DOCSLIB.ORG

Katedra Genetiky a Mikrobiologie

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Katedra Genetiky a Mikrobiologie Svoluji k zapůjčení své diplomové práce ke studijním účelům a prosím, aby byla vedena přesná evidence vypůjčovatelů. Převzaté údaje je vypůjčovatel povinen řádně ocitovat. 1 UNIVERZITA KARLOVA V PRAZE Přírodovědecká fakulta Katedra genetiky a mikrobiologie Studium minoritních kapsidových proteinů myšího polyomaviru Ondřej Vít Praha 2010 2 Vedoucí diplomové práce: Doc. RNDr. Jitka Forstová, Csc. Prohlašuji, že jsem tuto práci vypracoval samostatně, jen s použitím citované literatury a pod vedením vedoucí diplomové práce. Ondřej Vít 3 Na tomto místě bych chtěl poděkovat doc. RNDr. Jitce Forstové, CSc. neocenitelnou pomoc, rady a připomínky během vypracovávání této diplomové práce. Dále děkuji všem členům naší laboratoře za zaučení do metod a vytvoření příjemného pracovního prostředí. Děkuji Ondřeji Šebestovi za pomoc s konfokálním mikroskopem, Vojtěchu Žílovi za zhotovení elektronmikroskopických snímků a Ludmile Vítámvásové za pomoc s 2D elektroforézami. Děkuji svým rodičům za podporu, bez které bych nemohl tuto práci dokončit. Děkuji Evičce Vopálkové za toleranci, lásku a duševní podporu, bez které bych to nezvládl. 4 Předkládaná diplomová práce vznikla v letech 2007 – 2010 za podpory grantů 1M0508, MSM0021620858 a LC545 Ministerstva školství, mládeže a tělovýchovy České republiky. 5 Studium minoritních kapsidových proteinů myšího polyomaviru Abstrakt. Myší polyomavirus (MPyV) je malý neobalený virus. Jeho kapsida se skládá ze 72 pentamerů hlavního kapsidového proteinu VP1. Vnitřní prohlubeň každého pentameru VP1 obsahuje jeden minoritní kapsidový protein, buď VP2 nebo VP3. Minoritní proteiny nejsou potřeba ke vzniku kapsidy, avšak pro infekci hostitelské buňky jsou nezbytné, pravděpodobně kvůli jejich předpokládaným funkcím během vstupu viru do buňky. Po vstupu do buňky jsou viriony MPyV dopraveny do endoplazmatického retikula (ER). Předpokládá se, že VP2 a VP3 jsou zodpovědné za narušení membrány ER, které je potřeba pro následné doručení virové DNA do jádra. V sekvenci VP2 a VP3 byly předpovězeny tři hydrofobní domény. První, v unikátní N-koncové oblasti VP2, druhá a třetí ve společné části sekvence VP2 a VP3. Třetí doména je zároveň C-koncovým α-helixem, zodpovědným za vazbu VP1. V naší laboratoři bylo již dříve zjištěno, že VP2 a VP3 fúzované k N-konci EGFP mají při produkci v savčích buňkách obdobné VP2 a VP3 divokého typu, konkrétně afinitu k vnitrobuněčným membránám a vysokou cytotoxicitu. Aby bylo zjištěno, která z předpokládaných hydrofobních domén VP2 a VP3 je zodpovědná za afinitu k membránám a cytotoxicitu, byly připraveny expresní plazmidy, nesoucí mutované VP2 a VP3 fúzované k N-konci EGFP. Výsledky ukazují, že delece druhé hydrofobní domény vede ke ztrátě VP2 a VP3 interagovat s vnitrobuněčnými membránami a samotná unikátní N-koncová část VP2 (která obsahuje první hydrofobní doménu) nepostačuje k interakcím s vnitrobuněčnými membránami. Tyto výsledky naznačují, že druhá hydrofobní doména VP2 a VP3 je důležitá pro afinitu VP2 a VP3 k membránám. Dále byly pomocí SDS-PAGE pro analýzu hmotnostní spektrometrií (MS) separovány dvě izoformy VP2 a VP3, lišící se elektroforetickou mobilitou. MS analýzou byla nalezena acetylace N-koncového alaninu obou forem VP3. 2D elektroforézou byly detekovány nejméně čtyři izoformy VP2 a VP3, což je, společně s předběžnými výsledky MS analýz, náznakem existence několika fosforylací obou minoritních kapsidových proteinů. Klíčová slova: myší polyomavirus, VP2, VP3, kapsidové proteiny, posttranslační modifikace, vstup viru do buňky. 6 Studies of minor capsid proteins of the mouse polyomavirus Abstract. Mouse polyomavirus (MPyV) is a small non-enveloped virus. Its capsid consists of 72 pentamers of the major capsid protein VP1. The central cavity of each VP1 pentamer contains one minor capsid protein, either VP2, or VP3. The minor capsid proteins are dispensable for capsid formation, but their presence is required for infection of the host cell, presumably because of their anticipated functions during virus entry. After internalization, MPyV virions traffic to endoplasmic reticulum (ER). VP2 and VP3 have been proposed to function as factors responsible for penetration of ER membranes, which is required for subsequent delivery of the viral DNA into the nucleus, a key step of the early phase of MPyV infection. Three hydrophobic domains were predicted in the sequence of VP2 and VP3. First in the unique N-terminal part of VP2, second and third in the common part of VP2 and VP3. The third domain corresponds to C-terminal VP1-binding α-helix. It has been previously found in our laboratory, that VP2 and VP3 fused to N-terminus of EGFP, when expressed in mammalian cells, display properties similiar to the wild-type VP2 and VP3, namely affinity