DOCSLIB.ORG

Fidelity of Human Immunodeficiency Viruses Type 1 and Type 2 Reverse Transcriptases in DNA Synthesis Reactions Using DNA and RNA Templates

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Fidelity of Human Immunodeficiency Viruses Type 1 and Type 2 Reverse Transcriptases in DNA Synthesis Reactions Using DNA and RNA Templates UNIVERSIDAD AUTÓNOMA DE MADRID Programa de Doctorado en Biociencias Moleculares Fidelity of Human Immunodeficiency Viruses Type 1 and Type 2 Reverse Transcriptases in DNA Synthesis Reactions using DNA and RNA Templates Alba Sebastián Martín Madrid, noviembre, 2018 Universidad Autónoma de Madrid Facultad de Ciencias Departamento de Biología Molecular Programa de doctorado en Biociencias Moleculares Fidelity of Human Immunodeficiency Viruses Type 1 and Type 2 Reverse Transcriptases in DNA Synthesis Reactions using DNA and RNA Templates Memoria presentada por Alba Sebastián Martín, graduada en Biología, para optar al título de doctora en Biociencias Moleculares por la Universidad Autónoma de Madrid Director de la Tesis: Dr. Luis Menéndez Arias Este trabajo ha sido realizado en el Centro de Biología Molecular ‘Severo Ochoa’ (UAM-CSIC), con el apoyo de una beca de Formación de Profesorado Universitario, financiada por el Ministerio de Educación, Cultura y Deporte (FPU13/00693). Abbreviations 3TC 2’ 3’-dideoxy-3’-thiacytidine AIDS Acquired immunodeficiency syndrome AMV Avian myeloblastosis virus APOBEC Apolipoprotein B mRNA editing enzyme ATP Adenosine 5’ triphosphate AZT 3’-azido-2’, 3’-dideoxythymidine (zidovudine) AZT-MP 3´-azido-2´, 3´-dideoxythymidine monophosphate AZTppppA 3´azido-3´-deoxythymidine-(5´)-tetraphospho-(5´)-adenosine bp Base pair BSA Bovine serum albumin CA Capsid protein cDNA Complementary DNA Cir-Seq Circular sequencing CypA Cyclophilin A dATP 2’-deoxyadenoside 5’-triphosphate dCTP 2’-deoxycytidine 5’-triphosphate ddC 2’, 3’-dideoxycytidine ddI 2’, 3’-dideoxyinosine dGTP 2’-deoxyguanoside 5’-triphosphate DNA Deoxyribonucleic acid dNTP 2’-deoxynucleoside 5’-triphosphate dsDNA Double-stranded DNA DTT Dithiothreitol dTTP 2’-deoxythymidine 5’-triphosphate EDTA Ethylenediaminetetraacetic acid Env Envelope proteins ESCRT Endosomal sorting complex required for transport FeLV Feline leukemia virus fext Mispair extension ratio fins Misinsertion efficiency constant FIV Feline immunodeficiency virus FPCM Fundación Parque Científico de Madrid gRNA Genomic RNA HiRes-Seq High resolution sequencing HIV-1 Human immunodeficiency virus type 1 HIV-2 Human immunodeficiency virus type 2 HTLV-III Human T-lymphotropic virus type III ICTV International Committee on Taxonomy of Viruses IN Integrase IPTG Isopropyl β-D-1-thiogalactopyranoside kcat Steady-state turnover number Kd Dissociation equilibrium constant Km Michaelis constant kobs Apparent kinetic constant koff Dissociation rate constant kpol Nucleotide incorporation rate kpol/Kd Catalytic efficiency lacZ Gene encoding the β-galactosidase enzyme LAV Lymphoadenopathy-associated virus LEDGF Lens epithelium-derived growth factor LTR Long-terminal repeat MA Matrix protein MCS Multicloning site MLV Murine leukemia virus mRNA Messenger RNA NC Nucleocapsid protein Nef Negative regulating factor NGS Next-generation sequencing NNRTI Non-nucleoside reverse transcriptase inhibitor NRTI Nucleoside reverse transcriptase inhibitor PCR Polymerase chain