Discovery and Characterization of Novel Polyomaviruses in Humans Erica Anne Siebrasse Washington University in St

Total Page:16

File Type:pdf, Size:1020Kb

Discovery and Characterization of Novel Polyomaviruses in Humans Erica Anne Siebrasse Washington University in St Washington University in St. Louis Washington University Open Scholarship All Theses and Dissertations (ETDs) Summer 9-1-2014 Discovery and Characterization of Novel Polyomaviruses in Humans Erica Anne Siebrasse Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/etd Recommended Citation Siebrasse, Erica Anne, "Discovery and Characterization of Novel Polyomaviruses in Humans" (2014). All Theses and Dissertations (ETDs). 1347. https://openscholarship.wustl.edu/etd/1347 This Dissertation is brought to you for free and open access by Washington University Open Scholarship. It has been accepted for inclusion in All Theses and Dissertations (ETDs) by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Division of Biology and Biological Sciences Molecular Microbiology and Microbial Pathogenesis Dissertation Examination Committee: David Wang, Chair Jacco Boon Erika Crouch Michael S. Diamond Henry V. Huang Thaddeus S. Stappenbeck Discovery and Characterization of Novel Polyomaviruses in Humans by Erica Anne Siebrasse A dissertation presented to the Graduate School of Arts and Sciences of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy August 2014 Saint Louis, Missouri TABLE OF CONTENTS LIST OF FIGURES AND TABLES ............................................................................... iii LIST OF ABBREVIATIONS ......................................................................................... v ACKNOWLEDGEMENTS ........................................................................................... vii ABSTRACT ....................................................................................................................... x CHAPTER 1. Introduction .............................................................................................. 1 OVERVIEW .................................................................................................................................... 2 SECTION I: DISCOVERY OF NOVEL VIRUSES ................................................................................ 2 SECTION II: THE POLYOMAVIRIDAE ............................................................................................ 10 CONCLUSIONS............................................................................................................................. 16 REFERENCES ............................................................................................................................... 18 CHAPTER 2. Identification of MW polyomavirus, a novel polyomavirus in human stool 22 ABSTRACT .................................................................................................................................. 23 INTRODUCTION ........................................................................................................................... 24 MATERIALS AND METHODS ....................................................................................................... 26 RESULTS ..................................................................................................................................... 30 DISCUSSION ................................................................................................................................ 37 ACKNOWLEDGEMENTS ............................................................................................................... 39 REFERENCES ............................................................................................................................... 40 CHAPTER 3. Determination of the cell and tissue tropism of WU and KI polyomaviruses44 ABSTRACT .................................................................................................................................. 45 INTRODUCTION ........................................................................................................................... 46 MATERIALS AND METHODS ....................................................................................................... 47 RESULTS ..................................................................................................................................... 51 DISCUSSION ................................................................................................................................ 74 ACKNOWLEDGEMENTS ............................................................................................................... 80 REFERENCES ............................................................................................................................... 81 CHAPTER 4. Progress toward a cell culture system for WU polyomavirus ............ 84 ABSTRACT .................................................................................................................................. 85 INTRODUCTION ........................................................................................................................... 86 MATERIALS AND METHODS ....................................................................................................... 89 RESULTS ..................................................................................................................................... 93 DISCUSSION .............................................................................................................................. 106 ACKNOWLEDGEMENTS ............................................................................................................. 