to intracellular membranes and high cytotoxicity. Expression plasmids carrying mutated VP2 and VP3 fused to N-terminus of EGFP were prepared to determine the hydrophobic domain, responsible for previously observed membrane affinity and cytotoxicity. The results show, that deletion of the second hydrophobic domain results in loss of ability of VP2 and VP3 to interact with intracellular membranes, and that the unique part of VP2 (which contains the first hydrophobic domain) on its own is not sufficient for interaction with intracellular membranes. These results indicate that the second hydrophobic domain of is important for membrane affinity of VP2 and VP3. In addition, two isoforms of VP2 and VP3, differing in mobility on SDS-PAGE, were separated for mass spectrometry (MS) analysis and further characterized. The MS analysis showed acetylation of N-terminal alanine of both isoforms of VP3. At least four isoforms of VP2 and VP3 were identified by 2D electrophoresis, which, together with preliminary MS analysis results, indicate possible multiple phoshorylation sites on both minor capsid proteins. Key words: mouse polyomavirus, VP2, VP3, capsid proteins, posttranslational modifications, virus cell entry. 7 Obsah 1. ÚVOD .................................................................................................................................. 14 2. LITERÁRNÍ PŘEHLED ...................................................................................................... 16 2.1. Polyomaviry ................................................................................................................. 17 2.2. Myší polyomavirus ....................................................................................................... 18 2.2.1. Struktura kapsidy .................................................................................................. 18 2.2.1.1. Hlavní strukturní protein VP1 ....................................................................... 19 2.2.1.2. Minoritní strukturní proteiny VP2 a VP3 ...................................................... 20 2.2.2. Organizace genomu .............................................................................................. 24 2.2.3. Infekční cyklus MPyV .......................................................................................... 24 2.2.3.1. Vstup viru do buňky a transport do perinukleárního prostoru ....................... 25 2.2.3.2. Doručení virové DNA do buněčného jádra ................................................... 28 2.2.3.3. Časná transkripce .......................................................................................... 32 2.2.3.4. Časné proteiny .............................................................................................. 32 2.2.3.5. Replikace virové DNA ................................................................................. 33 2.2.3.6. Pozdní exprese .............................................................................................. 34 2.2.3.7. Morfogeneze virionů .................................................................................... 35 2.2.3.8. Smrt hostitelské buňky – role VP2 a VP3 ..................................................... 35 2.3. Virové kapsidové proteiny jiných virů podobné VP2 a VP3 polyomavirů ................... 38 2.3.1. VP4 poliovirů ....................................................................................................... 40 2.3.2. Protein γ nodavirů ................................................................................................. 42 2.3.3. Protein μ1 reovirů ................................................................................................. 42 2.3.4. VP5 rotavirů ......................................................................................................... 44 2.3.5. Kapsidové proteiny parvovirů .............................................................................. 45 2.3.6. L2 papillomavirů .................................................................................................. 46 2.3.7. Protein VI adenovirů ............................................................................................ 48 2.3.8. Viroporiny ............................................................................................................ 49 3. CÍLE PRÁCE ....................................................................................................................... 52 4. MATERIÁL A METODY .................................................................................................... 54 4.1. Materiál ........................................................................................................................ 55 4.1.1. Přístroje ...............................................................................................................