reaction PEG Polyethylene glycol PFV Prototype primate foamy virus PIC Pre-integration complex PMSF Phenylmethylsulfonyl fluoride PNK Polynucleotide kinase PPT Polypurine tract PR Protease RBS Ribosome binding site Rep-Seq Replicated sequencing RF Replicative form RNA Ribonucleic acid RNase H Ribonuclease H RNAP RNA polymerase RT Reverse transcriptase SAMHD1 Sterile α motif domain and histidine-aspartic domain-containing protein 1 SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SIV Simian immunodeficiency virus SSC Saline-sodium citrate ssDNA Single-stranded DNA ssRNA Single-stranded RNA SU Surface subunit TAM Thymidine analogue mutation TAM1 Thymidine analogue mutation pathway 1 TAM2 Thymidine analogue mutation pathway 2 TM Transmembrane subunit TRIM5α Tripartite motif-containing protein 5 T/P Template/primer U3 Unique 3’-region U5 Unique 5’-region Vif Viral infectivity factor Vpr Viral protein R Vpu Viral protein unique Vpx Viral protein X WT Wild-type (w/v) (weight/volume) X-Gal 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside XMRV Xenotropic murine leukemia virus-related virus Amino acids, one and three letters codes A Ala Alanine C Cys Cysteine D Asp Aspartic acid E Glu Glutamic acid F Phe Phenylalanine G Gly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine P Pro Proline Q Gln Glutamine R Arg Arginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan Y Tyr Tyrosine INDEX SUMMARY 1 RESUMEN 3 1. INTRODUCTION...................................................................................................... 7 1.1. Human immunodeficiency virus 7 1.2. HIV virion 9 1.3. Cycle of HIV infection 11 1.4. HIV RT 14 1.4.1. Structure 14 1.4.2. RT activities 16 1.4.2.1. DNA polymerization 16 1.4.2.2. RNase H 17 1.4.3. Fidelity of RTs 18 1.4.3.1. Experimental methods to estimate the accuracy of RTs in vitro 19 1.4.3.1.1. Biochemical assays 19 1.4.3.1.2. Genetic assays 21 1.4.3.1.3. Deep sequencing assays 22 1.4.3.2. DNA-dependent DNA synthesis fidelity of WT and multidrug-resistant HIV-2 RTs 24 1.4.3.3. RNA-dependent DNA synthesis fidelity of retroviral RTs 26 2. OBJECTIVES………………………………………………………………………. 31 3. MATERIALS AND METHODS…………………………………………………... 35 3.1. Enzymes and bacterial strains 35 3.2. RT purification 36 3.2.1. HIV-1 RTs 36 3.2.1.1. Site-directed mutagenesis 37 3.2.1.2. Expression of HIV-1 RT p66/p51 heterodimers 38 3.2.1.3. Lysis of bacteria 38 3.2.1.4. Cation-exchange chromatography 38 3.2.1.5. Affinity chromatography 39 3.2.1.6. Dialysis and concentration 39 3.2.2. Purification of HIV-2 RTs 40 3.2.2.1. Site-directed mutagenesis 40 3.2.2.2. Expression of HIV-2 RT p68/p54 heterodimers 42 3.2.2.3. Lysis of bacteria 42 3.2.2.4. Affinity chromatography 42 3.2.2.5. Cation-exchange chromatography 43 3.3. Purification of T7 RNA polymerase 43 3.3.1. Expression of T7 RNAP 44 3.3.2. Lysis of bacteria 44 3.3.3. Affinity chromatography 45 3.3.4. Dialysis and concentration 45 3.4. M13mp2 forward mutation assays 45 3.4.1. Determination of DNA-dependent DNA synthesis fidelity of RTs 45 3.4.1.1. Preparation of “gapped DNA” 45 3.4.1.1.1. Purification of phage M13mp2 ssDNA 46 3.4.1.1.2. Purification of phage M13mp2 dsDNA and digestion 47 3.4.1.1.3. Hybridization of ssDNA and 6789-bp dsDNA fragment 47 3.4.1.2. Gap-filling synthesis reaction 48 3.4.1.3. Preparation of competent cells 48 3.4.1.4. Electroporation and plating 