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1
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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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2
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Yu JM, Liang ZY, Fu YH, Peng XL, Zheng YP, Dong YJ, He JS. Coexistence of adeno-associated virus 2 with adenovirus 18 or herpesvirus may be associated with severe lingual papillomatosis in an immunocompromised individual. Virol Sin 2024; 39:705-707. [PMID: 38914259 PMCID: PMC11401458 DOI: 10.1016/j.virs.2024.06.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2023] [Accepted: 06/19/2024] [Indexed: 06/26/2024] Open
Affiliation(s)
- Jie-Mei Yu
- College of Life Sciences and Bioengineering, Beijing Jiaotong University, Beijing, 100044, China
| | - Ze-Yin Liang
- Department of Hematology, Peking University First Hospital, Beijing, 100034, China
| | - Yuan-Hui Fu
- College of Life Sciences and Bioengineering, Beijing Jiaotong University, Beijing, 100044, China
| | - Xiang-Lei Peng
- College of Life Sciences and Bioengineering, Beijing Jiaotong University, Beijing, 100044, China
| | - Yan-Peng Zheng
- College of Life Sciences and Bioengineering, Beijing Jiaotong University, Beijing, 100044, China
| | - Yu-Jun Dong
- Department of Hematology, Peking University First Hospital, Beijing, 100034, China.
| | - Jin-Sheng He
- College of Life Sciences and Bioengineering, Beijing Jiaotong University, Beijing, 100044, China.
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3
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Huo S, Hai Y, Guo Y, Nie L, Li H, Qiao P, Zong K, Li X, Guo Y, Song J, Zhao H, Lei W, Lan Y, Liu WJ, Gao GF. Intra-host variation and evolutionary dynamics of adenoviruses correlate to neutrophils in infected patients. J Med Virol 2022; 94:3863-3875. [PMID: 35355288 DOI: 10.1002/jmv.27744] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2022] [Revised: 03/26/2022] [Accepted: 03/30/2022] [Indexed: 11/10/2022]
Abstract
With deep sequencing of virus genomes within the hosts, intra-host single nucleotide variations (iSNVs) have been used for analyses of virus genome variation and evolution, which is indicated to correlate with viral pathogenesis and disease severity. Little is known about the features of iSNVs among DNA viruses. We performed the epidemiological and laboratory investigation of one outbreak of adenovirus. The whole genomes of viruses in both original oral swabs and cell-cultured virus isolates were deeply sequenced. We identified 737 iSNVs in the viral genomes sequenced from original samples and 46 viral iSNVs in cell cultured isolates, with 33 iSNVs shared by original samples and cultured isolates. Meanwhile, we found these 33 iSNVs were shared by different patients, among which, three hot-spot areas 6367-6401, 9213-9247 and 10584-10606 within the functional genes of the adenovirus genome were found. Notably, the substitution rates of iSNVs were closely correlated with the clinical and immune indicators of the patients. Especially a positive correlation to neutrophils was found, indicating a predictable biomarker of iSNV dynamics. Our findings demonstrated the neutrophil-correlated dynamic evolution features of the iSNVs within adenoviruses, which indicates a virus-host interaction during human infection of a DNA virus. This article is protected by copyright. All rights reserved.