Load more

Recommended publications
  • Inhibition of Polyomavirus DNA Replication by Nucleotide Analogs
    Provided by the author(s) and NUI Galway in accordance with publisher policies. Please cite the published version when available. Title Inhibition of polyomavirus DNA replication by nucleotide analogs Author(s) Onwubiko, Nichodemus Okechukwu Publication Date 2017-01-31 Item record http://hdl.handle.net/10379/6277 Downloaded 2021-09-27T20:27:15Z Some rights reserved. For more information, please see the item record link above. Inhibition of Polyomavirus DNA Replication by Nucleotide Analogs Nichodemus Okechukwu Onwubiko Supervised by Professor Heinz-Peter Nasheuer School of Natural Sciences, Biochemistry National University of Ireland, Galway A thesis submitted to the National University of Ireland, Galway for a degree of Doctor of Philosophy January, 2017 Table of Contents Table of contents List of Abbreviation…………………………………………………………….…….…....iv Acknowledgment…………………………………………………………………………..vii Abstract...….………………………………………………………………………………viii 1.0 Introduction.........……………………………………………………………………….1 1.1 Polyomaviruses.............…………………………………………………….………....2 1.1.1 Discovery, Classification and Disease Association of Human Polyomaviruses....2 1.1.1.1 JC Virus..….............……………………………………………………………3 1.1.1.1.1 Reactivation of JCV in Humans.......………..........…….....……………...4 1.1.1.2 BK Virus...……….............…………………………………...……………..….7 1.1.1.2.1 Disease Association of BKV...…………....……............………………...8 1.1.1.2.1.1 Polyomavirus-Associated Nephropathy...…………….................……..8 1.1.1.2.1.2 Polyomavirus-Assoiated Hemorrhagic Cystitis...……………..............10
    [Show full text]
  • Potent Inhibition of Hemangioma Formation in Rats by the Acyclic Nucleoside Phosphonate Analogue Cidofovir1
    (CANCER RESEARCH 58, 2562-2567. June 15. 1998| Potent Inhibition of Hemangioma Formation in Rats by the Acyclic Nucleoside Phosphonate Analogue Cidofovir1 Sandra Liekens,2 Gracida Andrei, Michel Vandeputte, Erik De Clercq, and Johan Neyts Rega Institute for Medical Research, Katholieke L/niversiteit Leuven, B-3000 Leuven, Belgium ABSTRACT genie therapy could improve the life span of hemangioma-bearing children. At present, IFN-a is being used with relative success for the The acyclic nucleoside phosphonate analogue cidofovir elicited a treatment of hemangiomas (7, 8). The mechanism underlying this marked protection against hemangioma growth in newborn rats that had antitumor action is, however, not completely understood. been infected i.p. with a high titer of murine polyomavirus. Untreated, We have developed a novel animal model for the study of strategies infected rats developed cutaneous, ¡.in.,and cerebral hemangiomas asso for the treatment of hemangiomas. Infection of newborn rats with a ciated with severe hemorrhage and anemia leading to death within 3 high titer of the Marseille strain of mPyV3 was found to induce weeks postinfection (p.i.). s.c. treatment with cidofovir at 25 mg/kg, once a week, resulted in a complete suppression of hemangioma development cutaneous, i.m., and intracerebral hemangiomas with a short latency and associated mortality when treatment was initiated at 3 days p.i. (100% period. The cerebral hemangiomas were associated with hemorrhage, survival compared with 0% for the untreated animals). Cidofovir still resulting in severe anemia and subsequent death of the animals within afforded 40% survival and a significant delay in tumor-associated mor 3 weeks postinfection (p.i.).
