DOCSLIB.ORG

HIV-1 INTERSUBTYPE RECOMBINATION WITHIN GP120 IMPOSES SEVERE FUNCTIONAL RESTRICTION on RESULTANT ENVELOPES by BERNARD SSENTALO

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
HIV-1 INTERSUBTYPE RECOMBINATION WITHIN GP120 IMPOSES SEVERE FUNCTIONAL RESTRICTION on RESULTANT ENVELOPES by BERNARD SSENTALO HIV-1 INTERSUBTYPE RECOMBINATION WITHIN GP120 IMPOSES SEVERE FUNCTIONAL RESTRICTION ON RESULTANT ENVELOPES by BERNARD SSENTALO BAGAYA Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis Advisors: Yong Gao, MD, PhD. Eric J. Arts, PhD. Department of Molecular Biology and Microbiology CASE WESTERN RESERVE UNIVERSITY MAY, 2015 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of BERNARD SSENTALO BAGAYA __________________________________________________________ Doctor of Philosophy candidate for the __________________________________________ degree*. David McDonald, Ph.D. Signed _______________________________________________________ (chair of the committee) Jonathan Karn, Ph.D. _______________________________________________________ Eric J. Arts, Ph.D. _______________________________________________________ Yong Gao, M.D, PhD. _______________________________________________________ (dissertation advisor) Neil Greenspan, M.D, PhD. _______________________________________________________ Carlos Subauste, M.D, PhD. _______________________________________________________ October 24, 2014 (date) _______________________________________________________ *We also certify that written approval has been obtained for any proprietary material contained therein 1 TABLE OF CONTENTS Content Page Approval Signatures…………………………………………................................. 1 Table of contents……………...…………………………....…………………........ 2 Dedication……………………..…………………………………………..……… 8 Acknowledgements…..……………………………………..…………..………… 9 List of figures………………………………………………………………..……. 11 List of tables………………………………………….……………..…………...... 14 List of Appendices……………………………………………..………………..... 15 List of abbreviations…………………………….…….………………………….. 16 Abstract…………………………………….…………………………..…………. 21 CHAPTER ONE: INTRODUCTION……………………….…..………………... 23 1.1. History of HIV-1 and AIDS…………………………………...... 23 1.2. The HIV-1 particle………………..……….……………...…….. 25 1.3. The HIV-1 genome and proteins…………………………..……. 25 1.3.1. Regulatory genes/elements………...…….…………. 27 1.3.1.1. The LTR sequences..……………….……... 27 1.3.1.2. Tat/rev……………………………...…….... 28 1.3.2. Accessory genes (vif, vpr, vpu, and nef)………...…. 28 1.3.3. The HIV-1 structural genes (gag, pol and Env)....…... 29 1.3.3.1. gag……………………….…………….….. 29 2 1.3.3.2. pol…………………………......................... 30 1.3.3.3. The Env gene……………………................ 31 1.4. The HIV-1 Envelope glycoprotein…………………..…..…….… 34 1.5. The HIV-1 life cycle………………………....……………..……. 38 1.5.1. HIV-1 entry………………………...………………. 38 1.5.2. Reverse transcription…………………..................... 43 1.5.3. Assembly, budding and maturation…………...…… 45 1.6. HIV-1 diversity…………………………..……………………….. 50 1.6.1. HIV-1 recombination………...…….…….................. 50 1.6.1.1. Models of HIV-1 recombinations…... 52 1.6.1.2. Consequences of HIV-1 intersubtype recombination…….............................. 59 CHAPTER TWO: MATERIALS AND METHODS……………………...……… 66 2.1. HIV-1 primary isolates………………….…........................................... 66 2.2. Mammalian cells………………….....…………………………......…... 66 2.3. Yeast and bacteria cell lines…………………………….…..………..… 67 2.4. Reagents and Media………………………...………..………………… 67 2.5 . Preparation of culture media and plates……..…..................................... 68 2.6 . Yeast gap repair homologous recombination-based cloning method….. 68 2.7 . Phenol:chloroform:Isoamyl alcohol plasmid DNA extraction from yeast………...…..……......…………………………………………... 69 3 2.8 . Bacterial transformation and plasmid extraction……............................. 