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Affiliation(s)
- Shuting Huo
- School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, 325035, China.,NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 100052, China
| | - Yan Hai
- Inner Mongolia Center for Disease Control and Prevention, Hohhot, 010031, China
| | - Yaxin Guo
- NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 100052, China
| | - Li Nie
- Tongliao Center for Disease Control and Prevention, Tongliao, 028000, China
| | - Hongmei Li
- School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, 325035, China.,NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 100052, China
| | - Peiwen Qiao
- NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 100052, China
| | - Kexin Zong
- School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, 325035, China.,NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 100052, China
| | - Xin Li
- School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, 325035, China.,NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 100052, China
| | - Yuanyuan Guo
- NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 100052, China.,School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, 211816, China
| | - Jingdong Song
- NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 100052, China.,State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 100052, China
| | - Honglan Zhao
- NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 100052, China
| | - Wenwen Lei
- NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 100052, China
| | - Yu Lan
- NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 100052, China
| | - William J Liu
- School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, 325035, China.,NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 100052, China
| | - George F Gao
- School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, 325035, China.,NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 100052, China.,CAS Key Laboratory of Pathogen Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China
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4
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Charman M, Herrmann C, Weitzman MD. Viral and cellular interactions during adenovirus DNA replication. FEBS Lett 2019; 593:3531-3550. [PMID: 31764999 DOI: 10.1002/1873-3468.13695] [Citation(s) in RCA: 59] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2019] [Revised: 11/14/2019] [Accepted: 11/15/2019] [Indexed: 12/30/2022]
Abstract
Adenoviruses represent ubiquitous and clinically significant human pathogens, gene-delivery vectors, and oncolytic agents. The study of adenovirus-infected cells has long been used as an excellent model to investigate fundamental aspects of both DNA virus infection and cellular biology. While many key details supporting a well-established model of adenovirus replication have been elucidated over a period spanning several decades, more recent findings suggest that we have only started to appreciate the complex interplay between viral genome replication and cellular processes. Here, we present a concise overview of adenovirus DNA replication, including the biochemical process of replication, the spatial organization of replication within the host cell nucleus, and insights into the complex plethora of virus-host interactions that influence viral genome replication. Finally, we identify emerging areas of research relating to the replication of adenovirus genomes.
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Affiliation(s)
- Matthew Charman
- Division of Protective Immunity and Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, PA, USA.,Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA
| | - Christin Herrmann
- Division of Protective Immunity and Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, PA, USA.,Cell and Molecular Biology Graduate Group, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA
| | - Matthew D Weitzman
- Division of Protective Immunity and Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, PA, USA.,Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA
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5
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Identification of a novel bipartite nuclear localization signal in the DNA polymerase of the betabaculovirus Pieris rapae granulovirus. Arch Virol 2018; 164:839-845. [PMID: 30506470 DOI: 10.1007/s00705-018-4115-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2018] [Accepted: 10/15/2018] [Indexed: 10/27/2022]
Abstract
DNA polymerase (DNApol) is highly conserved in all baculoviruses and plays an essential role in viral DNA replication. Previous results showed that the DNApol of the betabaculovirus Pieris rapae granulovirus (PiraGV) can localize in the nucleus. However, it is not clear how the DNApol is transported into the nucleus. Bioinformatic and GFP localization analysis showed that PiraGV DNApol contains a nuclear localization signal (NLS) at aa 4-25 (LFKRKLDEPPTDHTLVKAIKLS) of the N-terminus that does not match either the classical monopartite or the bipartite NLS consensus sequence. Multiple-point-substitution analysis confirmed that the NLS is required for transport of PiraGV DNApol into the nucleus. We also substituted the NLS of the PiraGV DNApol for that of the alphabaculovirus Spodoptera litura nuclear polyhedrosis virus (SpltNPV) DNApol. A viral growth curve and quantitative real-time PCR revealed that the substitution impaired viral DNA replication and resulted in a reduction in virus production. Together, our results show that PiraGV contains a novel NLS and that the NLS cannot efficiently replace that of SpltNPV DNApol for viral DNA synthesis and virus production.