108 REFERENCES ............................................................................................................................. 108 CHAPTER 5. Discussion ............................................................................................. 111 OVERVIEW ................................................................................................................................ 112 SECTION I: ARE THERE ADDITIONAL, NOVEL POLYOMAVIRUSES INFECTING HUMANS? ......... 112 SECTION II: DO THESE POLYOMAVIRUSES CAUSE DISEASE IN THEIR HUMAN HOSTS? ............ 119 FINAL CONCLUSIONS ................................................................................................................ 123 REFERENCES ............................................................................................................................. 124 APPENDIX I ................................................................................................................. 126 CURRICULUM VITAE ............................................................................................... 135 ii LIST OF FIGURES AND TABLES CHAPTER 1. Introduction Figure 1.1 Virus detection and discovery pipeline 7 Figure 1.2 Schematic representation of the WUPyV genome 11 Figure 1.3 The polyomavirus life cycle 12 Figure 1.4 Timeline of human polyomavirus discoveries 14 Table 1.1 Top 10 causes of death worldwide in 2011 3 Table 1.2 The human cancer viruses 5 CHAPTER 2. Identification of MW polyomavirus, a novel polyomavirus in human stool Figure 2.1 Genome organization of MWPyV 31 Figure 2.2 Phylogenetic analysis of MWPyV 35 Table 2.1 Putative proteins encoded by MWPyV (strain MA095) 32 Table 2.2 Specimens and patients testing positive for MWPyV 36 CHAPTER 3. Determination of the cell and tissue tropism of WU and KI polyomaviruses Figure 3.1 Validation of anti-KI-VP1 monoclonal antibody specificity 53 Figure 3.2 Validation of anti-WU-VP1 monoclonal antibody specificity 54 Figure 3.3 Electron micrographs of polyomavirus-like particles in a human lung 57 Figure 3.4 Nucleotide sequence of the 3’ end of the large T antigen gene from WUPyV strain Rochester-7029 58 Figure 3.5 Detection of WU-VP1 in the human respiratory tract 59 Figure 3.6 Detection of WU-VP1 in human alveolar macrophages 60 Figure 3.7 Detection of WU-VP1 in close proximity to MUC5AC-positive cells in the trachea 61 Figure 3.8 WU-VP1 detected in a BAL from a lung transplant recipient with Job’s syndrome 63 Figure 3.9 WU-VP1 detected within respiratory epithelial cells 64 Figure 3.10 WU-VP1 detected in a lung from an HIV-positive patient 66 Figure 3.11 IHC of human lung with a KI-VP1 monoclonal antibody 67 Figure 3.12 Detection of KI-VP1 in cells of hematopoietic origin in the lung 69 Figure 3.13 Detection of KI-VP1 in alveolar macrophages 70 Figure 3.14 Double IHC of the lung with monoclonal antibodies against KI-VP1 and cytokeratin at 600x 70 Figure 3.15 IHC of human spleen and lung with a KI-VP1 monoclonal antibody 73 Figure 3.16 Additional dIHC stains of human spleen 74 Table 3.1 Summary of findings 58 Table 3.2 Summary of results 75 CHAPTER 4. Progress toward a cell culture system for WU polyomavirus Figure 4.1 Establishment of a WUPyV replication system 88 Figure 4.2 Determination of the density of rWUPyV viral particles 95 Figure 4.3 Viral DNA detected in supernatants from hNECs infected with rWUPyV 97 Figure 4.4 Viral DNA detected in supernatants from hTECs infected with rWUPyV 98 iii Figure 4.5 Viral DNA detected in supernatants from hNECs infected with rWUPyV 99 Figure 4.6 Viral DNA detected in supernatants from hNECs infected with lysate passaged from a rWUPyV hNEC infection 99 Figure 4.7 Viral DNA detected in supernatants from immortal cell lines infected with rWUPyV at a MOI of ~100 101 Figure 4.8 Passage of rWUPyV-infected THP-1 cell lysates into fresh THP-1 cells
Recommended publications
  • Enhanced Expression of Vascular Endothelial Growth Factor (VEGF) Plays a Critical Role in the Tumor Progression Potential Induced by Simian Virus 40 Large T Antigen
    Oncogene (2002) 21, 2896 ± 2900 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc Enhanced expression of vascular endothelial growth factor (VEGF) plays a critical role in the tumor progression potential induced by simian virus 40 large T antigen Alfonso Catalano1, Mario Romano*,2, Stefano Martinotti3 and Antonio Procopio*,1 1Institute of Experimental Pathology, University of Ancona, Faculty of Medicine, Via Ranieri, 60131 Ancona, Italy; 2Department of Human Pathology, University of Messina, 98125 Messina, Italy; 3Department of Oncology and Neuroscience, `G. D'Annunzio' University, Chieti, Italy Vascular endothelial growth factor (VEGF), an impor- disorders, including mesotheliomas (Kumar-Singh et tant angiogenic factor, regulates cell proliferation, al., 1999). Among these, VEGF, whose crucial role in dierentiation, and apoptosis through activation of its tumor angiogenesis is well established (Hanahan and tyrosine-kinase receptors, such as Flt-1 and Flk-1/Kdr. Folkman., 1996), acts also as a potent mitogen and Human malignant mesothelioma cells (HMC), which survival factor for human malignant mesothelioma have wild-type p53, express VEGF and exhibit cell cells (HMC). In fact, VEGF regulates HMC prolifera- growth increased by VEGF. Here, we demonstrate that tion through its tyrosine-kinase receptors activation early transforming proteins of simian virus (SV) 40, large (i.e. Flt-1 and KDR/¯k-1) (Strizzi et al., 2001). In tumor antigen (Tag) and small tumor antigen (tag), addition, VEGF is the main eector of 5-lipoxygenase which have been associated with mesotheliomas, en- action on HMC survival (Romano et al., 2001). Thus, hanced HMC proliferation by inducing VEGF expres- VEGF plays a key role by regulating multiple sion.