49 3.4.1.5. Selection and analysis of mutant plaques 49 3.4.2. Determination of RNA-dependent DNA synthesis fidelity of RTs 51 3.4.2.1. Purification of RNA template 51 3.4.2.2. Synthesis of complementary DNA 53 3.4.2.3. Phosphorylation and hybridization of cDNA with “gapped DNA” 54 3.4.2.4. Plating, selection and analysis of mutant plaques 54 3.5. Promoter-dependent transcription assays 54 3.6. Promoter-independent single-nucleotide transcription assays 55 3.7. Binding affinity of T7 RNAP for template-primer VSR10 57 3.8. Deep sequencing assays 58 3.8.1. Reverse transcription 58 3.8.2. Amplification and sequencing 59 3.8.3. Analysis of sequences 60 3.8.4. Calculation of error rates 61 4. RESULTS………………………………………………………………………….. 65 4.1. Determination of DNA-dependent DNA synthesis fidelity of WT HIV-2ROD RT in comparison with drug-resistant mutants K65RROD and K65R/V75IROD RTs, and WT HIV-1BH10 RT 65 4.2. Determination of RNA-dependent DNA synthesis fidelity of six retroviral RTs 74 4.2.1. Analysis of mutant frequencies and error rates of wild-type MLV, AMV, HIV-1BH10 and HIV-1ESP49 RTs, as well as mutant RTs K65RESP49 and K65R/V75IESP49 using RNA templates 74 4.2.2. Analysis of bacteriophage T7 RNA polymerase transcription fidelity 84 4.2.2.1. Fidelity of promoter-dependent transcription under different conditions 84 4.2.2.2. Nucleotide incorporation kinetics under different conditions 86 4.2.2.3. Template-primer binding affinity of T7 RNAP under different conditions 88 4.2.3. Estimates of RNA-dependent DNA synthesis fidelity of HIV-1 RTs using more faithful RNA templates 89 4.3. Determination of RNA-dependent DNA synthesis fidelity of RTs by deep sequencing experiments based on the use of Primer IDs 97 5. DISCUSSION………………………………………………………………………. 101 5.1. Fidelity of WT and drug-resistant HIV-2ROD RTs 103 5.2. RNA-dependent DNA synthesis fidelity of retroviral RTs 108 5.2.1. Fidelity of retroviral RTs in reactions carried out with RNA templates 108 5.2.2. Mechanistic insights of RT-catalyzed DNA polymerization reaction with RNA or DNA templates 109 5.2.3. Influence of transcription inaccuracy in forward mutation assays 112 6. CONCLUSIONS (Y CONCLUSIONES) 119 7. REFERENCES 125 Summary Summary Human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2, respectively) are the causative agent of acquired immunodeficiency syndrome (AIDS). Most of the available clinically approved antiretroviral drugs directed against HIV inhibit the viral reverse transcriptase (RT), a multifunctional enzyme with RNA- and DNA-dependent DNA polymerase activity, as well as ribonuclease H, strand-transfer, and strand displacement activities. Nucleoside RT inhibitors (NRTIs) constitute the backbone of current therapies against both types of HIV, although the acquisition of NRTI resistance is mediated by the development of different mutational pathways. In HIV-2, resistance to all approved nucleoside analogues is conferred by the combination of RT substitutions K65R, Q151M and M184V. Interestingly, in highly divergent HIV-1 RTs, K65R alone confers >8-fold increased accuracy of DNA synthesis. In this Thesis, we have determined the intrinsic fidelity of DNA synthesis of wild-type (WT) HIV-2 group A (strain ROD) RT and mutants K65R and K65R/Q151M/M184V. Our results show that in HIV-2ROD RT those changes have a relatively small impact on nucleotide selectivity.