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6
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Feghoul L, Mercier-Delarue S, Salmona M, Ntsiba N, Dalle JH, Baruchel A, Klonjkowski B, Richardson J, Simon F, LeGoff J. Genetic diversity of the human adenovirus species C DNA polymerase. Antiviral Res 2018; 156:1-9. [PMID: 29842914 DOI: 10.1016/j.antiviral.2018.05.011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2017] [Revised: 03/28/2018] [Accepted: 05/24/2018] [Indexed: 10/16/2022]
Abstract
BACKGROUND Human Adenovirus (HAdV) are responsible for severe infections in hematopoietic stem cells transplant (HSCT) recipient, species C viruses being the most commonly observed in this population. There is no approved antiviral treatment yet. Cidofovir (CDV), a cytidine analog, is the most frequently used and its lipophilic conjugate, brincidofovir (BCV), is under clinical development. These drugs target the viral DNA polymerase (DNA pol). Little is known about the natural polymorphism of HAdV DNA pol in clinical strains. METHODS We assessed the inter- and intra-species variability of the whole gene coding for HAdV DNA pol of HAdV clinical strains of species C. The study included 60 species C HAdV (21 C1, 27 C2 and 12 C5) strains isolated from patients with symptomatic infections who had never experienced CDV or BCV treatments and 20 reference strains. We also evaluated the emergence of mutations in thrirteen patients with persistent HAdV infection despite antiviral treatment. RESULTS We identified 356 polymorphic nucleotide positions (9.9% of the whole gene), including 102 positions with nonsynonymous mutations (28.0%) representing 8.7% of all amino acids. The mean numbers of nucleotide and amino acid mutations per strain were 23.1 (±6.2) and 5.2 (±2.4) respectively. Most of amino acid substitutions (60.6%) were observed in one instance only. A minority (13.8%) were observed in more than 10% of all strains. The most variable region was the NH2 terminal domain (44.2% of amino acid mutations). Mutations in the exonuclease domain accounted for 27.8%. The binding domains for the terminal protein (TPR), TPR1 and TPR2, presented a limited number of mutations, which were nonetheless frequently observed (62.5% and 58.8% of strains for TPR1 and TPR2, respectively). None of the mutations associated with CDV or BCV resistance were detected. In patients receieving antiviral drugs with persistent HAdV replication, we identified a new mutation in the NH2 terminal region. CONCLUSIONS Our study shows a high diversity in HAdV DNA pol sequences in clinical species C HAdV and provides a comprehensive mapping of its natural polymorphism. These data will contribute to the interpretation of HAdV DNA pol mutations selected in patients receiving antiviral treatments.
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Affiliation(s)
- Linda Feghoul
- Paris Diderot University, Pres Sorbone Paris Cité, Inserm U941, Microbiology Laboratory, Hôpital Saint-Louis, APHP, Paris, France
| | - Séverine Mercier-Delarue
- Paris Diderot University, Pres Sorbone Paris Cité, Inserm U941, Microbiology Laboratory, Hôpital Saint-Louis, APHP, Paris, France
| | - Maud Salmona
- Paris Diderot University, Pres Sorbone Paris Cité, Inserm U941, Microbiology Laboratory, Hôpital Saint-Louis, APHP, Paris, France
| | - Nora Ntsiba
- Paris Diderot University, Pres Sorbone Paris Cité, Inserm U941, Microbiology Laboratory, Hôpital Saint-Louis, APHP, Paris, France
| | - Jean-Hugues Dalle
- Paris Diderot University, Pres Sorbone Paris Cité, Hematology Department, Hôpital Robert Debré, APHP, Paris, France
| | - André Baruchel
- Paris Diderot University, Pres Sorbone Paris Cité, Hematology Department, Hôpital Robert Debré, APHP, Paris, France
| | | | | | - François Simon
- Paris Diderot University, Pres Sorbone Paris Cité, Inserm U941, Microbiology Laboratory, Hôpital Saint-Louis, APHP, Paris, France
| | - Jérôme LeGoff
- Paris Diderot University, Pres Sorbone Paris Cité, Inserm U941, Microbiology Laboratory, Hôpital Saint-Louis, APHP, Paris, France.
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7
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Yang X, Wang Q, Liang B, Wu F, Li H, Liu H, Sheng C, Ma Q, Yang C, Xie J, Li P, Jia L, Wang L, Du X, Qiu S, Song H. An outbreak of acute respiratory disease caused by a virus associated RNA II gene mutation strain of human adenovirus 7 in China, 2015. PLoS One 2017; 12:e0172519. [PMID: 28225804 PMCID: PMC5321423 DOI: 10.1371/journal.pone.0172519] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2016] [Accepted: 02/06/2017] [Indexed: 01/17/2023] Open
Abstract
Human adenovirus 7 (HAdV-7) strains are a major cause of acute respiratory disease (ARD) among adults and children, associated with fatal pneumonia. An ARD outbreak caused by HAdV-7 that involved 739 college students was reported in this article. To better understand the underlying cause of this large-scale epidemic, virus strains were isolated from infected patients and sequence variations of the whole genome sequence were detected. Evolutionary trees and alignment results indicated that the major capsid protein genes hexon and fibre were strongly conserved among serotype 7 strains in China at that time. Instead, the HAdV-7 strains presented three thymine deletions in the virus associated RNA (VA RNA) II terminal region. We also found that the mutation might lead to increased mRNA expression of an adjacent gene, L1 52/55K, and thus promoted faster growth. These findings suggest that sequence variation of VA RNA II gene was a potential cause of such a severe HAdV-7 infection and this gene should be a new-emerging factor to be monitored for better understanding of HAdV-7 infection.