    [Show full text]
  • Journal of Virology
    JOURNAL OF VIROLOGY Volume 61 October 1987 No. 10 ANIMAL VIRUSES Analysis of Pseudorabies Virus Glycoprotein glll Localization and Modification by Using Novel Infectious Viral Mutants Carrying Unique EcoRI Sites. J. Patrick Ryan, Mary E. Whealy, Alan K. Robbins, and Lynn W. Enquist 2962-2972 Effects of Position and Orientation of the 72-Base-Pair-Repeat Transcriptional Enhancer on Replication from the Simian Virus 40 Core Origin. Settara C. Chandrasekharappa and Kiranur N. Subramanian .................... 2973-2980 Mutants of the Rous Sarcoma Virus Envelope Glycoprotein That Lack the Transmembrane Anchor and Cytoplasmic Domains: Analysis of Intracellular Transport and Assembly into Virions. Lautaro G. Perez, Gary L. Davis, and Eric Hunter....................................... 2981-2988 Sequences of Herpes Simplex Virus Type 1 That Inhibit Formation of Stable TK+ Transformants. Daniel H. Farkas, Timothy M. Block, Paul B. Hart, and Robert G. Hughes, Jr................................................ 2989-2996 Primer-Dependent Synthesis of Covalently Linked Dimeric RNA Molecules by Poliovirus Replicase. John M. Lubinski, Lynn J. Ransone, and Asim Dasgupta ........................................................... 2997-3003 Adaptor Plasmids Simplify the Insertion of Foreign DNA into Helper-Independent Retroviral Vectors. Stephen H. Hughes, Jack J. Greenhouse, Christos J. Petropoulos, and Pramod Sutrave ..................................... 3004-3012 Point Mutation in the S Gene of Hepatitis B Virus for a d/y or w/r Subtypic Change in Two Blood Donors Carrying a Surface Antigen of Compound Subtype adyr or adwr. Hiroaki Okamoto, Mitsunobu Imai, Fumio Tsuda, Takeshi Tanaka, Yuzo Miyakawa, and Makoto Mayumi ................. 3030-3034 Hepatitis A Virus cDNA and Its RNA Transcripts Are Infectious in Cell Culture. Jeffrey 1. Cohen, John R. Ticehurst, Stephen M.
    [Show full text]
  • JOURNAL of VIROLOGY Volume 52 January 1985 No
    JOURNAL OF VIROLOGY Volume 52 January 1985 No. 1 ANIMAL VIRUSES Differential Stability of Host mRNAs in Friend Erythroleukemia Cells Infected with Herpes Simplex Virus Type 1. Barbara A. Mayman and Yutaka Nishioka ............................................................ 1-6 Myristic Acid, a Rare Fatty Acid, Is the Lipid Attached to the Transforming Pro- tein of Rous Sarcoma Virus and Its Cellular Homolog. Janice E. Buss and Bartholomew M. Sefton ............. ................................. 7-12 Genome Organization of Herpesvirus Aotus Type 2. Pawel G. Fuchs, Rudiger Ruger, Herbert Pfister, and Bernhard Fleckenstein ...... ................ 13-18 Isolation and Structural Mapping of a Human c-src Gene Homologous to the Transforming Gene (v-src) of Rous Sarcoma Virus. Carol P. Gibbs, Akio Tanaka, Stephen K. Anderson, Janet Radul, Joseph Baar, Anthony Ridgway, Hsing-Jien Kung, and Donald J. Fujita ........................ 19-24 Molecular Basis of Host Range Variation in Avian Retroviruses. Andrew J. Dorner, Jonathan P. Stoye, and John M. Coffin ......................... 32-39 Assignment of the Temperature-Sensitive Lesion in the Replication Mutant Al of Vesicular Stomatitis Virus to the N Gene. M. David Marks, Jennifer Kennedy-Morrow, and Judith A. Lesnaw .............................. 44-51 Mapping of the Structural Gene of Pseudorabies Virus Glycoprotein A and Identi- fication of Two Non-Glycosylated Precursor Polypeptides. Thomas C. Mettenleiter, Noemi Lukacs, and Hanns-Joachim Rziha ..... ............ 52-57 Preliminary Characterization of an Epitope Involved in Neutralization and Cell Attachment That Is Located on the Major Bovine Rotavirus Glycoprotein. Marta Sabara, James E. Gilchrist, G. R. Hudson, and L. A. Babiuk ...... 58-66 Use of a Bacterial Expression Vector to Map the Varicella-Zoster Virus Major Glycoprotein Gene, gC.