70 2.9 . The complementation system for production of HIV-1 in-vitro; the pREC_nfl_HIV and the pCMV_5’LTR_cplt plasmids........................ 71 2.10 . Transfection and infection for virus propagation...……………...……. 72 2.11 . Reverse Transcriptase-activity assay (RT-Assay)……………..……... 73 2.12 . In-house gp140/160 ELISA…………………………..………………. 74 2.13 . Veritrop assay………………………………………...……………...... 75 2.14 . Production of chimeric HIV-1………………………………………... 77 2.15 . The Dual infection method for production of HIV-1 recombinants.... 79 2.16. Design and construction of the HIV-1 Env recombination system.…... 80 2.17 . HIV-1 Env cloning……………………………………………………. 81 2.18 . Clonal sequencing………………….…………………………………. 82 2.1 9. Downloading HIV-1 recombinant Env sequences from database.......... 83 2.20 . Design and construction of the HIV-1 pol recombination system......... 84 2.21 . 454 sequencing…………..….………………………............................ 86 CHAPTER THREE: A NOVEL SYSTEM FOR GENERATION OF PURE AND FUNCTIONAL HIV-1 POL AND ENV INTERSUBTYPE RECOMBINANTS IN VITRO………...…………………………......................... 89 3.1. PREFACE………………………………………………..………. 90 3.2. INTRODUCTION………………………………………..………. 92 3.3. RESULTS…………………………….………………..………… 94 3.3.1. Establishment of the yeast based recombination/HIV-1 cloning system..................................................................... 94 3.3.2. The complementation system for production of chimeric infectious HIV-1 in cell cultures………………................. 96 4 3.3.3. Establishment of the HIV-1 Env recombination system….. 103 3.3.4. Production of replicating intersubtype A/D Envelope recombinant HIV-1 by the Env recombination system…... 104 3.3.5. Optimization of the HIV-1 intersubtype Env recombination system…………………………………….. 110 3.3.6. The HIV-1 intersubtype Env recombination system produces 100% recombinants……………………………. 113 3.3.7. The HIV-1 pol intersubtype recombination system………. 117 3.3.8. The pol recombination system produces infectious replicating virus…………………………………………... 118 3.4. Discussion…………………………………….………………..... 122 CHAPTER FOUR: RECOMBINATION BREAKPOINTS IN GP120 IMPOSE SEVERE FUNCTIONAL RESTRICTION ON RESULTANT ENVELOPE GLYCOPROTEINS………………………………..…………......... 126 4.1. PREFACE……...……...…………….…………………………... 127 4.2. INTRODUCTION……………………………..………................ 128 4.3. RESULTS……………………………….…………..……...……. 131 4.3.1. Production of HIV-1 Env recombinants using the novel Env recombination system that enriches for functional recombinants……………………………………………… 131 4.3.2. Clonal sequence analysis shows an absence of gp120 recombination breakpoints in recombination system virus population…………..…………………………………….. 136 4.3.3. The dual infection method generates gp120 breakpoints, ruling out lack of adequate sequence similarity to support homologous recombination…............................................. 138 4.3.4. gp120 breakpoints are present in sequence database- 5 derived recombinant Envs, overwhelmingly concentrated in the C1 domain and very few elsewhere………………... 139 4.3.5. Pyro-sequencing shows that HIV-1 Env recombination system is not defective in generating gp120 breakpoints… 143 4.4. DISCUSSION………………...…………….…………………… 147 4.5. CONCLUSION………………………………………………….. 150 CHAPTER FIVE: BOTTLENECKS FOR GENERATION OF FUNCTIONAL HIV-1 ENV RECOMBINANTS IN VITRO……………………. 151 5.1. PREFACE……………...…….………..…………….................... 152 5.2. INTRODUCTION………………….………………..................... 153 5.3. RESULTS………………………...………….…………………... 155 5.3.1. Generation of Intersubtype A/D gp120 recombinants….... 155 5.3.2. Full length HIV-1 A/D recombinant Envelopes with breakpoints in gp120 are non-functional when cloned into a neutral NL4-3 backbone…………...…………..…...…... 156 5.3.3. Replacement of HIV-1 NL4-3 tat1/rev1 sequence with D109 tat1/rev1 restores function in 2 of the 21 EnvA/D recombinants……………………………………………… 161 5.3.4. Cloning only gp120 region does not overcome the functional restriction of EnvA/Ds with breakpoints in gp120……………………………………………………... 