    [Show full text]
  • A Polyoma Mutant That Encodessmall T Antigen but Not Middle T Antigen
    MOLECULAR AND CELLULAR BIOLOGY, Dec. 1984, p. 2774-2783 Vol. 4, No. 12 0270-7306/84/122774-10$02.00/0 Copyright © 1984, American Society for Microbiology A Polyoma Mutant That Encodes Small T Antigen But Not Middle T Antigen Demonstrates Uncoupling of Cell Surface and Cytoskeletal Changes Associated with Cell Transformation T. JAKE LIANG, GORDON G. CARMICHAEL,t AND THOMAS L. BENJAMIN* Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115 Received 2 July 1984/Accepted 31 August 1984 The hr-t gene of polyoma virus encodes both the small and middle T (tumor) antigens and exerts pleiotropic effects on cells. By mutating the 3' splice site for middle T mRNA, we have constructed a virus mutant, Py8O8A, which fails to express middle T but encodes normal small and large T proteins. The mutant failed to induce morphological transformation or growth in soft agar, but did stimulate postconfluent growth of normal cells. Cells infected by Py8O8A became fully agglutinable by lectins while retaining normal actin cable architecture and normal levels of extracellular fibronectin. These properties of Py8O8A demonstrated the separability of structural changes at the cell surface from those in the cytoskeleton and extracellular matrix, parameters which have heretofore been linked in the action of the hr-t and other viral oncogenes. The early region of polyoma encodes three proteins de- substitution of phenylalanine for tyrosine at position 315 in tected as tumor (T) antigens. These polypeptides of 100,000 middle T has been made and studied in two laboratories, daltons (100K; large T), 56K (middle T), and 22K (small T) with different results.
    [Show full text]
  • Review Article Human Polyomaviruses in Skin Diseases
    SAGE-Hindawi Access to Research Pathology Research International Volume 2011, Article ID 123491, 12 pages doi:10.4061/2011/123491 Review Article Human Polyomaviruses in Skin Diseases Ugo Moens,1 Maria Ludvigsen,1 and Marijke Van Ghelue2 1 Institute of Medical Biology, Faculty of Health Sciences, University of Tromsø, 9037 Tromsø, Norway 2 Department of Medical Genetics, University Hospital of Northern-Norway, 9038 Tromsø, Norway Correspondence should be addressed to Ugo Moens, [email protected] Received 28 February 2011; Accepted 29 June 2011 Academic Editor: Gerardo Ferrara Copyright © 2011 Ugo Moens et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Polyomaviruses are a family of small, nonenveloped viruses with a circular double-stranded DNA genome of ∼5,000 base pairs protected by an icosahedral protein structure. So far, members of this family have been identified in birds and mammals. Until 2006, BK virus (BKV), JC virus (JCV), and simian virus 40 (SV40) were the only polyomaviruses known to circulate in the human population. Their occurrence in individuals was mainly confirmed by PCR and the presence of virus-specific antibodies. Using the same methods, lymphotropic polyomavirus, originally isolated in monkeys, was recently shown to be present in healthy individuals although with much lower incidence than BKV, JCV, and SV40. The use of advanced high-throughput sequencing and improved rolling circle amplification techniques have identified the novel human polyomaviruses KI, WU, Merkel cell polyomavirus, HPyV6, HPyV7, trichodysplasia spinulosa-associated polyomavirus, and HPyV9.