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Affiliation(s)
- Xiaoxia Yang
- Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China
| | - Qiongshu Wang
- Deprtment of Infection Control, Wuhan General Hospital of Guangzhou Military Command, Wuhan, China
| | - Beibei Liang
- Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China
| | - Fuli Wu
- Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China
| | - Hao Li
- Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China
| | - Hongbo Liu
- Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China
| | - Chunyu Sheng
- Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China
| | - Qiuxia Ma
- Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China
| | - Chaojie Yang
- Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China
| | - Jing Xie
- Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China
| | - Peng Li
- Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China
| | - Leili Jia
- Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China
| | - Ligui Wang
- Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China
| | - Xinying Du
- Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China
| | - Shaofu Qiu
- Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China
- * E-mail: (HS); (SQ)
| | - Hongbin Song
- Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China
- * E-mail: (HS); (SQ)
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8
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Interregional Coevolution Analysis Revealing Functional and Structural Interrelatedness between Different Genomic Regions in Human Mastadenovirus D. J Virol 2015; 89:6209-17. [PMID: 25833048 DOI: 10.1128/jvi.00515-15] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2015] [Accepted: 03/25/2015] [Indexed: 01/22/2023] Open
Abstract
UNLABELLED Human mastadenovirus D (HAdV-D) is exceptionally rich in type among the seven human adenovirus species. This feature is attributed to frequent intertypic recombination events that have reshuffled orthologous genomic regions between different HAdV-D types. However, this trend appears to be paradoxical, as it has been demonstrated that the replacement of some of the interacting proteins for a specific function with other orthologues causes malfunction, indicating that intertypic recombination events may be deleterious. In order to understand why the paradoxical trend has been possible in HAdV-D evolution, we conducted an interregional coevolution analysis between different genomic regions of 45 different HAdV-D types and found that ca. 70% of the genome has coevolved, even though these are fragmented into several pieces via short intertypic recombination hot spot regions. Since it is statistically and biologically unlikely that all of the coevolving fragments have synchronously recombined between different genomes, it is probable that these regions have stayed in their original genomes during evolution as a platform for frequent intertypic recombination events in limited regions. It is also unlikely that the same genomic regions have remained almost untouched during frequent recombination events, independently, in all different types, by chance. In addition, the coevolving regions contain the coding regions of physically interacting proteins for important functions. Therefore, the coevolution of these regions should be attributed at least in part to natural selection due to common biological constraints operating on all types, including protein-protein interactions for essential functions. Our results predict additional unknown protein interactions. IMPORTANCE Human mastadenovirus D, an exceptionally type-rich human adenovirus species and causative agent of different diseases in a wide variety of tissues, including that of ocular region and digestive tract, as well as an opportunistic infection in immunocompromised patients, is known to have highly diverged through frequent intertypic recombination events; however, it has also been demonstrated that the replacement of a component protein of a multiprotein system with a homologous protein causes malfunction. The present study solved this apparent paradox by looking at which genomic parts have coevolved using a newly developed method. The results revealed that intertypic recombination events have occurred in limited genomic regions and been avoided in the genomic regions encoding proteins that physically interact for a given function. This approach detects purifying selection against recombination events causing the replacement of partial components of multiprotein systems and therefore predicts physical and functional interactions between different proteins and/or genomic elements.