162 5.3.5. Re-insertion of NL4-3 gp120 C1 and C5 domains back into non-functional recombinant gp120 chimeric plasmids restored function in 4 of 9 recombinants……..… 163 5.4. DISCUSSION……………………………….………………… 171 CHAPTER SIX: GENERAL CONCLUSIONS AND FUTURE DIRECTIONS... 176 6 6.1. General conclusions……….…...……………………...…...…... 176 6.2 . Future direction…..………………………...…………...…….... 178 6.2. 1. Study on HIV-1 intersubtype recombinant Envelope function restriction factors................................................ 178 6.2.2. Study on anti-HIV-1 drug resistance……….….…..….... 179 6.2.3. Development of anti-HIV-1 vaccines with intersubtype Env recombinants...…..………………………………… 181 APPENDICES………………………...……………….………………................ 183 REFERENCES…………………………….…………………...………………... 193 7 DEDICATION This thesis is dedicated to my darling wife Lillian L. Bagaya. You have been my strength in times of weakness and my guiding star when all seemed dark. You made the old English proverb of “behind every successful man, there is a woman”, come to reality. To you I am forever indebted. 8 ACKNOWLEDGEMENTS To my father who, despite of having had no opportunity to receive an education, knew its value, toiled and unreservedly spent his hard earned income to pay for my education so I wouldn’t have to earn a living through hard labor like he did; you are my hero. To my mother who has been a never drying fountain of encouragement and wisdom, I will always revere you. Great thanks to my wife Lillian and to my little Angels Daniella, Diella and Atuwaire for the sacrifices you have made for us to achieve this. You have been patient and encouraging, I have almost been missing in your lives but you endured, thank you for understanding and persevering. I thank all the current and former members of the Arts/Gao laboratories at CWRU School of Medicine for the help, friendship and humor we shared. It would have been a lot harder without you around. I am grateful
Load more

Recommended publications
  • Repression of Viral Gene Expression and Replication by the Unfolded Protein Response Effector Xbp1u Florian Hinte1, Eelco Van Anken2,3, Boaz Tirosh4, Wolfram Brune1*
    RESEARCH ARTICLE Repression of viral gene expression and replication by the unfolded protein response effector XBP1u Florian Hinte1, Eelco van Anken2,3, Boaz Tirosh4, Wolfram Brune1* 1Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany; 2Division of Genetics and Cell Biology, San Raffaele Scientific Institute, Milan, Italy; 3Universita` Vita-Salute San Raffaele, Milan, Italy; 4Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University, Jerusalem, Israel Abstract The unfolded protein response (UPR) is a cellular homeostatic circuit regulating protein synthesis and processing in the ER by three ER-to-nucleus signaling pathways. One pathway is triggered by the inositol-requiring enzyme 1 (IRE1), which splices the X-box binding protein 1 (Xbp1) mRNA, thereby enabling expression of XBP1s. Another UPR pathway activates the activating transcription factor 6 (ATF6). Here we show that murine cytomegalovirus (MCMV), a prototypic b-herpesvirus, harnesses the UPR to regulate its own life cycle. MCMV activates the IRE1-XBP1 pathway early post infection to relieve repression by XBP1u, the product of the unspliced Xbp1 mRNA. XBP1u inhibits viral gene expression and replication by blocking the activation of the viral major immediate-early promoter by XBP1s and ATF6. These findings reveal a redundant function of XBP1s and ATF6 as activators of the viral life cycle, and an unexpected role of XBP1u as a potent repressor of both XBP1s and ATF6-mediated activation. *For correspondence: [email protected] Introduction The endoplasmic reticulum (ER) is responsible for synthesis, posttranslational modification, and fold- Competing interest: See ing of a substantial portion of cellular proteins.
