Rei-Koren Z, Krispel S, Shmueli E, Peretz Y, Gutter B, Gallili GE, Michael A, Goldberg D. The complete DNA sequence and genome organization of the avian adenovirus, hemorrhagic enteritis virus. Virology 1998; 249:307-15. [PMID: 9791022 DOI: 10.1006/viro.1998.9336] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Hemorrhagic enteritis virus (HEV) belongs to the Adenoviridae family, a subgroup of adenoviruses (Ads) that infect avian species. In this article, the complete DNA sequence and the genome organization of the virus are described. The full-length of the genome was found to be 26,263 bp, shorter than the DNA of any other Ad described so far. The G + C content of the genome is 34.93%. There are short terminal repeats (39 bp), as described for other Ads. Genes were identified by comparison of the DNA and predicted amino acid sequences with published sequences of other Ads. The organization of the genome in respect to late genes (52K, IIIa, penton base, core protein, hexon, endopeptidase, 100K, pVIII, and fiber), early region 2 genes (polymerase, terminal protein, and DNA binding protein), and intermediate gene IVa2 was found to be similar to that of other human and avian Ad genomes. No sequences similar to E1 and E4 regions were found. Very low similarity to ovine E3 region was found. Open reading frames were identified with no similarity to any published Ad sequence.
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Affiliation(s)
- J Pitcovski
- South Industrial Zone, MIGAL, Kiryat Shmona, 10200, Israel.
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31
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Angeletti PC, Engler JA. Adenovirus preterminal protein binds to the CAD enzyme at active sites of viral DNA replication on the nuclear matrix. J Virol 1998; 72:2896-904. [PMID: 9525610 PMCID: PMC109735 DOI: 10.1128/jvi.72.4.2896-2904.1998] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/1997] [Accepted: 12/18/1997] [Indexed: 02/06/2023] Open
Abstract
Adenovirus (Ad) replicative complexes form at discrete sites on the nuclear matrix (NM) via an interaction mediated by the precursor of the terminal protein (pTP). The identities of cellular proteins involved in these complexes have remained obscure. We present evidence that pTP binds to a multifunctional pyrimidine biosynthesis enzyme found at replication domains on the NM. Far-Western blotting identified proteins of 150 and 240 kDa that had pTP binding activity. Amino acid sequencing of the 150-kDa band revealed sequence identity to carbamyl phosphate synthetase I (CPS I) and a high degree of homology to the related trifunctional enzyme known as CAD (for carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase). Western blotting with an antibody directed against CAD detected a 240-kDa band that comigrated with that detected by pTP far-Western blotting. Binding experiments showed that a pTP-CAD complex was immunoprecipitable from cell extracts in which pTP was expressed by a vaccinia virus recombinant. Additionally, in vitro-translated epitope-tagged pTP and CAD were immunoprecipitable as a complex, indicating the occurrence of a protein-protein interaction. Confocal fluorescence microscopy of Ad-infected NM showed that pTP and CAD colocalized in nuclear foci. Both pTP and CAD were confirmed to colocalize with active sites of replication detected by bromodeoxyuridine incorporation. These data support the concept that the pTP-CAD interaction may allow anchorage of Ad replication complexes in the proximity of required cellular factors and may help to segregate replicated and unreplicated viral DNA.
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Affiliation(s)
- P C Angeletti
- Department of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham, 35294-0005, USA
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32
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Webster A, Leith IR, Nicholson J, Hounsell J, Hay RT. Role of preterminal protein processing in adenovirus replication. J Virol 1997; 71:6381-9. [PMID: 9261355 PMCID: PMC191911 DOI: 10.1128/jvi.71.9.6381-6389.1997] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
Preterminal protein (pTP), the protein primer for adenovirus DNA replication, is processed at two sites by the virus-encoded protease to yield mature terminal protein (TP). Here we demonstrate that processing to TP, via an intermediate (iTP), is conserved in all serotypes sequenced to date; and in determining the sites cleaved in Ad4 pTP, we extend the previously published substrate specificity of human adenovirus proteases to include a glutamine residue at P4. Furthermore, using monoclonal antibodies raised against pTP, we show that processing to iTP and TP are temporally separated in the infectious cycle, with processing to iTP taking place outside the virus particles. In vitro and in vivo studies of viral DNA replication reveal that iTP can act as a template for initiation and elongation and argue against a role for virus-encoded protease in switching off DNA replication. Virus DNA with TP attached to its 5' end (TP-DNA) has been studied extensively in in vitro DNA replication assays. Given that in vivo pTP-DNA, not TP-DNA, is the template for all but the first round of replication, the two templates were compared in vitro and shown to have different properties. Immunofluorescence studies suggest that a region spanning the TP cleavage site is involved in defining the subnuclear localization of pTP. Therefore, a likely role for the processing of pTP-DNA is to create a distinct template for early transcription (TP-DNA), while the terminal protein moiety, be it TP or pTP, serves to guide the template to the appropriate subcellular location through the course of infection.