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9
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Gonzalez G, Koyanagi KO, Aoki K, Kitaichi N, Ohno S, Kaneko H, Ishida S, Watanabe H. Intertypic modular exchanges of genomic segments by homologous recombination at universally conserved segments in human adenovirus species D. Gene 2014; 547:10-7. [PMID: 24726548 DOI: 10.1016/j.gene.2014.04.018] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2013] [Revised: 03/28/2014] [Accepted: 04/08/2014] [Indexed: 11/20/2022]
Abstract
Human adenovirus species D (HAdV-D), which is composed of clinically and epidemiologically important pathogens worldwide, contains more taxonomic "types" than any other species of the genus Mastadenovirus, although the mechanisms accounting for the high level of diversity remain to be disclosed. Recent studies of known and new types of HAdV-D have indicated that intertypic recombination between distant types contributes to the increasing diversity of the species. However, such findings raise the question as to how homologous recombination events occur between diversified types since homologous recombination is suppressed as nucleotide sequences diverge. In order to address this question, we investigated the distribution of the recombination boundaries in comparison with the landscape of intergenomic sequence conservation assessed according to the synonymous substitution rate (dS). The results revealed that specific genomic segments are conserved between even the most distantly related genomes; we call these segments "universally conserved segments" (UCSs). These findings suggest that UCSs facilitate homologous recombination, resulting in intergenomic segmental exchanges of UCS-flanking genomic regions as recombination modules. With the aid of such a mechanism, the haploid genomes of HAdV-Ds may have been reshuffled, resulting in chimeric genomes out of diversified repertoires in the HAdV-D population analogous to the MHC region reshuffled via crossing over in vertebrates. In addition, some HAdVs with chimeric genomes may have had the opportunity to avoid host immune responses thereby causing epidemics.
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Affiliation(s)
- Gabriel Gonzalez
- Graduate School of Information Science and Technology, Hokkaido University, Sapporo 060-0814, Japan
| | - Kanako O Koyanagi
- Graduate School of Information Science and Technology, Hokkaido University, Sapporo 060-0814, Japan
| | - Koki Aoki
- Department of Ophthalmology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan
| | - Nobuyoshi Kitaichi
- Department of Ophthalmology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan; Department of Ophthalmology, Health Sciences University of Hokkaido, Sapporo 002-8072, Japan
| | - Shigeaki Ohno
- Department of Ophthalmology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan
| | - Hisatoshi Kaneko
- Department of Ophthalmology, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan; Hobara Eye clinic, Date 960-0612, Japan
| | - Susumu Ishida
- Department of Ophthalmology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan
| | - Hidemi Watanabe
- Graduate School of Information Science and Technology, Hokkaido University, Sapporo 060-0814, Japan.
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10
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Abstract
Adenoviruses have attracted much attention as probes to study biological processes such as DNA replication, transcription, splicing, and cellular transformation. More recently these viruses have been used as gene-transfer vectors and oncolytic agents. On the other hand, adenoviruses are notorious pathogens in people with compromised immune functions. This article will briefly summarize the basic replication strategy of adenoviruses and the key proteins involved and will deal with the new developments since 2006. In addition, we will cover the development of antivirals that interfere with human adenovirus (HAdV) replication and the impact of HAdV on human disease.
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Affiliation(s)
- Rob C Hoeben
- Department of Molecular Cell Biology, Leiden University Medical Centre, 2333 ZC Leiden, The Netherlands.
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11
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Selective modification of adenovirus replication can be achieved through rational mutagenesis of the adenovirus type 5 DNA polymerase. J Virol 2012; 86:10484-93. [PMID: 22811532 DOI: 10.1128/jvi.00739-12] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023] Open
Abstract
Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1 DNA polymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We therefore tested whether mutations in regions of the adenovirus type 5 (Ad5) DNA polymerase that interact with the dNTP substrate or DNA template could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2- to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viral DNA polymerase utilizes dNTP substrates.