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2004/0023207 A1 Polansky (43) Pub
    US 20040023207A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0023207 A1 Polansky (43) Pub. Date: Feb. 5, 2004 (54) ASSAYS FOR DRUG DISCOVERY BASED ON Publication Classification MICROCOMPETITION WITH A FOREIGN POLYNUCLEOTIDE (51) Int. Cl." .............................. C12O 1/70; C12O 1/68; C12N 15/85; C12N 15/86 (52) U.S. Cl. .................. 435/5; 435/6; 435/455; 435/456 (76) Inventor: Hanan Polansky, Rochester, NY (US) (57) ABSTRACT Correspondence Address: A recent discovery showed that microcompetition between a Hanan Polansky foreign polynucleotide and a cellular polynucleotide is a risk 3159 S. Winton Rd. factor for Some of the major chronic diseases. The invention Rochester, NY 14623 (US) uses this novel discovery to present assays for Screening compounds based on their effectiveness in modulating Such microcompetition. The effective compounds can be used in (21) Appl. No.: 10/211,295 treatment of these chronic diseases. The invention also presents assays for Screening compounds that can be used in treatment of chronic diseaseS resulting from other foreign (22) Filed: Aug. 1, 2002 polynucleotide-type disruptions. Patent Application Publication Feb. 5, 2004 Sheet 1 of 6 US 2004/0023207 A1 s e 2 9 s H c 2 4 competitor plasmid/test plasmid Figure 1 1 O 09% 1 2 3 4 competitor plasmid/test plasmid -H Ltk- (pSV2Neo) -A-ML (pSV2neo) . A- - - Ltk- (pA1 Oneo) - - - - - - Mill (pA1 Oneo) Figure 2 Patent Application Publication Feb. 5, 2004 Sheet 2 of 6 US 2004/0023207 A1 e 2 r U H c O s S 2 3. Molar ratio (pSV2NeolhMT-ilA-CAT) Figure 3 Patent Application Publication Feb.
    [Show full text]
  • Tumor Antigen (Hybrid Trp-Lac Promoter/Actin Cable Effect) ILAN BIKEL*, THOMAS M
    Proc. NatL Acad. Sci. USA Vol. 80, pp. 906-910, February 1983 Biochemistry Purification of biologically active simian virus 40 small tumor antigen (hybrid trp-lac promoter/actin cable effect) ILAN BIKEL*, THOMAS M. ROBERTS*, MARILUCI T. BLADON*, ROBERT GREEN*, EGON AMANNt, AND DAVID M. LIVINGSTON* *The Sidney Farber Cancer Institute and The Departments of Medicine and Pathology, Harvard Medical School, Boston, Massachusetts 02115; and tDepartment of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138 Communicated by Morris Friedkin, October 29, 1982 The simian virus 40 small tumor antigen (t antigen) gene has been were grown in L broth containing ampicillin (40 Ag/ml) to an cloned downstream from a hybrid Escherichia coli trp-lac pro- ODW of0.7 and then incubated for 2 hr with 5 mM isopropyl moter anda suitable ribosome binding site. Abacterial clone (865i) f3-D-thiogalactopyranoside. Radiolabeling of proteins with transformed by such a plasmid (pTR865) expresses this gene and, [3S]methionine was performed as described (31). under optimal conditions, can produce .5% ofits total protein as t Antigen and Protein Assays. t antigen was detected by Na- t antigen. Soluble extracts ofsuch a clone were relatively depleted DodSO4/polyacrylamide slab gel electrophoresis (32) using 4% in t antigen, which was found in the initial pellet fraction. The stacking and 15% separating gels. Marker proteins were bovine protein was recovered from this fraction in a significantly purified serum albumin (Mr 68,000), carbonic anhydrase (29,000), form byextraction with urea-containing buffer. After gel filtration concentrations were deter- of such t antigen-enriched solutions, highly purified protein was and myoglobin (17,000).