    [Show full text]
  • Impact of Natural HIV-1 Nef Alleles and Polymorphisms on SERINC3/5 Downregulation
    Impact of natural HIV-1 Nef alleles and polymorphisms on SERINC3/5 downregulation by Steven W. Jin B.Sc., Simon Fraser University, 2016 Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Master of Science Program Faculty of Health Sciences © Steven W. Jin 2019 SIMON FRASER UNIVERSITY Spring 2019 Copyright in this work rests with the author. Please ensure that any reproduction or re-use is done in accordance with the relevant national copyright legislation. Approval Name: Steven W. Jin Degree: Master of Science Title: Impact of natural HIV-1 Nef alleles and polymorphisms on SERINC3/5 downregulation Examining Committee: Chair: Kanna Hayashi Assistant Professor Mark Brockman Senior Supervisor Associate Professor Masahiro Niikura Supervisor Associate Professor Ralph Pantophlet Supervisor Associate Professor Lisa Craig Examiner Professor Department of Molecular Biology and Biochemistry Date Defended/Approved: April 25, 2019 ii Ethics Statement iii Abstract HIV-1 Nef is a multifunctional accessory protein required for efficient viral pathogenesis. It was recently identified that the serine incorporators (SERINC) 3 and 5 are host restriction factors that decrease the infectivity of HIV-1 when incorporated into newly formed virions. However, Nef counteracts these effects by downregulating SERINC from the cell surface. Currently, there lacks a comprehensive study investigating the impact of primary Nef alleles on SERINC downregulation, as most studies to date utilize lab- adapted or reference HIV strains. In this thesis, I characterized and compared SERINC downregulation from >400 Nef alleles isolated from patients with distinct clinical outcomes and subtypes. I found that primary Nef alleles displayed a dynamic range of SERINC downregulation abilities, thus allowing naturally-occurring polymorphisms that modulate this activity to be identified.
    [Show full text]
  • Recombinant DNA and Elements Utilizing Recombinant DNA Such As Plasmids and Viral Vectors, and the Application of Recombinant DNA Techniques in Molecular Biology
    Fact Sheet Describing Recombinant DNA and Elements Utilizing Recombinant DNA Such as Plasmids and Viral Vectors, and the Application of Recombinant DNA Techniques in Molecular Biology Compiled and/or written by Amy B. Vento and David R. Gillum Office of Environmental Health and Safety University of New Hampshire June 3, 2002 Introduction Recombinant DNA (rDNA) has various definitions, ranging from very simple to strangely complex. The following are three examples of how recombinant DNA is defined: 1. A DNA molecule containing DNA originating from two or more sources. 2. DNA that has been artificially created. It is DNA from two or more sources that is incorporated into a single recombinant molecule. 3. According to the NIH guidelines, recombinant DNA are molecules constructed outside of living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell, or molecules that result from their replication. Description of rDNA Recombinant DNA, also known as in vitro recombination, is a technique involved in creating and purifying desired genes. Molecular cloning (i.e. gene cloning) involves creating recombinant DNA and introducing it into a host cell to be replicated. One of the basic strategies of molecular cloning is to move desired genes from a large, complex genome to a small, simple one. The process of in vitro recombination makes it possible to cut different strands of DNA, in vitro (outside the cell), with a restriction enzyme and join the DNA molecules together via complementary base pairing. Techniques Some of the molecular biology techniques utilized during recombinant DNA include: 1.