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12
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Joshi A, Tang J, Kuzma M, Wagner J, Mookerjee B, Filicko J, Carabasi M, Flomenberg N, Flomenberg P. Adenovirus DNA polymerase is recognized by human CD8+ T cells. J Gen Virol 2009; 90:84-94. [PMID: 19088277 DOI: 10.1099/vir.0.002493-0] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Donor lymphocytes have potential as a treatment for adenovirus (Ad) disease in haematopoietic stem cell transplant (SCT) recipients, but better understanding of Ad-specific T-cell responses is required. Most healthy adults exhibit memory T-cell responses to hexon, a capsid protein synthesized late after infection. However, since the Ad E3-19k downregulates major histocompatibility complex (MHC) class I molecules, cytotoxic T cells (CTLs) targeted to early viral proteins may be more effective in eliminating Ad-infected cells in vivo. Here we show that Ad-specific CTLs recognize the early region 2 proteins DNA polymerase (Pol) and DNA-binding protein (DBP). Firstly, memory Ad-specific CD8(+) T cells were amplified from healthy donors by in vitro stimulation with Ad-infected dendritic cells and found to exhibit MHC-restricted cytotoxicity to targets expressing Pol and DBP. Secondly, gamma interferon responses to HLA A2-binding motif peptides from Pol and DBP were directly detected in peripheral blood mononuclear cells (PBMCs) from a recently infected normal donor. Peptide-specific CTLs generated to Pol and DBP epitopes were confirmed to exhibit HLA A2-restricted killing of targets expressing Pol or DBP. Lastly, Pol-epitope-specific T cells were detected at similar or higher frequencies than hexon and DBP in three of three SCT recipients recovering from invasive Ad disease. Pol epitopes were well conserved among different Ad serotypes. Therefore, Pol is a promising target for immunotherapy of Ad disease.
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Affiliation(s)
- Amita Joshi
- Department of Microbiology, Thomas Jefferson University, Philadelphia, PA 19107, USA
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13
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Chmielewicz B, Nitsche A, Schweiger B, Ellerbrok H. Development of a PCR-based assay for detection, quantification, and genotyping of human adenoviruses. Clin Chem 2005; 51:1365-73. [PMID: 15951322 DOI: 10.1373/clinchem.2004.045088] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
BACKGROUND Adenoviruses (AdVs) can cause serious disease in immunosuppressed patients, particularly those undergoing allogeneic stem cell transplantation. A method for virus quantification in clinical specimens is essential for monitoring patient adenoviral loads and evaluating new therapeutic approaches. METHODS We developed a PCR-based assay that combines detection and genotyping of human AdVs, targeting a highly conserved region of the adenoviral genome coding for the DNA polymerase (AdV DPol PCR). We tested the diagnostic applicability of this PCR-based assay by analyzing 159 clinical specimens from children with respiratory disease and comparing the results with those obtained by nested PCR analysis. RESULTS The PCR assay detected all currently known AdV serotypes, with a detection limit of approximately 10 genome equivalents per reaction for 49 of 51 serotypes. No cross-reactivity to human DNA or other DNA viruses was observed. In addition, genotyping of PCR-positive samples was achieved within minutes by fluorescence curve melting analysis in a LightCycler instrument using 6 pairs of hybridization probes, each specific for a single AdV species. Results for clinical specimens were in good concordance with those obtained by nested PCR. CONCLUSION The presented assay is a suitable tool for the detection and genotyping of human AdVs in clinical samples.
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Affiliation(s)
- Barbara Chmielewicz
- Robert Koch-Institut, Zentrum für Biologische Sicherheit 1, FG12 'Virale Infektionen', Berlin, Germany.
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14
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Wanrooij S, Luoma P, van Goethem G, van Broeckhoven C, Suomalainen A, Spelbrink JN. Twinkle and POLG defects enhance age-dependent accumulation of mutations in the control region of mtDNA. Nucleic Acids Res 2004; 32:3053-64. [PMID: 15181170 PMCID: PMC434440 DOI: 10.1093/nar/gkh634] [Citation(s) in RCA: 95] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2004] [Revised: 04/06/2004] [Accepted: 05/12/2004] [Indexed: 01/07/2023] Open
Abstract
Autosomal dominant and/or recessive progressive external ophthalmoplegia (ad/arPEO) is associated with mtDNA mutagenesis. It can be caused by mutations in three nuclear genes, encoding the adenine nucleotide translocator 1, the mitochondrial helicase Twinkle or DNA polymerase gamma (POLG). How mutations in these genes result in progressive accumulation of multiple mtDNA deletions in post- mitotic tissues is still unclear. A recent hypothesis suggested that mtDNA replication infidelity could promote slipped mispairing, thereby stimulating deletion formation. This hypothesis predicts that mtDNA of ad/arPEO patients will contain frequent mutations throughout; in fact, our analysis of muscle from ad/arPEO patients revealed an age-dependent, enhanced accumulation of point mutations in addition to deletions, but specifically in the mtDNA control region. Both deleted and non-deleted mtDNA molecules showed increased point mutation levels, as did mtDNAs of patients with a single mtDNA deletion, suggesting that point mutations do not cause multiple deletions. Deletion breakpoint analysis showed frequent breakpoints around homopolymeric runs, which could be a signature of replication stalling. Therefore, we propose replication stalling as the principal cause of deletion formation.