    [Show full text]
  • PP2A-Dependent Disruption of Centrosome Replication and Cytoskeleton Organization in Drosophila by SV40 Small Tumor Antigen
    Oncogene (2008) 27, 6334–6346 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc ORIGINAL ARTICLE PP2A-dependent disruption of centrosome replication and cytoskeleton organization in Drosophila by SV40 small tumor antigen S Kotadia1,LRKao1, SA Comerford2, RT Jones1, RE Hammer3 and TL Megraw1 1Department of Pharmacology, The Cecil and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA; 2Department of Molecular Genetics, The Cecil and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA and 3Department of Biochemistry, The Cecil and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA Viruses of the DNA tumor virus family share the ability to cell-cycle and apoptosis regulation, viral oncoproteins transform vertebrate cells through the action of virus- transform cells. Investigation of DNA tumor virus encoded tumor antigens that interfere with normal cell oncoproteins has led to the identification of many physiology. They accomplish this very efficiently by fundamental mechanisms of tumor suppression (Lavia inhibiting endogenous tumor suppressor proteins that et al., 2003; Ahuja et al., 2005). By altering the activity control cell proliferation and apoptosis. Simian virus 40 of p53, retinoblastoma protein and serine/threonine (SV40) encodes two oncoproteins, large tumor antigen, protein phosphatase 2A (PP2A), three key tumor which directly inhibits the tumor suppressors p53 and Rb, suppressors, SV40 can cause tumor formation in and small tumor antigen (ST), which interferes with transgenic mouse models (Ahuja et al., 2005; Arroyo serine/threonine protein phosphatase 2A (PP2A).
    [Show full text]
  • Antibodies Response to Polyomaviruses Primary Infection: High Seroprevalence of Merkel Cell
    Antibodies response to Polyomaviruses primary infection: high seroprevalence of Merkel Cell Polyomavirus and lymphoid tissues involvement. Carolina Cason1, Lorenzo Monasta2, Nunzia Zanotta2, Giuseppina Campisciano2, Iva Maestri3, Massimo Tommasino4, Michael Pawlita5, Sonia Villani6, Manola Comar1,2, Serena Delbue6,*. Authors affiliations: 1 Department of Medical Sciences, University of Trieste, Piazzale Europa 1, 34127 Trieste, Italy. 2Institute for Maternal and Child Health - IRCCS "Burlo Garofolo", Via dell' Istria 65/1, 34137 Trieste, Italy. 3Department of Experimental and Diagnostic Medicine, Pathology Unit of Pathologic Anatomy, Histology and Cytology University of Ferrara, Via Luigi Borsari 46, 44121 Ferrara, Italy. 4Infections and Cancer Biology Group, International Agency for Research on Cancer, Cours Albert Thomas 150, 69372 Lyon, France. 5German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. 6Department of Biomedical, Surgical & Dental Sciences, University of Milano, Via Pascal 36, 20100 Milano, Italy. * Corresponding author: [email protected], +390250315070 1 ABSTRACT Human polyomaviruses (HPyVs) asymptomatically infect the human population establishing latency in the host and their seroprevalence can reach 90% in healthy adults. Few studies have focused on the pediatric population and there are no reports regarding the seroprevalence of all the newly isolated HPyVs among Italian children. Therefore, we investigated the frequency of serum antibodies against 12 PyVs in 182 immunocompetent children from Northeast Italy, by means of a multiplex antibody detection system. Additionally, secondary lymphoid tissues were collected to analyze the presence of HPyVs DNA sequences using a specific Real Time PCRs or PCRs. Almost 100% of subjects were seropositive for at least one PyV. Seropositivity ranged from 3% for antibodies against Simian virus 40 (SV40) in children from 0 to 3 years, to 91% for antibodies against WU polyomavirus (WUPyV) and HPyV10 in children from 8 to 17 years.