Load more

Recommended publications
  • RANDY SCHEKMAN Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, USA
    GENES AND PROTEINS THAT CONTROL THE SECRETORY PATHWAY Nobel Lecture, 7 December 2013 by RANDY SCHEKMAN Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, USA. Introduction George Palade shared the 1974 Nobel Prize with Albert Claude and Christian de Duve for their pioneering work in the characterization of organelles interrelated by the process of secretion in mammalian cells and tissues. These three scholars established the modern field of cell biology and the tools of cell fractionation and thin section transmission electron microscopy. It was Palade’s genius in particular that revealed the organization of the secretory pathway. He discovered the ribosome and showed that it was poised on the surface of the endoplasmic reticulum (ER) where it engaged in the vectorial translocation of newly synthesized secretory polypeptides (1). And in a most elegant and technically challenging investigation, his group employed radioactive amino acids in a pulse-chase regimen to show by autoradiograpic exposure of thin sections on a photographic emulsion that secretory proteins progress in sequence from the ER through the Golgi apparatus into secretory granules, which then discharge their cargo by membrane fusion at the cell surface (1). He documented the role of vesicles as carriers of cargo between compartments and he formulated the hypothesis that membranes template their own production rather than form by a process of de novo biogenesis (1). As a university student I was ignorant of the important developments in cell biology; however, I learned of Palade’s work during my first year of graduate school in the Stanford biochemistry department.
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2004/0058321 A1 Brunkow Et Al
    US 200400.58321A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0058321 A1 BrunkOW et al. (43) Pub. Date: Mar. 25, 2004 (54) COMPOSITIONS AND METHODS FOR Related U.S. Application Data INCREASING BONE MINERALIZATION (63) Continuation of application No. 09/449,218, filed on (75) Inventors: Mary E. Brunkow, Seattle, WA (US); Nov. 24, 1999, now Pat. No. 6,395,511. David J. Galas, Claremont, CA (US); Brian Kovacevich, Renton, WA (US); (60) Provisional application No. 60/110,283, filed on Nov. John T. Mulligan, Seattle, WA (US); 27, 1998. Bryan Paeper, Seattle, WA (US); Jeffrey Van Ness, Claremont, CA (US); Publication Classification David G. Winkler, Seattle, WA (US) (51) Int. Cl. ............................ C12O 1/68; CO7H 21/04; Correspondence Address: A61K 39/395; C12P 21/02; SEED INTELLECTUAL PROPERTY LAW C12N 5/06; CO7K 16/22 GROUP PLLC (52) U.S. Cl. ......................... 435/6; 435/69.1; 435/320.1; 701 FIFTHAVE 435/325; 530/388.25; 424/145.1; SUTE 6300 536/23.5 SEATTLE, WA 98104-7092 (US) (57) ABSTRACT (73) Assignee: Darwin Discovery Ltd., Slough (GB) A novel class or family of TGF-B binding proteins is (21) Appl. No.: 10/095,248 disclosed. Also disclosed are assays for Selecting molecules for increasing bone mineralization and methods for utilizing (22) Filed: Mar. 7, 2002 Such molecules. Patent Application Publication Mar. 25, 2004 Sheet 1 of 6 US 2004/0058321 A1 Common Cysteine Backbone 1. 50 human-gremlin.pro human-Cerberus pro MHLLLFOLLY LLPLGKTTRH ODGRONOSSL SPYLLPRNOR ELPTGNHEEA human-dan pro re-asawwara reserwrwarrara's swarara-as-a-Wiswe sawsWawswaas awaawawa-a-a-ay human-beer pro 51 100 human-gremlin.pro MSRTAYTVGALLLLLGTLLPA AEGKKKGSOG human-CerberuS.