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Affiliation(s)
- A Webster
- School of Biological and Medical Science, University of St. Andrews, Fife, Scotland
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33
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van Leeuwen HC, Rensen M, van der Vliet PC. The Oct-1 POU homeodomain stabilizes the adenovirus preinitiation complex via a direct interaction with the priming protein and is displaced when the replication fork passes. J Biol Chem 1997; 272:3398-405. [PMID: 9013582 DOI: 10.1074/jbc.272.6.3398] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
Initiation of adenovirus DNA replication is strongly enhanced by two cellular transcription factors, NFI and Oct-1, which bind to the auxiliary origin and tether the viral precursor terminal protein-DNA polymerase (pTP.pol) complex to the core origin. NFI acts through a direct contact with the DNA polymerase, but the mode of action of Oct 1 is unknown. Employing glutathione S-transferase-POU pull-down assays and protein affinity chromatography, we have established that the POU domain contacts pTP rather than pol. The POU homeodomain is responsible for this interaction. The protein-protein contacts lead to increased binding of pTP-pol to the core origin, which is caused by a reduced off-rate. The enhanced formation of a pTP.pol.POU complex on the origin correlates with stimulation of replication. Using an immobilized replication system, we have studied the kinetics of dissociation of the Oct-1 POU domain during replication. In contrast to NFI, which dissociates very early in initiation, Oct-1 dissociates only when the binding site is rendered single-stranded upon translocation of the replication fork. Our data indicate that NFI and Oct-1 enhance initiation synergistically by touching different targets in the preinitiation complex and dissociate independently after initiation.
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Affiliation(s)
- H C van Leeuwen
- Laboratory for Physiological Chemistry, Utrecht University, Stratenum, P. O. Box 80042, 3508 TA Utrecht, The Netherlands
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34
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Abstract
In adenovirus-infected cells, the virus-encoded preterminal protein and DNA polymerase form a heterodimer that is directly involved in initiation of DNA replication. Monoclonal antibodies were raised against preterminal protein, and epitopes recognized by the antibodies were identified by using synthetic peptides. Partial proteolysis of preterminal protein reveals that it has a tripartite structure, with the three domains being separated by two protease-sensitive areas, located at sites processed by adenovirus protease. These areas of protease sensitivity are probably surface-exposed loops, as they are the sites, along with the C-terminal region of preterminal protein, recognized by the monoclonal antibodies. Preterminal protein is protected from proteolytic cleavage when bound to adenovirus DNA polymerase, suggesting either multiple contact points between the proteins or a DNA polymerase-induced conformational change in preterminal protein. Two of the preterminal protein-specific antibodies induced dissociation of the preterminal protein-adenovirus DNA polymerase heterodimer and inhibited initiation of adenovirus DNA replication in vitro. Antibodies binding close to the primary processing sites of adenovirus protease inhibited DNA binding, consistent with UV cross-linking results which reveal that an N-terminal, protease-resistant domain of preterminal protein contacts DNA. Monoclonal antibodies recognizing epitopes within the C-terminal 60 amino acids of preterminal protein stimulate DNA binding, an effect mediated through a decrease in the dissociation rate constant. These results suggest that preterminal protein contains a large, noncontiguous surface required for interaction with DNA polymerase, an N-terminal DNA binding domain, and a C-terminal regulatory domain.
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Affiliation(s)
- A Webster
- School of Biological and Medical Science, University of St. Andrews, Fife, Scotland
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35
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Abstract
Pyridoxal phosphate modification of adenovirus DNA polymerase results in loss of DNA polymerase activity, whereas the 3' --> 5' exonuclease activity is unaffected. Inhibition by pyridoxal phosphate is time-dependent, displays saturation kinetics, and is reversible in the presence of excess primary amine unless the pyridoxal phosphate-enzyme adduct is first reduced with NaBH4. Thus, inhibition is the consequence of Schiff base formation between the aldehyde moiety of pyridoxal phosphate and primary amino groups on the enzyme. In addition to inhibiting DNA polymerase activity, pyridoxal phosphate also inhibited the ability of the enzyme to initiate viral DNA replication, by transfer of dCMP onto the preterminal protein. Neither template-primer nor dNTP protect against pyridoxal phosphate inhibition, but the combination of template-primer and complementary substrate dNTP protected both initiation and DNA polymerase activities. Thus, it is likely that both the dCMP transfer activity required for initiation and DNA polymerase activity are carried out at the same site of the enzyme.