    [Show full text]
  • Interactions Between Polyomavirus Large T Antigen and the Viral Replication Origin Dna: How and Why
    INTERACTIONS BETWEEN POLYOMAVIRUS LARGE T ANTIGEN AND THE VIRAL REPLICATION ORIGIN DNA: HOW AND WHY by Yu-Cai Peng Department of Microbiology and Immunology McGill University, Montreal April, 1999 A thesis submitted to the Faculty of Craduate Studies and Re3earch in partial fulfullmeat of the requirements of the degree of doctor of philosophy @Yu-Cai Peng, 1999 National Library Biblioth ue nationale 1+1 of Canada du Cana7 a Acquisitions and Acquisitions et Bibliographie Srvices services bibliographiques 395 Wellmgton Street 395, nie Weliingîm Ottawa ON KIA ON4 OrtawaON K1AON4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive Licence ailowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfichelfilm, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thèse ni des extraits substantiels may be printed or othenvise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. TABLE OF CONTENTS Page Tableofcontents................................................... I Abstract .......................................................... VI Resumé........................................................... VI11 Acknowledgements ................................................. X Claim of contribution to kaowledge ................................... XI Listoffigures ...................................................... XllI Guidelines regarding doctoral thesis ................................... XV CHAPTER 1. INTRODUCTION .................................... 1 1. Overview: Life cycle of polyomavirus and simian virus 40 ..........
    [Show full text]
  • Advances in Human Polyomaviruses Field
    rren : Cu t R y es g e lo a o r r c i h V Virology: Current Research Ciotti, Virol Curr Res 2017, 1:1 Editorial Open Access Advances in Human Polyomaviruses Field Marco Ciotti* Laboratory of Molecular Virology, Polyclinic Tor Vergata Foundation, Viale Oxford 81, 00133 Rome, Italy *Corresponding author: Ciotti M, Laboratory of Molecular Virology, Polyclinic Tor Vergata Foundation, Viale Oxford 81, 00133 Rome, Italy, Tel: +390620902087; E-mail: [email protected] Received date: February 27, 2017; Accepted date: March 02, 2017; Published date: March 02, 2017 Copyright: © 2017 Ciotti M. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editorial References Polyomaviruses are small non-enveloped DNA viruses with a 1. Gardner SD, Field AM, Coleman DV, Hulme B (1971) New human circular double stranded genome of about 5 Kb in length. The genome papovavirus (B.K.) isolated from urine after renal transplantation. Lancet is contained in a capsid with icosahedral structure of about 45 nm in 1: 1253-1257. diameter. 2. Padgett BL, Walker DL, ZuRhein GM, Eckroade RJ, Dessel BH (1971) Cultivation of papova-like virus from human brain with progressive Up to 2007, two human polyomaviruses BK (BKPyV) and JC multifocal leucoencephalopathy. Lancet 1: 1257-1260. (JCPyV) were known and named after the initials of the patients where 3. Allander T, Andreasson K, Gupta S, Bjerkner A, Bogdanovic G, et al. they were first isolated. BKV was isolated from the urine of a kidney (2007) Identification of a Third Human Polyomavirus.
    [Show full text]
  • Polyoma Virus Small Tumor Antigen Pre-Mrna Splicing Requires Cooperation Between Two 3' Splice Sites Hui GE, JONATHAN NOBLE, JOHN COLGAN, and JAMES L
    Proc. Nati. Acad. Sci. USA Vol. 87, pp. 3338-3342, May 1990 Biochemistry Polyoma virus small tumor antigen pre-mRNA splicing requires cooperation between two 3' splice sites Hui GE, JONATHAN NOBLE, JOHN COLGAN, AND JAMES L. MANLEY Department of Biological Sciences, Columbia University, New York, NY 10027 Communicated by Joan A. Steitz, January 25, 1990 (receivedfor review December 13, 1989) ABSTRACT We have studied splicing ofthe polyoma virus proteins also likely play a role in the splicing reaction (e.g., early region pre-mRNA in vitro. This RNA is alternatively refs. 19-22). spliced in vivo to produce mRNA encoding the large, middle- The complexity and size of the splicing apparatus suggests sized (MTAg), and small (StAg) tumor antigens. Our primary that steric constraints may play a role in splicing of some interest was to learn how the 48-nucleotide StAg intron is pre-mRNAs. Consistent with this proposal, several studies excised, because the length of this intron is significantly less have shown that introns in higher eukaryotes have a mini- than the apparent minimum established for mammalian in- mum size requirement. Roughly 45 nucleotides (nt) must trons. Although the products ofall three splices are detected in separate the 5' splice site and branch point (23-27), and the vitro, characterization of the pathway and sequence require- minimum distance between the branch point and 3' splice site ments of StAg splicing suggests that splicing factors interact appears to be approximately 18 nt (26, 28). The simian virus with the precursor RNA in an unexpected way to catalyze 40 small tumor antigen pre-mRNA contains an intron of66 nt removal ofthis intron.