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Affiliation(s)
- Sjoerd Wanrooij
- Institute of Medical Technology and Tampere University Hospital, Tampere, Finland
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15
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Shen Y, Tang XF, Yokoyama H, Matsui E, Matsui I. A 21-amino acid peptide from the cysteine cluster II of the family D DNA polymerase from Pyrococcus horikoshii stimulates its nuclease activity which is Mre11-like and prefers manganese ion as the cofactor. Nucleic Acids Res 2004; 32:158-68. [PMID: 14704353 PMCID: PMC373266 DOI: 10.1093/nar/gkh153] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Family D DNA polymerase (PolD) is a new type of DNA polymerase possessing polymerization and 3'-5' exonuclease activities. Here we report the characterization of the nuclease activity of PolD from Pyrococcus horikoshii. By site-directed mutagenesis, we verified that the putative Mre11-like nuclease domain in the small subunit (DP1), predicted according to computer analysis and structure inference reported previously, is the catalytic domain. We show that D363, H365 and H454 are the essential residues, while D407, N453, H500, H563 and H565 are critical residues for the activity. We provide experimental evidence demonstrating that manganese, rather than magnesium, is the preferable metal ion for the nuclease activity of PolD. We also show that DP1 alone is insufficient to perform full catalysis, which additionally requires the formation of the PolD complex and manganese ion. We found that a 21 amino acid, subunit-interacting peptide of the sequence from cysteine cluster II of the large subunit (DP2) stimulates the exonuclease activity of DP1 and the internal deletion mutants of PolD lacking the 21-aa sequence. This indicates that the putative zinc finger motif of the cysteine cluster II is deeply involved in the nucleolytic catalysis.
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Affiliation(s)
- Yulong Shen
- Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305, Japan
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16
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Van Goethem G, Martin JJ, Van Broeckhoven C. Progressive external ophthalmoplegia characterized by multiple deletions of mitochondrial DNA: unraveling the pathogenesis of human mitochondrial DNA instability and the initiation of a genetic classification. Neuromolecular Med 2003; 3:129-46. [PMID: 12835509 DOI: 10.1385/nmm:3:3:129] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2003] [Accepted: 03/19/2003] [Indexed: 12/15/2022]
Abstract
Over the last decade, many sporadic and familial cases have been reported with multiple deletions of mitochondrial DNA (mtDNA) in postmitotic tissues. Most patients suffer from progressive external ophthalmoplegia (PEO) and may have a nuclear gene defect that predisposes to the accumulation of mtDNA deletions. Recently, positional cloning has led to the discovery of mutations in four such nuclear genes. Some mutations are dominant and others recessive. In all autosomal mutations, defective mtDNA replication and/or repair are probably responsible for the generation of secondary mtDNA deletions. There are also data suggestive of a prominent pathogenic role for disturbed nucleotide metabolism. We here present a tentative genotype-phenotype correlation. Since clinical presentations are heterogeneous and overlap with different previously described clinical syndromes, we advocate the use of a genetic, instead of a clinical, classification of disorders with multiple mtDNA deletions.