    [Show full text]
  • THE POSSIBLE ROLE of ENDOGENOUS RETROVIRUSES in TUMOUR DEVELOPMENT & INNATE SIGNALLING by EMMANUEL ATANGANA MAZE a Thesis Su
    University of Plymouth PEARL https://pearl.plymouth.ac.uk 04 University of Plymouth Research Theses 01 Research Theses Main Collection 2018 THE POSSIBLE ROLE OF ENDOGENOUS RETROVIRUSES IN TUMOUR DEVELOPMENT AND INNATE SIGNALLING Atangana Maze, Emmanuel http://hdl.handle.net/10026.1/13081 University of Plymouth All content in PEARL is protected by copyright law. Author manuscripts are made available in accordance with publisher policies. Please cite only the published version using the details provided on the item record or document. In the absence of an open licence (e.g. Creative Commons), permissions for further reuse of content should be sought from the publisher or author. THE POSSIBLE ROLE OF ENDOGENOUS RETROVIRUSES IN TUMOUR DEVELOPMENT & INNATE SIGNALLING by EMMANUEL ATANGANA MAZE A thesis submitted to the University of Plymouth in partial fulfilment for the degree of DOCTOR OF PHILOSOPHY School of Biomedical Sciences 2018 COPYRIGHT STATEMENT This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognize that its copyright rests with its author and that no quotation from the thesis and no information derived from it may be published without the author’s prior consent. 2 THE POSSIBLE ROLE OF ENDOGENOUS RETROVIRUSES IN TUMOUR DEVELOPMENT & INNATE SIGNALLING by EMMANUEL ATANGANA MAZE UNIVERSITY OF PLYMOUTH School of Biomedical Sciences 2018 3 “You are the light of the world. A town built on a hill cannot be hidden. Neither do people light a lamp and put it under a bowl. Instead they put it on its stand, and it gives light to everyone in the house.
    [Show full text]
  • Origins and Evolution of the Global RNA Virome
    bioRxiv preprint doi: https://doi.org/10.1101/451740; this version posted October 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Origins and Evolution of the Global RNA Virome 2 Yuri I. Wolfa, Darius Kazlauskasb,c, Jaime Iranzoa, Adriana Lucía-Sanza,d, Jens H. 3 Kuhne, Mart Krupovicc, Valerian V. Doljaf,#, Eugene V. Koonina 4 aNational Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, USA 5 b Vilniaus universitetas biotechnologijos institutas, Vilnius, Lithuania 6 c Département de Microbiologie, Institut Pasteur, Paris, France 7 dCentro Nacional de Biotecnología, Madrid, Spain 8 eIntegrated Research Facility at Fort Detrick, National Institute of Allergy and Infectious 9 Diseases, National Institutes of Health, Frederick, Maryland, USA 10 fDepartment of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon, USA 11 12 #Address correspondence to Valerian V. Dolja, [email protected] 13 14 Running title: Global RNA Virome 15 16 KEYWORDS 17 virus evolution, RNA virome, RNA-dependent RNA polymerase, phylogenomics, horizontal 18 virus transfer, virus classification, virus taxonomy 1 bioRxiv preprint doi: https://doi.org/10.1101/451740; this version posted October 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 19 ABSTRACT 20 Viruses with RNA genomes dominate the eukaryotic virome, reaching enormous diversity in 21 animals and plants. The recent advances of metaviromics prompted us to perform a detailed 22 phylogenomic reconstruction of the evolution of the dramatically expanded global RNA virome.
    [Show full text]
  • Meta-Transcriptomic Detection of Diverse and Divergent RNA Viruses