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Affiliation(s)
- A Monaghan
- School of Biological and Medical Sciences, Irvine Building, University of St. Andrews, Fife KY16 9AL, Scotland, United Kingdom
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36
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Ramachandra M, Sasaguri Y, Nakano R, Padmanabhan R. Heterologous expression, purification, and characterization of adenovirus DNA polymerase and preterminal protein. Methods Enzymol 1996; 275:168-94. [PMID: 9026638 DOI: 10.1016/s0076-6879(96)75012-7] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Affiliation(s)
- M Ramachandra
- Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892, USA
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37
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Caravokyri C, Leppard KN. Human adenovirus type 5 variants with sequence alterations flanking the E2A gene: effects on E2 expression and DNA replication. Virus Genes 1996; 12:65-75. [PMID: 8879122 DOI: 10.1007/bf00370002] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
The human adenovirus type 5 (Ad5) E2 transcription unit is divided into a promoter-proximal region, E2A, and a distal region, E2B, each with its own polyadenylation site. Together these regions encode the three virus-derived proteins necessary for genome replication. Ad5 variants were produced that carried linker insertion mutations immediately 5' and/or 3' to the coding sequence for the E2A gene DNA binding protein (DBP). Two variants carrying solely a 5' lesion showed decreased usage of the adjacent 3' splice site, via which the DBP mRNA is produced, and an increased usage of the alternative downstream splice sites in the E2B region, wherein viral DNA polymerase and terminal protein precursor are encoded; these viruses showed somewhat reduced growth. A variant carrying a 3' lesion showed a marginal increase in DBP expression and slightly accelerated growth. When lesions 5' and 3' to the DBP coding sequence were combined in cis, the resulting virus was severely defective for growth and expressed E2B products to the virtual exclusion of E2A DBP. These data indicate that interactions must occur between the E2A 3' splice site and polyadenylation site before this region can be treated as an exon by the RNA processing machinery, and that a sequence alteration at the polyadenylation site that alone has only minor effects on the pattern of RNA processing can drastically affect terminal exon usage when placed in cis with a mutation that reduces splicing efficiency at the upstream 3' splice site. The data further indicate that, in vivo, Ad5 DNA replication is limited by prevailing DBP levels rather than by levels of polymerase or terminal protein precursor.
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Affiliation(s)
- C Caravokyri
- Department of Biological Sciences, University of Warwick, Coventry, UK
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38
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Affiliation(s)
- L A Lucher
- Department of Biological Sciences, Illinois State, University, Normal 61761, USA
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39
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Affiliation(s)
- P C Van der Vliet
- Laboratory for Physiological Chemistry, University of Utrecht, The Netherlands
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40
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Yoshida K, Higashino F, Fujinaga K. Transcriptional regulation of the adenovirus E1A gene. Curr Top Microbiol Immunol 1995; 199 ( Pt 3):113-30. [PMID: 7555073 DOI: 10.1007/978-3-642-79586-2_6] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Affiliation(s)
- K Yoshida
- Department of Molecular Biology, Sapporo Medical University, School of Medicine, Japan
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41
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Russell WC, Kemp GD. Role of adenovirus structural components in the regulation of adenovirus infection. Curr Top Microbiol Immunol 1995; 199 ( Pt 1):81-98. [PMID: 7555062 DOI: 10.1007/978-3-642-79496-4_6] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Affiliation(s)
- W C Russell
- School of Biological and Medical Sciences, Division of Cell and Molecular Biology, University of St. Andrews, Fife, Scotland, UK
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42
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Ramachandra M, Padmanabhan R. Expression, Nuclear Transport, and Phosphorylation of Adenovirus DNA Replication Proteins. Curr Top Microbiol Immunol 1995. [DOI: 10.1007/978-3-642-79499-5_3] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
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43
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Hay RT, Freeman A, Leith I, Monaghan A, Webster A. Molecular interactions during adenovirus DNA replication. Curr Top Microbiol Immunol 1995; 199 ( Pt 2):31-48. [PMID: 7555069 DOI: 10.1007/978-3-642-79499-5_2] [Citation(s) in RCA: 25] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Affiliation(s)
- R T Hay
- School of Biological and Medical Sciences, University of St. Andrews, Scotland, UK
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44
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Kusukawa J, Ramachandra M, Nakano R, Padmanabhan R. Phosphorylation-dependent interaction of adenovirus preterminal protein with the viral origin of DNA replication. J Biol Chem 1994. [DOI: 10.1016/s0021-9258(17)42153-3] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022] Open