    [Show full text]
  • A Novel Ebola Virus VP40 Matrix Protein-Based Screening for Identification of Novel Candidate Medical Countermeasures
    viruses Communication A Novel Ebola Virus VP40 Matrix Protein-Based Screening for Identification of Novel Candidate Medical Countermeasures Ryan P. Bennett 1,† , Courtney L. Finch 2,† , Elena N. Postnikova 2 , Ryan A. Stewart 1, Yingyun Cai 2 , Shuiqing Yu 2 , Janie Liang 2, Julie Dyall 2 , Jason D. Salter 1 , Harold C. Smith 1,* and Jens H. Kuhn 2,* 1 OyaGen, Inc., 77 Ridgeland Road, Rochester, NY 14623, USA; [email protected] (R.P.B.); [email protected] (R.A.S.); [email protected] (J.D.S.) 2 NIH/NIAID/DCR/Integrated Research Facility at Fort Detrick (IRF-Frederick), Frederick, MD 21702, USA; courtney.fi[email protected] (C.L.F.); [email protected] (E.N.P.); [email protected] (Y.C.); [email protected] (S.Y.); [email protected] (J.L.); [email protected] (J.D.) * Correspondence: [email protected] (H.C.S.); [email protected] (J.H.K.); Tel.: +1-585-697-4351 (H.C.S.); +1-301-631-7245 (J.H.K.) † These authors contributed equally to this work. Abstract: Filoviruses, such as Ebola virus and Marburg virus, are of significant human health concern. From 2013 to 2016, Ebola virus caused 11,323 fatalities in Western Africa. Since 2018, two Ebola virus disease outbreaks in the Democratic Republic of the Congo resulted in 2354 fatalities. Although there is progress in medical countermeasure (MCM) development (in particular, vaccines and antibody- based therapeutics), the need for efficacious small-molecule therapeutics remains unmet. Here we describe a novel high-throughput screening assay to identify inhibitors of Ebola virus VP40 matrix protein association with viral particle assembly sites on the interior of the host cell plasma membrane.
    [Show full text]
  • An Autonomous Polyomavirus T Antigen Amino-Terminal Domain
    JOURNAL OF VIROLOGY, Aug. 1997, p. 6068–6074 Vol. 71, No. 8 0022-538X/97/$04.0010 Copyright © 1997, American Society for Microbiology Tiny T Antigen: an Autonomous Polyomavirus T Antigen Amino-Terminal Domain 1 2 1 1 MICHAEL I. RILEY, * WANGDON YOO, NOMUSA Y. MDA, AND WILLIAM R. FOLK Department of Biochemistry, University of Missouri—Columbia, Columbia, Missouri 65121,1 and Cheil Food & Chemicals Inc., Research & Development Center, Kyonggi-do, Korea2 Received 23 December 1996/Accepted 14 May 1997 Three mRNAs from the murine polyomavirus early region encode the three well-characterized tumor anti- gens. We report the existence of a fourth alternatively spliced mRNA which encodes a fourth tumor antigen, tiny T antigen, which comprises the amino-terminal domain common to all of the T antigens but is extended by six unique amino acid residues. The amount of tiny T antigen in infected cells is small because of its short half-life. Tiny T antigen stimulates the ATPase activity of Hsc70, most likely because of its DnaJ-like motif. The common amino-terminal domain may interface with chaperone complexes to assist the T antigens in carrying out their diverse functions of replication, transcription, and transformation in the appropriate cellular com- partments. The small, middle, and large T antigens expressed by the NIH 3T3 cells were transfected with DNA by calcium phosphate coprecipita- m murine polyomavirus (muPy) are formed of a common amino- tion and glycerol shock as previously described (83) or with Lipofectamine (20 l of Lipofectamine/60-mm-diameter plate) per directions by Gibco-BRL. MOP- terminal sequence juxtaposed to unique carboxy-terminal se- 3T3 and MOP-3T6 cells were transfected with DNA with Lipofectamine, and quences (33, 65).
    [Show full text]
  • FARE2021WINNERS Sorted by Institute
    FARE2021WINNERS Sorted By Institute Swati Shah Postdoctoral Fellow CC Radiology/Imaging/PET and Neuroimaging Characterization of CNS involvement in Ebola-Infected Macaques using Magnetic Resonance Imaging, 18F-FDG PET and Immunohistology The Ebola (EBOV) virus outbreak in Western Africa resulted in residual neurologic abnormalities in survivors. Many case studies detected EBOV in the CSF, suggesting that the neurologic sequelae in survivors is related to viral presence. In the periphery, EBOV infects endothelial cells and triggers a “cytokine stormâ€. However, it is unclear whether a similar process occurs in the brain, with secondary neuroinflammation, neuronal loss and blood-brain barrier (BBB) compromise, eventually leading to lasting neurological damage. We have used in vivo imaging and post-necropsy immunostaining to elucidate the CNS pathophysiology in Rhesus macaques infected with EBOV (Makona). Whole brain MRI with T1 relaxometry (pre- and post-contrast) and FDG-PET were performed to monitor the progression of disease in two cohorts of EBOV infected macaques from baseline to terminal endpoint (day 5-6). Post-necropsy, multiplex fluorescence immunohistochemical (MF-IHC) staining for various cellular markers in the thalamus and brainstem was performed. Serial blood and CSF samples were collected to assess disease progression. The linear mixed effect model was used for statistical analysis. Post-infection, we first detected EBOV in the serum (day 3) and CSF (day 4) with dramatic increases until euthanasia. The standard uptake values of FDG-PET relative to whole brain uptake (SUVr) in the midbrain, pons, and thalamus increased significantly over time (p<0.01) and positively correlated with blood viremia (p≤0.01).