    bioRxiv preprint doi: https://doi.org/10.1101/2020.06.08.141184; this version posted June 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Meta-transcriptomic detection of diverse and divergent 2 RNA viruses in green and chlorarachniophyte algae 3 4 5 Justine Charon1, Vanessa Rossetto Marcelino1,2, Richard Wetherbee3, Heroen Verbruggen3, 6 Edward C. Holmes1* 7 8 9 1Marie Bashir Institute for Infectious Diseases and Biosecurity, School of Life and 10 Environmental Sciences and School of Medical Sciences, The University of Sydney, 11 Sydney, Australia. 12 2Centre for Infectious Diseases and Microbiology, Westmead Institute for Medical 13 Research, Westmead, NSW 2145, Australia. 14 3School of BioSciences, University of Melbourne, VIC 3010, Australia. 15 16 17 *Corresponding author: 18 Marie Bashir Institute for Infectious Diseases and Biosecurity, School of Life and 19 Environmental Sciences and School of Medical Sciences, 20 The University of Sydney, 21 Sydney, NSW 2006, Australia. 22 Tel: +61 2 9351 5591 23 Email: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.08.141184; this version posted June 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
    [Show full text]
  • Table of Contents
    Copyright by William Paul Robins 2008 The Dissertation Committee for William Paul Robins certifies that this is the approved version of the following dissertation ANTITERMINATION IS OPERATIVE IN BACTERIOPHAGE T7 AND IS LARGELY DEPENDENT ON ONE PROMOTER Committee: ____________________________________ Ian J. Molineux , Supervisor ____________________________________ Whitney Yin ____________________________________ Tanya Paull ____________________________________ Richard Meyer ____________________________________ Charles Earhart ANTITERMINATION IS OPERATIVE IN BACTERIOPHAGE T7 AND IS LARGELY DEPENDENT ON ONE PROMOTER by William Paul Robins, B.S. DISSERTATION Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY The University of Texas at Austin August 2008 DEDICATION This Dissertation is dedicated to the memory of my grandfather, Paul Rogers. His profound love of nature influenced my desire to study Science. ACKNOWLEDGEMENTS I would like to thank my supervisor Ian Molineux for his support and encouragement. His rigorous, thorough, and enthusiastic approach to science has been a valuable experience. I expect his mentoring will be a very positive influence on my future endeavors. I am also grateful to Pricilla Kemp for additional help and assistance in my work. I would also like to acknowledge Dhruti Savalia for investigating and analyzing my promoter mutations using abortive transcription assays and primer extension experiments. Her biochemical approach on T7 gene 2 was a very useful complement to the phage genetics in our lab. Finally, I need to thank my family; especially my wife Hannah Robins, my son Jefferson Pinkus and my parents Sam Robins and Gail Levansellar. v ANTITERMINATION IS OPERATIVE IN BACTERIOPHAGE T7 AND IS LARGELY DEPENDENT ON ONE PROMOTER William Paul Robins, Ph.D.
    [Show full text]
  • (Λ) Rex Phenotype
    Isolation and Characterization of Host mutations that Supress the Bacteriophage Lambda (λ) Rex Phenotype by Hibah Alattas A thesis presented to the University of Waterloo in fulfillment of the thesis requirement for the degree of Master of Science in Pharmacy Waterloo, Ontario, Canada, 2015 ©Hibah Alattas 2015 Author’s Declaration I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii Abstract The Bacteriophage lambda (λ) T4rII exclusion (Rex) phenotype is defined as the inability of T4rII mutant bacteriophage to form plaques on a lawn of E. coli lysogenized by bacteriophage λ. More than six decades have passed following the discovery of Rex by Seymour Benzer in 1955, yet the mechanism behind this elusive exclusion system remains a mystery. The Rex system is encoded by two genes of λ (rexA, and rexB), the expression of which, is primarily regulated by the repressor gene cI from the PM promoter. The onset of the Rex phenotype, somehow triggered by T4rII infection of a Rex+ lysogen, results in rapid membrane depolarization and a harsh cellular environment that in many ways resembles the stationary phase in metabolism and morphology. In addition, the disruption of the RexA:RexB balance, particularly the over expression of rexA to rexB, can lead to same manifestations without infection, indicating that stoichiometry of RexA:RexB is important. Despite some cell killing, infected lysogens can to some extent recover from Rex activation.