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45
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Hatfield L, Hearing P. The NFIII/OCT-1 binding site stimulates adenovirus DNA replication in vivo and is functionally redundant with adjacent sequences. J Virol 1993; 67:3931-9. [PMID: 8510211 PMCID: PMC237760 DOI: 10.1128/jvi.67.7.3931-3939.1993] [Citation(s) in RCA: 42] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
The inverted terminal repeat (ITR) of adenovirus type 5 (Ad5) is 103 bp in length and contains the origin of DNA replication. Cellular transcription factors NFI/CTF and NFIII/OCT-1 bind to sites within the ITR and participate in the initiation of viral DNA replication in vitro. The ITR also contains multiple copies of two conserved sequence motifs that bind the cellular transcription factors SP1 and ATF. We have analyzed a series of viruses that carry deletions at the left terminus of Ad5. A virus carrying a deletion of the NFIII/OCT-1, SP1, and ATF sites within the ITR (mutant dl309-44/107) was wild type for virus growth. However, the deletion of these elements in addition to sequences immediately flanking the ITR (mutant dl309-44/195) resulted in a virus that grew poorly. The analysis of growth parameters of these and other mutants demonstrate that the NFIII/OCT-1 and adjacent SP1 sites augment the accumulation of viral DNA following infection. The function of these elements was most evident in coinfections with a wild-type virus, suggesting that these sites enhance the ability of a limiting trans-acting factor(s), that stimulates viral DNA replication, to interact with the ITR. The results of these analyses indicate functional redundancy between different transcription elements at the left terminus of the Ad5 genome and demonstrate that the NFIII/OCT-1 site and adjacent SP1 site, previously thought to be nonessential for adenovirus growth, play a role in viral DNA replication in vivo.
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Affiliation(s)
- L Hatfield
- Department of Microbiology, State University of New York, Stony Brook 11794-7621
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46
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Abstract
In common with many other viruses, adenoviruses code for a protease essential for the development of infectivity. Recombinant adenovirus protease was active in crude in vitro complementation assays but was inactive with peptide or purified protein substrates. Activity was reconstituted by a component of adenovirus virions, which was identified as GVQSLKRRRCF, a peptide derived from the virus protein pVI. Synthetic peptides were used to demonstrate that the cysteine is essential and that the disulphide-linked dimer is required for activity. It is proposed that the adenovirus protease is a cysteine protease and that its activation by the peptide involves thiol-disulphide interchange, which serves to expose the active site cysteine. This represents a novel strategy for controlling the activity of a protease that is required for virus maturation.
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Affiliation(s)
- A Webster
- Division of Biochemistry and Molecular Biology, School of Biological and Medical Sciences, University of St. Andrews, Scotland
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47
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Joung I, Engler JA. Mutations in two cysteine-histidine-rich clusters in adenovirus type 2 DNA polymerase affect DNA binding. J Virol 1992; 66:5788-96. [PMID: 1527843 PMCID: PMC241454 DOI: 10.1128/jvi.66.10.5788-5796.1992] [Citation(s) in RCA: 24] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Several point and linker insertion mutations in two Cys-His-rich regions of adenovirus (Ad) DNA polymerase (Pol) gene have been expressed in recombinant vaccinia virus. The resulting mutant enzymes were analyzed in vitro for their effects on DNA synthesis activity, on Ad-specific initiation assays, on gel shifts of Ad origin sequences, and on interactions with adenovirus preterminal protein (pTP) and nuclear factor I (NFI). In general, mutants in downstream Cys-His sequences had a pronounced effect in these assays. Mutants in the upstream Cys-His region had a moderate effect on DNA synthesis and elongation but failed to make dCMP-pTP initiation complexes and failed to make specific shifted complexes in a gel retardation assay. These mutants could still bind to pTP and NFI in a coimmunoprecipitation experiment, suggesting that this upstream Cys-His region of Ad Pol is involved either in specific Ad DNA origin binding or in nonspecific DNA binding. Changing residues within Cys doublets in the downstream Cys-His region had pronounced effects on many Ad Pol functions such as DNA synthesis, DNA binding, and in vitro initiation; however, these mutants showed little reduction in binding to pTP and NFI; mutants at other cysteines or histidines within this region of Ad Pol did not appear to have an effect on enzyme function. This observation suggests that the downstream Cys-His region of Ad Pol is important for DNA binding and might fold into a Zn finger motif.
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Affiliation(s)
- I Joung
- Department of Biochemistry, School of Medicine, University of Alabama, Birmingham 35294-0005
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