    [Show full text]
  • Biologie Studijní Obor
    Univerzita Karlova Přírodovědecká fakulta Studijní program: Biologie Studijní obor: Genetika, molekulární biologie a virologie Bc. Barbora Číhařová Studium účinku modifikace virových částic polyhistidinem na jejich intracelulární lokalizaci a dopravu genů do jádra Effect of polyhistidine modification of viral particles on their intracellular localization and gene delivery to the nucleus Diplomová práce Školitel: RNDr. Hana Španielová, Ph.D. Praha, 2021 Prohlášení Prohlašuji, že jsem závěrečnou práci zpracovala samostatně a že jsem uvedla všechny použité informační zdroje a literaturu. Tato práce ani její podstatní část nebyla předložena k získaní jiného nebo stejného akademického titulu. V Praze, 26.4. 2021 ……..….…………………………………. Barbora Číhařová Poděkovaní Moc ráda bych poděkovala všem, kteří mě během mého studia a při psaní diplomové práce podporovali, věnovali mi svůj čas a vytvářeli příjemné prostředí pro dokončení tohoto projektu. Jmenovitě moc děkuji mé školitelce paní doktorce Haně Španielové za inspirativní přístup k vědě, trpělivost, spoustu rad a především za její čas strávený nad plánováním tohoto projektu a následnou kontrolou diplomové práce. Také děkuji magistře Janě Váňové, která zajišťuje každodenní úsměv všem členům laboratoře, za neuvěřitelnou trpělivost, zaučení v laboratoři a neocenitelnou pomoc v průběhu celého studia. Moc děkuji také Mgr. et Mgr. Alžbětě Hejtmánkové za řadu důležitých rad, přátelský přístup a zaučení při práci s elektronovým mikroskopem. Paní docentce Forstové děkuji za přijetí do laboratoře a možnost naučit se techniky molekulární biologie. Velký dík také patří všem současným i bývalým členům laboratoře, kteří vytvářeli přátelské prostředí, ve kterém byla radost pracovat. Mnohokrát děkuji mé rodině za nesmírnou podporu, poskytnutí zázemí a pochopení po celou dobu studia. V neposlední řadě moc děkuji Bc.
    [Show full text]
  • Deletion of Proline-Rich Domain in Polyomavirus T Antigens Results in Virus Partially Defective in Transformation and Tumorigenesis
    VIROLOGY 248, 420±431 (1998) ARTICLE NO. VY989246 View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Deletion of Proline-Rich Domain in Polyomavirus T Antigens Results in Virus Partially Defective in Transformation and Tumorigenesis Xiande Yi and Robert Freund1 Department of Microbiology and Immunology, University of Maryland School of Medicine, 655 W. Baltimore Street, Baltimore, Maryland 21201 Received March 25, 1998; returned to author for revision April 21, 1998; accepted May 20, 1998 Polyomavirus productively infects mouse cells, transforms rat fibroblasts in culture, and induces a broad spectrum of tumors when inoculated into newborn mice. The expression of large, middle, and small T antigen are necessary for virus growth and oncogenic transformation. We have generated a small deletion in a region common to both large and middle T antigen that encodes three consecutive prolines. In this report we characterize this mutant virus in terms of its ability to replicate in mouse cells, transform rat fibroblasts, and induce tumors in the mouse. We find that the virus immortalizes primary cells and that viral DNA replication is not impaired, indicating that these functions of large T antigen are not altered. However, the ability of the virus to transform rat fibroblasts is defective. The mutant virus makes fewer foci and the foci are weaker in appearance. The mutant middle T still associates with PI 3-kinase and shc, suggesting that the overall structure of the protein has not been disrupted. When inoculated into newborn C3H mice, the mutant virus induces fewer overall tumors with a longer latency than wild-type virus.