    [Show full text]
  • Participation of Multifunctional RNA in Replication, Recombination and Regulation of Endogenous Plant Pararetroviruses (Eprvs)
    fpls-12-689307 June 18, 2021 Time: 16:5 # 1 MINI REVIEW published: 21 June 2021 doi: 10.3389/fpls.2021.689307 Participation of Multifunctional RNA in Replication, Recombination and Regulation of Endogenous Plant Pararetroviruses (EPRVs) Katja R. Richert-Pöggeler1*, Kitty Vijverberg2,3, Osamah Alisawi4, Gilbert N. Chofong1, J. S. (Pat) Heslop-Harrison5,6 and Trude Schwarzacher5,6 1 Julius Kühn-Institut, Federal Research Centre for Cultivated Plants, Institute for Epidemiology and Pathogen Diagnostics, Braunschweig, Germany, 2 Naturalis Biodiversity Center, Evolutionary Ecology Group, Leiden, Netherlands, 3 Radboud University, Institute for Water and Wetland Research (IWWR), Nijmegen, Netherlands, 4 Department of Plant Protection, Faculty of Agriculture, University of Kufa, Najaf, Iraq, 5 Department of Genetics and Genome Biology, University of Leicester, Leicester, United Kingdom, 6 Key Laboratory of Plant Resources Conservation and Sustainable Utilization, Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China Edited by: Jens Staal, Pararetroviruses, taxon Caulimoviridae, are typical of retroelements with reverse Ghent University, Belgium transcriptase and share a common origin with retroviruses and LTR retrotransposons, Reviewed by: Jie Cui, presumably dating back 1.6 billion years and illustrating the transition from an RNA Institut Pasteur of Shanghai (CAS), to a DNA world. After transcription of the viral genome in the host nucleus, viral DNA China Marco Catoni, synthesis occurs in the cytoplasm on the generated terminally redundant RNA including University of Birmingham, inter- and intra-molecule recombination steps rather than relying on nuclear DNA United Kingdom replication. RNA recombination events between an ancestral genomic retroelement with *Correspondence: exogenous RNA viruses were seminal in pararetrovirus evolution resulting in horizontal Katja R.
    [Show full text]
  • Escherichia Coli
    Escherichia coli “E. coli” redirects here. For the protozoan commensal, 1 Biology and biochemistry see Entamoeba coli. This article is about Escherichia coli as a species. For E. coli in medicine, see Pathogenic Escherichia coli. For E. coli in molecular biology, see Escherichia coli (molecular biology). Escherichia coli (/ˌɛʃᵻˈrɪkiə ˈkoʊlaɪ/;[1] also known as E. coli) is a Gram-negative, facultatively anaerobic, rod- shaped bacterium of the genus Escherichia that is com- monly found in the lower intestine of warm-blooded or- ganisms (endotherms).[2] Most E. coli strains are harm- less, but some serotypes can cause serious food poisoning in their hosts, and are occasionally responsible for product recalls due to food contamination.[3][4] The harmless strains are part of the normal flora of the gut, and can [5] benefit their hosts by producing vitamin K2, and pre- venting colonization of the intestine with pathogenic bac- teria.[6][7] E. coli is expelled into the environment within Model of successive binary fission in E. coli fecal matter. The bacterium grows massively in fresh fe- cal matter under aerobic conditions for 3 days, but its numbers decline slowly afterwards.[8] E. coli and other facultative anaerobes constitute about 0.1% of gut flora,[9] and fecal–oral transmission is the major route through which pathogenic strains of the bac- terium cause disease. Cells are able to survive outside the body for a limited amount of time, which makes them potential indicator organisms to test environmental sam- ples for fecal contamination.[10][11] A growing body of re- search, though, has examined environmentally persistent E.
    [Show full text]
  • Phage Lambda P Protein: Trans-Activation, Inhibition Phenotypes and Their Suppression
    Viruses 2013, 5, 619-653; doi:10.3390/v5020619 OPEN ACCESS viruses ISSN 1999-4915 www.mdpi.com/journal/viruses Article Phage Lambda P Protein: Trans-Activation, Inhibition Phenotypes and their Suppression Sidney Hayes *, Craig Erker, Monique A. Horbay, Kristen Marciniuk, Wen Wang and Connie Hayes Department of Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, S7N 5E5 Canada; E-Mails: [email protected] (S.H.); [email protected] (C.E.); [email protected] (M.A.H.); [email protected] (K.M.); [email protected] (W.W.); [email protected] (C.H.) * Author to whom correspondence should be addressed; [email protected]; Tel.: +1-306-966-4307; Fax: +1-306-966-4298. Received: 4 December 2012; in revised form: 21 January 2013 / Accepted: 29 January 2013 / Published: 6 February 2013 Abstract: The initiation of bacteriophage λ replication depends upon interactions between the oriλ DNA site, phage proteins O and P, and E. coli host replication proteins. P exhibits a high affinity for DnaB, the major replicative helicase for unwinding double stranded DNA. The concept of P-lethality relates to the hypothesis that P can sequester DnaB and in turn prevent cellular replication initiation from oriC. Alternatively, it was suggested that P-lethality does not involve an interaction between P and DnaB, but is targeted to DnaA. P-lethality is assessed by examining host cells for transformation by ColE1-type plasmids that can express P, and the absence of transformants is attributed to a lethal effect of P expression. The plasmid we employed enabled conditional expression of P, where under permissive conditions, cells were efficiently transformed.