    [Show full text]
  • Drosophila As a Model for Infectious Diseases
    International Journal of Molecular Sciences Review Drosophila as a Model for Infectious Diseases J. Michael Harnish 1,2 , Nichole Link 1,2,3,† and Shinya Yamamoto 1,2,4,5,* 1 Department of Molecular and Human Genetics, Baylor College of Medicine (BCM), Houston, TX 77030, USA; [email protected] (J.M.H.); [email protected] (N.L.) 2 Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, TX 77030, USA 3 Howard Hughes Medical Institute, Houston, TX 77030, USA 4 Department of Neuroscience, BCM, Houston, TX 77030, USA 5 Development, Disease Models and Therapeutics Graduate Program, BCM, Houston, TX 77030, USA * Correspondence: [email protected]; Tel.: +1-832-824-8119 † Current Affiliation: Department of Neurobiology, University of Utah, Salt Lake City, UT 84112, USA. Abstract: The fruit fly, Drosophila melanogaster, has been used to understand fundamental principles of genetics and biology for over a century. Drosophila is now also considered an essential tool to study mechanisms underlying numerous human genetic diseases. In this review, we will discuss how flies can be used to deepen our knowledge of infectious disease mechanisms in vivo. Flies make effective and applicable models for studying host-pathogen interactions thanks to their highly con- served innate immune systems and cellular processes commonly hijacked by pathogens. Drosophila researchers also possess the most powerful, rapid, and versatile tools for genetic manipulation in multicellular organisms. This allows for robust experiments in which specific pathogenic proteins can be expressed either one at a time or in conjunction with each other to dissect the molecular functions of each virulent factor in a cell-type-specific manner.
    [Show full text]
  • Expression of the Hepatitis B Virus X Gene in Mammalian Cells
    Proc. Nati. Acad. Sci. USA Vol. 84, pp. 2513-2517, April 1987 Microbiology Expression of the hepatitis B virus X gene in mammalian cells (in vivo transcription/chloramphenicol acetyltransferase assay/protein analysis/cell fractionation/imnmunofluorescence) ALEEM SIDDIQUI, SHAHID JAMEEL, AND JOHN MAPOLES Department of Microbiology and Immunology, University of Colorado School of Medicine, Denver, CO 80262 Communicated by Theodore T. Puck, December 23, 1986 ABSTRACT The hepatitis B virus (HBV) DNA contains an (National Institutes of Health). Two types of recombinant open reading frame (ORF) designated X, which has the vectors were constructed for X gene expression (Fig. 1B). capacity to encode a protein of 16,560 Da (subtype adw). Such Plasmid pMNX contains an Acc I/Taq I [1076-2020 nucle- a protein has not been identified in either HBV particles or otides (nt)] fragment, which has -300 base pairs (bp) of 5' infected human livers, and therefore its role in the viral life flanking sequences, including the enhancer element (12, 13), cycle remains unknown. We report here the expression of the the X ORF (1380-1842 nt), followed by a polyadenylylation HBV X ORF in cultured cells using recombinant vectors. A signal sequence (1920 nt). This plasmid was designed to protein of 16 kDa was identified by means of an antiserum investigate the possible existence of a promoter upstream of prepared against a synthetic peptide and with human antisera the X region. In plasmid pAdX, the X ORF was placed under from hepatitis B patients as well as those with hepatocellular the control of the adenovirus major late promoter, simian carcinoma.
    [Show full text]