    [Show full text]
  • MS Ritgerð Aðalbjörg Aðalbjörnsdóttir
    The Vif protein of maedi-visna virus Protein interaction and new roles Aðalbjörg Aðalbjörnsdóttir Thesis for the degree of Master of Science University of Iceland Faculty of medicine School of Health Sciences Vif prótein mæði-visnuveiru Prótein tengsl og ný hlutverk Aðalbjörg Aðalbjörnsdóttir Ritgerð til meistaragráðu í Líf og læknavísindum Umsjónarkennari: Valgerður Andrésdóttir Meistaranámsnefnd: Stefán Ragnar Jónsson og Ólafur S. Andrésson Læknadeild Heilbrigðisvísindasvið Háskóla Íslands Júní 2016 The Vif protein of maedi-visna virus Protein interaction and new roles Aðalbjörg Aðalbjörnsdóttir Thesis for the degree of Master of Science Supervisor: Valgerður Andrésdóttir Masters committee: Stefán Ragnar Jónsson and Ólafur S. Andrésson Faculty of Medicine School of Health Sciences June 2016 Ritgerð þessi er til meistaragráðu í Líf og læknavísindum og er óheimilt að afrita ritgerðina á nokkurn hátt nema með leyfi rétthafa. © Aðalbjörg Aðalbjörnsdóttir 2016 Prentun: Háskólaprent Reykjavík, Ísland 2016 Ágrip Mæði-visnuveira (MVV) er lentiveira af ættkvísl retróveira. Hún veldur hæggengri lungnabólgu (mæði) og heilabólgu (visnu) í kindum. Aðalmarkfrumur veirunnar eru mónocytar/makrófagar. Veiran er náskyld HIV og hefur verið notuð sem módel fyrir HIV sýkingar. Stöðug vopnakapphlaup milli veira og fruma hafa leitt af sér fjölda sértækra aðferða í vörnum hýsilsfrumu gegn veirusýkingum. Fruman hefur þróað með sér innrænar varnir gegn ýmsum sýkingum. Þessar varnir geta verið mjög sérhæfðar og tjáning þeirra spilar stórt hlutverk í hvaða frumur er hægt að sýkja og hverjar ekki. Dæmi um slíkan frumubundinn þátt eru APOBEC3 próteinin. APOBEC3 próteinin eru fjölskylda cytósín deaminasa sem geta hindrað retróveirur og retróstökkla. Þetta gera þau með því að afaminera cýtósín í úrasil í einþátta DNA á meðan á víxlritun stendur og valda þar með G-A stökkbreytingum í forveirunni.
    [Show full text]
  • 718 HIV Disorders of the Brain; Pathology and Pathogenesis Luis
    [Frontiers in Bioscience 11, 718-732, January 1, 2006] HIV disorders of the brain; pathology and pathogenesis Luis Del Valle and Sergio Piña-Oviedo Center for Neurovirology and Cancer Biology, Laboratory of Neuropathology and Molecular Pathology, Temple University, 1900 North 12th Street, Suite 240, Philadelphia, Pennsylvania 19122 USA TABLE OF CONTENTS 1. Abstract 2. AIDS-Encephalopathy 2.1. Definition 2.2. HIV-1 Structure 2.3. Histopathology 2.4. Clinical Manifestations 2.5. Physiopathology 3. Progressive Multifocal Leukoencephalopathy 3.1. Definition 3.2. JC Virus Biological Considerations 3.3. JC Virus Structure 3.4. Histopathology 3.5. Clinical Manifestations 3.6. Physiopathology 4. Cryptococcosis 5.1. Definition 5.2. Cryptococcus neoformans Structure 5.3. Histopathology 5.4. Clinical Manifestations 5.5. Physiopathology 5. Toxoplasmosis 5.1. Definition 5.2. Toxoplasma godii Structure 5.3. Histopathology 5.4. Clinical Manifestations 5.5. Physiopathology 6. Primary CNS Lymphomas 6.1. Definition 6.2. Histopathology 6.3. Clinical Manifestations 6.4. Physiopathology 7. Acknowledgments 8. References 1. ABSTRACT Infection with HIV-1 has spread exponentially in still present in approximately 70 to 90% of patients and recent years to reach alarming proportions. It is estimated can be the result of HIV itself or of opportunistic than more than 33 million adults and 1.3 million children infections. Here we briefly review the pathology and are infected worldwide. Approximately 16,000 new cases pathophysiology of AIDS-Encephalopathy, of some of are diagnosed every day and almost 3 million people die the significant opportunistic infections affecting the every year from AIDS, making it the fourth leading brain in the context of AIDS, including Progressive cause of death in the world.