    [Show full text]
  • Machine Learning Using Intrinsic Genomic Signatures for Rapid Classification of Novel Pathogens: COVID-19 Case Study
    bioRxiv preprint doi: https://doi.org/10.1101/2020.02.03.932350. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license. Machine learning using intrinsic genomic signatures for rapid classification of novel pathogens: COVID-19 case study Gurjit S. Randhawa1Y*, Maximillian P.M. Soltysiak2Y, Hadi El Roz2, Camila P.E. de Souza3, Kathleen A. Hill2, Lila Kari4 1 Department of Computer Science, The University of Western Ontario, London, ON, Canada 2 Department of Biology, The University of Western Ontario, London, ON, Canada 3 Department of Statistical and Actuarial Sciences, The University of Western Ontario, London, ON, Canada 4 School of Computer Science, University of Waterloo, Waterloo, ON, Canada YThese authors contributed equally to this work. * [email protected] Abstract As of February 20, 2020, the 2019 novel coronavirus (renamed to COVID-19) spread to 30 countries with 2130 deaths and more than 75500 confirmed cases. COVID-19 is being compared to the infamous SARS coronavirus, which resulted, between November 2002 and July 2003, in 8098 confirmed cases worldwide with a 9:6% death rate and 774 deaths. Though COVID-19 has a death rate of 2:8% as of 20 February, the 75752 confirmed cases in a few weeks (December 8, 2019 to February 20, 2020) are alarming, with cases likely being under-reported given the comparatively longer incubation period. Such outbreaks demand elucidation of taxonomic classification and origin of the virus genomic sequence, for strategic planning, containment, and treatment. This paper identifies an intrinsic COVID-19 genomic signature and uses it together with a machine learning-based alignment-free approach for an ultra-fast, scalable, and highly accurate classification of whole COVID-19 genomes.
    [Show full text]
  • Structure of the Ty3/Gypsy Retrotransposon Capsid and the Evolution of Retroviruses
    Structure of the Ty3/Gypsy retrotransposon capsid and the evolution of retroviruses Svetlana O. Dodonovaa,b, Simone Prinza,1, Virginia Bilanchonec, Suzanne Sandmeyerc, and John A. G. Briggsa,d,2 aStructural and Computational Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany; bDepartment of Molecular Biology, Max Planck Institute for Biophysical Chemistry, 37077 Gottingen, Germany; cDepartment of Biological Chemistry, University of California, Irvine, CA 92697; and dStructural Studies Division, MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, CB2 0QH Cambridge, United Kingdom Edited by Wesley I. Sundquist, University of Utah Medical Center, Salt Lake City, UT, and approved March 29, 2019 (received for review January 21, 2019) Retroviruses evolved from long terminal repeat (LTR) retrotranspo- a bilobar capsid domain with structural similarity to retroviral CA sons by acquisition of envelope functions, and subsequently rein- proteins (11, 12). Arc was recently shown to form capsid-like vaded host genomes. Together, endogenous retroviruses and LTR structures, which are implicated in neuronal function and mem- retrotransposons represent major components of animal, plant, and ory (13, 14). fungal genomes. Sequences from these elements have been exapted The GAG gene in retroviruses and retrotransposons is initially to perform essential host functions, including placental development, expressed as Gag and Gag-Pol precursor polyproteins. Gag is synaptic communication, and transcriptional regulation. They encode greatly abundant over Gag-Pol and forms the structural basis of a Gag polypeptide, the capsid domains of which can oligomerize to immature particle assembly and genome packaging (15, 16). In form a virus-like particle. The structures of retroviral capsids have retroviruses, the conserved domains of Gag are MA (the matrix been extensively described.
    [Show full text]