    [Show full text]
  • Convergent Perturbation of the Human Domain-Resolved Interactome by Viruses and Mutations Inducing Similar Disease Phenotypes
    RESEARCH ARTICLE Convergent perturbation of the human domain-resolved interactome by viruses and mutations inducing similar disease phenotypes Yangchun Frank ChenID, Yu XiaID* Department of Bioengineering, McGill University, Montreal, Quebec, Canada a1111111111 a1111111111 * [email protected] a1111111111 a1111111111 a1111111111 Abstract An important goal of systems medicine is to study disease in the context of genetic and envi- ronmental perturbations to the human interactome network. For diseases with both genetic OPEN ACCESS and infectious contributors, a key postulate is that similar perturbations of the human inter- actome by either disease mutations or pathogens can have similar disease consequences. Citation: Chen YF, Xia Y (2019) Convergent perturbation of the human domain-resolved This postulate has so far only been tested for a few viral species at the level of whole pro- interactome by viruses and mutations inducing teins. Here, we expand the scope of viral species examined, and test this postulate more rig- similar disease phenotypes. PLoS Comput Biol 15 orously at the higher resolution of protein domains. Focusing on diseases with both genetic (2): e1006762. https://doi.org/10.1371/journal. and viral contributors, we found significant convergent perturbation of the human domain- pcbi.1006762 resolved interactome by endogenous genetic mutations and exogenous viral proteins induc- Editor: Andrey Rzhetsky, University of Chicago, ing similar disease phenotypes. Pan-cancer, pan-oncovirus analysis further revealed that UNITED STATES domains of human oncoproteins either physically targeted or structurally mimicked by Received: September 17, 2018 oncoviruses are enriched for cancer driver rather than passenger mutations, suggesting Accepted: January 7, 2019 convergent targeting of cancer driver pathways by diverse oncoviruses.
    [Show full text]
  • Proquest Dissertations
    Conformational Changes of Polyomavirus during Cell Entry by Marjan Dolatshahi Department of Anatomy and Cell Biology McGill University Montreal, Quebec, Canada Submitted in February2008 A thesis submitted to McGill University in partial fulfillment of the role of the requirements of the degree of Master of Science Copyright © Marjan Dolatshahi 2008 Library and Archives Biblioth&que et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l'6dition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-66908-2 Our file Notre r6f6rence ISBN: 978-0-494-66908-2 NOTICE: AVIS: The author has granted a non- L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduce, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lntemet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non- support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimis ou autrement printed or otherwise reproduced reproduits sans son autorisation.
    [Show full text]
  • ISG15 Inhibits Ebola VP40 VLP Budding in an L-Domain-Dependent Manner by Blocking Nedd4 Ligase Activity
    ISG15 inhibits Ebola VP40 VLP budding in an L-domain-dependent manner by blocking Nedd4 ligase activity Atsushi Okumura*, Paula M. Pitha†, and Ronald N. Harty*‡ *Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104; and †The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, 1650 Orleans Street, Baltimore, MD 21231 Edited by Diane E. Griffin, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, and approved January 15, 2008 (received for review November 9, 2007) Ebola virus budding is mediated by the VP40 matrix protein. effect; however, the mechanism of action of ISG15 remains to be VP40 can bud from mammalian cells independent of other viral determined. proteins, and efficient release of VP40 virus-like particles (VLPs) Ebola virus (Zaire; EBOZ) is a member of the Filoviridae requires interactions with host proteins such as tsg101 and family of negative-sense RNA viruses, and the VP40 matrix Nedd4, an E3 ubiquitin ligase. Ubiquitin itself is thought to be protein is a key structural protein critical for virion egress. exploited by Ebola virus to facilitate efficient virus egress. Late-budding domains (L-domains) present in VP40 mediate Disruption of VP40 function and thus virus budding remains an interactions with host proteins to facilitate VLP and virus release attractive target for the development of novel antiviral thera- (23–39). pies. Here, we investigate the effect of ISG15 protein on the For example, Ebola VP40 contains overlapping L-domains release of Ebola VP40 VLPs. ISG15 is an IFN-inducible, ubiquitin- (7PTAP10 and 10PPEY13), which interact with members of the like protein expressed after bacterial or viral infection.
    [Show full text]