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Affiliation(s)
- Gert Van Goethem
- Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology (VIB), Born-Bunge Foundation (BBS), University of Antwerp (UIA), Antwerpen, Belgium
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17
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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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18
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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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19
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de Jong RN, Meijer LAT, van der Vliet PC. DNA binding properties of the adenovirus DNA replication priming protein pTP. Nucleic Acids Res 2003; 31:3274-86. [PMID: 12799455 PMCID: PMC162239 DOI: 10.1093/nar/gkg405] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
The precursor terminal protein pTP is the primer for the initiation of adenovirus (Ad) DNA replication and forms a heterodimer with Ad DNA polymerase (pol). Pol can couple dCTP to pTP directed by the fourth nucleotide of the viral genome template strand in the absence of other replication proteins, which suggests that pTP/pol binding destabilizes the origin or stabilizes an unwound state. We analyzed the contribution of pTP to pTP/pol origin binding using various DNA oligonucleotides. We show that two pTP molecules bind cooperatively to short DNA duplexes, while longer DNA fragments are bound by single pTP molecules as well. Cooperative binding to short duplexes is DNA sequence independent and most likely mediated by protein/protein contacts. Furthermore, we observed that pTP binds single-stranded (ss)DNA with a minimal length of approximately 35 nt and that random ssDNA competed 25-fold more efficiently than random duplex DNA for origin binding by pTP. Remarkably, short DNA fragments with two opposing single strands supported monomeric pTP binding. pTP did not stimulate, but inhibited strand displacement by the Ad DNA binding and unwinding protein DBP. These observations suggest a mechanism in which the ssDNA affinity of pTP stabilizes Ad pol on partially unwound origin DNA.
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Affiliation(s)
- R N de Jong
- Department of Physiological Chemistry, University Medical Center Utrecht and Center for Biomedical Genetics, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands
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20
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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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21
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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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22
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Van Goethem G, Dermaut B, Löfgren A, Martin JJ, Van Broeckhoven C. Mutation of POLG is associated with progressive external ophthalmoplegia characterized by mtDNA deletions. Nat Genet 2001; 28:211-2. [PMID: 11431686 DOI: 10.1038/90034] [Citation(s) in RCA: 564] [Impact Index Per Article: 23.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Progressive external ophthalmoplegias (PEO) characterized by accumulation of large-scale mitochondrial DNA (mtDNA) deletions are rare human diseases. We mapped a new locus for dominant PEO at 15q22-q26 in a Belgian pedigree and identified a heterozygous mutation (Y955C) in the polymerase motif B of the mtDNA polymerase gamma (POLG). We identified three additional POLG missense mutations compatible with recessive PEO In two nuclear families. POLG is the only DNA polymerase responsible for mtDNA replication.
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Affiliation(s)
- G Van Goethem
- Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology, Born-Bunge Foundation, University of Antwerp, Universiteitsplein 1; B-2610 Antwerpen, Belgium
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23
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Bao K, Cohen SN. Terminal proteins essential for the replication of linear plasmids and chromosomes in Streptomyces. Genes Dev 2001; 15:1518-27. [PMID: 11410532 PMCID: PMC312717 DOI: 10.1101/gad.896201] [Citation(s) in RCA: 90] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
Linear plasmids and chromosomes of the bacterial genus Streptomyces have proteins of unknown characteristics and function linked covalently to their 5' DNA termini. We purified protein attached to the end of the pSLA2 linear plasmid of Streptomyces rochei, determined the N-terminal amino acid sequence, and used this information to clone corresponding genes from a S. rochei cosmid library. Three separate terminal protein genes (here designated as tpgR1, tpgR2, and tpgR3), which map to the S. rochei chromosome and to 100-kb and 206-kb linear plasmids contained in S. rochei, were isolated and found to encode a family of similar but distinct 21-kD proteins. Using tpgR1 to probe a genomic DNA library of Streptomyces lividans ZX7, whose linear chromosome can undergo transition to a circular form, we isolated a S. lividans chromosomal gene (tpgL) that we found specifies a protein closely related to, and functionally interchangeable with, TpgR proteins for pSLA2 maintenance in S. lividans. Mutation of tpgL precluded propagation of the pSLA2 plasmid in a linear form and also prevented propagation of S. lividans cells that contain linear, but not circular, chromosomes, indicating a specific and essential role for tpg genes in linear DNA replication. Surprisingly, Tpg proteins were observed to contain a reverse transcriptase-like domain rather than sequences in common with proteins that attach covalently to the termini of linear DNA replicons.
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Affiliation(s)
- K Bao
- Department of Genetics, Stanford University School of Medicine, Stanford, California 94305-5120, USA
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