    [Show full text]
  • Gibson Assembly Cloning Guide, Second Edition
    Gibson Assembly® CLONING GUIDE 2ND EDITION RESTRICTION DIGESTFREE, SEAMLESS CLONING Applications, tools, and protocols for the Gibson Assembly® method: • Single Insert • Multiple Inserts • Site-Directed Mutagenesis #DNAMYWAY sgidna.com/gibson-assembly Foreword Contents Foreword The Gibson Assembly method has been an integral part of our work at Synthetic Genomics, Inc. and the J. Craig Venter Institute (JCVI) for nearly a decade, enabling us to synthesize a complete bacterial genome in 2008, create the first synthetic cell in 2010, and generate a minimal bacterial genome in 2016. These studies form the framework for basic research in understanding the fundamental principles of cellular function and the precise function of essential genes. Additionally, synthetic cells can potentially be harnessed for commercial applications which could offer great benefits to society through the renewable and sustainable production of therapeutics, biofuels, and biobased textiles. In 2004, JCVI had embarked on a quest to synthesize genome-sized DNA and needed to develop the tools to make this possible. When I first learned that JCVI was attempting to create a synthetic cell, I truly understood the significance and reached out to Hamilton (Ham) Smith, who leads the Synthetic Biology Group at JCVI. I joined Ham’s team as a postdoctoral fellow and the development of Gibson Assembly began as I started investigating methods that would allow overlapping DNA fragments to be assembled toward the goal of generating genome- sized DNA. Over time, we had multiple methods in place for assembling DNA molecules by in vitro recombination, including the method that would later come to be known as Gibson Assembly.
    [Show full text]
  • UC Merced UC Merced Undergraduate Research Journal
    UC Merced UC Merced Undergraduate Research Journal Title Antiviral Drugs Targeting Host Proteins an Efficient Strategy Permalink https://escholarship.org/uc/item/66f5b4m0 Journal UC Merced Undergraduate Research Journal, 9(2) Author Karmonphet, Arrada Publication Date 2017 DOI 10.5070/M492034789 Undergraduate eScholarship.org Powered by the California Digital Library University of California Antiviral Drugs Targeting Host Proteins an Efficient Strategy Arrada Karmonphet University of California, Merced Keywords: Proteins, Viruses, Drugs 1 Abstract Viruses have the ability to spread rapidly because the proteins and enzymes from the host cell help in the development of viruses. Although there are many vaccines that can prevent some viruses from infecting the body, the antiviral drugs today have not been effective in combating viruses from the start of spreading. This is due to the fact that the processes inside a virus are still being studied. However, host proteins proved to be valuable factors responsible for viral replication and spreading. It was found that certain functions such as capsid formation of the virus utilized a biochemical pathway that involved host proteins and some proteins of the host cell were evolutionarily conserved. When the important host proteins were altered, or removed the viruses weren’t able to replicate as effectively. It was concluded that targeting the host proteins had a significant effect in viral replication. This approach can stop viral replication from the start, create less viral resistance, and help find new antiviral drugs that work for many different types of viruses. This review will analyze five research articles about protein interactions in viruses and how monitoring the proteins and biochemical pathways can lead to the discovery of druggable targets during development.
    [Show full text]
  • Patent Document US 06872395
    I 1111111111111111 11111 111111111111111 1111111111 1111111111 lll111111111111111 US006872395B2 (12) United States Patent (10) Patent No.: US 6,872,395 B2 Kawaoka (45) Date of Patent: Mar.29,2005 (54) VIRUSES COMPRISING MUTANT ION Mena, I., et al., "Rescue of a Synthetic Choramphenicol CHANNEL PROTEIN Acetyltransferase RNA into influenza Virus-Like Particles obtained from recombinant plasmids", J. of Virology, vol. (75) Inventor: Yoshihiro Kawaoka, Madison, WI 70, No. 8, XP002150091, 5016-5024, (Aug. 1996). (US) Neumann, G., et al., "Generation of influenza A Viruses entirely from cloned cDNAs", Proc. of the Nat'l Aca. of (73) Assignee: Wisconsin Alumni Research Sciences, USA, vol. 96, XP002150093, 9345-9350, (Aug. Foundation, Madison, WI (US) 1999). ( *) Notice: Subject to any disclaimer, the term of this Neumann, G., et al., "Plasmid-Driven Formation of Influ­ patent is extended or adjusted under 35 enza Virus-Like Particles", J. of Virology, vol. 74, No. 1, U.S.C. 154(b) by O days. XP002150094, 547-551, (Jan. 2000). Neumann, G., et al., "RNA Polymerase I-Mediated Expres­ sion of Influenza Viral RNA Molecules", Virology, vol. 202, (21) Appl. No.: 09/834,095 No. 1, XP000952667, 477-479, (Jul. 1994). (22) Filed: Apr. 12, 2001 Neirynck, S., et al., "A universal influenza A vaccine based on the extracellular domain of the M2 protein", Nature (65) Prior Publication Data Medicine, 5 (10), pp. 1157-1163, (Oct. 1999). US 2003/0194694 Al Oct. 16, 2003 Piller, S. C., et al., "Vpr protein of human immunodeficiency virus type 1 forms cation-selective channels in planar lipid Related U.S. Application Data bilayers", PNAS, 93, pp.
    [Show full text]
  • 19-Kilodalton Tumor Antigen T
    JOURNAL OF VIROLOGY, Nov. 1984, p. 336-343 Vol. 52, No. 2 0022-538X/84/110336-08$02.00/0 Copyright ©D 1984, American Society for Microbiology Adenovirus cyt+ Locus, Which Controls Cell Transformation and Tumorigenicity, Is an Allele of Ip+ Locus, Which Codes for a 19-Kilodalton Tumor Antigen T. SUBRAMANIAN,' MOHAN KUPPUSWAMY,1 STANLEY MAK,2 AND G. CHINNADURAI1* Institute for Molecular Virology, St. Louis University Medical Center, St. Louis, Missouri 63110,1 and Department of Biology, McMaster University, Hamilton, Ontario, Canada2 Received 30 April 1984/Accepted 19 July 1984 The early region Elb of adenovirus type 2 (Ad2) codes for two major tumor antigens of 53 and 19 kilodaltons (kd). The adenovirus Ip+ locus maps within the 19-kd tumor antigen-coding region (G. Chinnadurai, Cell 33:759-766, 1983). We have now constructed a large-plaque deletion mutant (d1250) of Ad2 that has a specific lesion in the 19-kd tumor antigen-coding region. In contrast to most other Ad2 lp mutants (G. Chinnadurai, Cell 33:759-766, 1983), mutant d1250 is cytocidal (cyt) on infected KB cells, causing extensive cellular destruction. Cells infected with Ad2 wt or most of these other Ad2 Ip mutants are rounded and aggregated without cell lysis (cyt+). The cyt phenotype of d1250 resembles the cyt mutants of highly oncogenic Adl2, isolated by Takemori et al. (Virology 36:575-586, 1968). By intertypic complementation analysis, we showed that the Adl2 cyt mutants indeed map within the 19-kd tumor antigen-coding region. The transforming potential of d1250 was assayed on an established rat embryo fibroblast cell line, CREF, and on primary rat embryo fibroblasts and baby rat kidney cells.
    [Show full text]
  • The Hepatitis C Virus P7 Protein Forms an Ion Channel That Is Inhibited by Long-Alkyl-Chain Iminosugar Derivatives
    The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives Davor Pavlovic´*, David C. A. Neville*, Olivier Argaud*, Baruch Blumberg†, Raymond A. Dwek*, Wolfgang B. Fischer*, and Nicole Zitzmann*‡ *Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom; and †Fox Chase Cancer Center, Philadelphia, PA 19111 Contributed by Baruch Blumberg, March 17, 2003 We show that hepatitis C virus (HCV) p7 protein forms ion channels data have been obtained by introducing mutations into an in black lipid membranes. HCV p7 ion channels are inhibited by infectious cDNA clone of BVDV. An in-frame deletion of the long-alkyl-chain iminosugar derivatives, which have antiviral ac- entire p7 does not affect RNA replication but leads to the tivity against the HCV surrogate bovine viral diarrhea virus. HCV p7 production of noninfectious virions. However, infective viral presents a potential target for antiviral therapy. particles can be generated by complementing p7 in trans (9), which suggests that the pestivirus p7 is essential for the epatitis C virus (HCV) is the major cause of chronic production of infective progeny virus. Interestingly, noninfec- Hhepatitis with a significant risk of end-stage liver cirrhosis tive BVDV particles also are created by treatment with and hepatocellular carcinoma (1). HCV belongs to the family long-alkyl-chain iminosugar derivatives (3). Flaviviridae, which consists of three genera: flaviviruses, pesti- Recently, HCV p7 has been shown to be a polytopic mem- viruses, and hepaciviruses. In the absence of both a suitable small brane protein that crosses the membrane twice and has its N and animal model and a reliable in vitro infectivity assay for HCV, C termini oriented toward the extracellular environment (10).
    [Show full text]
  • A Comparative Look at High-Throughput Cloning Methods
    Downloaded from genome.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press Review Many Paths to Many Clones: A Comparative Look at High-Throughput Cloning Methods Gerald Marsischky1 and Joshua LaBaer Institute of Proteomics, Harvard Medical School, Department of Biological Chemistry and Molecular Pharmacology, Boston, Massachusetts 02115, USA The creation of genome-scale clone resources is a difficult and costly process, making it essential to maximize the efficiency of each step of clone creation. In this review, we compare the available commercial and open-source recombinational cloning methods with regard to their use in creating comprehensive open reading frame (ORF) clone collections with an emphasis on the properties requisite to use in a high-throughput setting. The most efficient strategy to the creation of ORF clone resources is to build a master clone collection that serves as a quality validated source for producing collections of expression clones. We examine the methods for recombinational cloning available for both the creation of master clones and their conversion into expression clones. Alternative approaches to creating clones involving mixing of cloning methods, including gap-repair cloning, are also explored. Functional genomics and proteomics offer the promise of exam- sequence validated. Most importantly, once constructed, the ining the roles of all genes and proteins in an organism in a clones are effectively locked into the configuration of the original controlled format. These studies depend on the availability of vector. Moving the ORFs to a different vector would require start- cloned copies of the genes in a format conducive to protein ex- ing again at the PCR step, with its inherent incorporation errors pression.
    [Show full text]
  • Random-Sequence Genetic Oligomer Pools Display an Innate Potential For
    RESEARCH ARTICLE Random-sequence genetic oligomer pools display an innate potential for ligation and recombination Hannes Mutschler1†‡*, Alexander I Taylor1†, Benjamin T Porebski1, Alice Lightowlers1§, Gillian Houlihan1, Mikhail Abramov2, Piet Herdewijn2, Philipp Holliger1* 1MRC Laboratory of Molecular Biology, Cambridge, United Kingdom; 2REGA Institute, Katholieke Universiteit Leuven, Leuven, Belgium Abstract Recombination, the exchange of information between different genetic polymer strands, is of fundamental importance in biology for genome maintenance and genetic diversification and is mediated by dedicated recombinase enzymes. Here, we describe an innate capacity for non-enzymatic recombination (and ligation) in random-sequence genetic oligomer pools. Specifically, we examine random and semi-random eicosamer (N20) pools of RNA, DNA and *For correspondence: the unnatural genetic polymers ANA (arabino-), HNA (hexitol-) and AtNA (altritol-nucleic acids). Correspondence to: ph1@mrc- While DNA, ANA and HNA pools proved inert, RNA (and to a lesser extent AtNA) pools displayed lmb.cam.ac.uk; [email protected] diverse modes of spontaneous intermolecular recombination, connecting recombination mechanistically to the vicinal ring cis-diol configuration shared by RNA and AtNA. Thus, the † These authors contributed chemical constitution that renders both susceptible to hydrolysis emerges as the fundamental equally to this work determinant of an innate capacity for recombination, which is shown to promote a concomitant Present address:
    [Show full text]