DOCSLIB.ORG

The Polyomavirus Major Capsid Protein VP1 Interacts with the Nuclear Matrix Regulatory Protein

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Polyomavirus Major Capsid Protein VP1 Interacts with the Nuclear Matrix Regulatory Protein FEBS 23316 FEBS Letters 467 (2000) 359^364 View metadata, citation and similar papers at core.ac.uk brought to you by CORE The polyomavirus major capsid protein VP1 interacts with theprovided nuclearby Elsevier - Publisher Connector matrix regulatory protein YY1 Zdena Palkova¨a;b, Hana Sípanielova¨b, Vanesa Gottifredia;1, Dana Hollanderova¨b, Jitka Forstova¨b, Paolo Amatia;* aInstituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Biotecnologie Cellulari ed Ematologia, Sezione di Genetica Molecolare, Universita© di Roma La Sapienza, Viale Regina Elena 324, 00161 Rome, Italy bDepartment of Genetics and Microbiology, Faculty of Natural Sciences, Charles University, Vinie©na¨ 5, 12844 Prague 2, Czech Republic Received 17 December 1999 Edited by Hans-Dieter Klenk The external loops are likely to be the structures involved in Abstract Polyomavirus reaches the nucleus in a still encapsi- dated form, and the viral genome is readily found in association cell receptor recognition by virions [4] and, therefore, to be with the nuclear matrix. This association is thought to be the main antigenic determinants. The £exible C-terminal arms essential for viral replication. In order to identify the protein(s) form interpentameric contacts and the basic amino acids of involved in the virus-nuclear matrix interaction, we focused on the N terminal arm are responsible for the non-speci¢c DNA- the possible roles exerted by the multifunctional cellular nuclear binding activity of VP1 [6,7]. matrix protein Yin Yang 1 (YY1) and by the viral major capsid The necessity for viral genomes to associate to the nuclear protein VP1. In the present work we report on the in vivo matrix (NM) in order to be expressed, has been demonstrated association between YY1 and VP1. Using the yeast two-hybrid in some DNA tumor viruses such as papilloma and adenovi- system we demonstrate that the VP1 and YY1 proteins ruses; this association has been shown to be mediated by physically interact through the D-E region of VP1 and the proteins encoded by the viral genome [8,9]. For the small activation domain of YY1. z 2000 Federation of European Biochemical Societies. polyomaviruses no such function has been found, although it has been shown that infecting polyomavirus reaches the Key words: Polyomavirus; VP1; Nuclear matrix; Yin Yang 1 nucleus encapsidated and its genomic DNA readily associates to the NM [10,11], and this association is thought to deter- mine the ability of the incoming viral minichromosome to be transcribed and replicated in the permissive host. Further- 1. Introduction more, it has been shown that in vivo expressed VP1 associates to the NM [12]. The mouse polyomavirus (Py) genome encodes three early Yin Yang 1 (YY1), a multifunctional cellular transcription- tumor antigens (LT, MT and sT) and three late structural al regulatory phosphoprotein with predicted molecular weight proteins VP1, VP2 and VP3 which make up the viral capsid. 44 kDa, migrating on SDS gels as 68 kDa protein, contains Polyoma late proteins, together with viral DNA and cellular four C2H2-type zinc ¢ngers at the C-terminus responsible for histones (except H1) are assembled in the host cell nuclei into the sequence-speci¢c DNA binding activity, a glycine-rich re- virions [1]. The icosahedral capsid is organized by 72 exter- pression domain and a N-terminal activation domain. De- nally exposed VP1 pentamers with one VP2 or VP3 minor pending on the context, YY1 can activate or repress the tran- capsid proteins associated internally with each pentamer [2]. scription of number a of both cellular and viral genes Py VP1 mutants have been isolated and characterized for (reviewed in [13]). Moreover, YY1 has been found to bind modi¢ed host range or tumorigenesis. All of these mutations DNA in speci¢c sites, also in the enhancer region of Py are located in the outfacing loops of VP1. The major outfac- [14]. The protein-protein interaction has an important func- ing protein of the capsid (VP1) has been characterized with tion in controlling the activity of YY1 and a number of pro- regard to conformational organization and interaction with teins have been identi¢ed which form complexes with it. An- the cell membrane receptor [3^5]. According to X-ray di¡rac- other aspect of YY1 function has been unravelled by tion studies of its tertiary structure [3], VP1 can be divided demonstration that YY1 is identical to the NM binding pro- into three modules: an N-terminal arm, an antiparallel tein NMP-1 [15]. These data imply that YY1 may also be L-sandwich core and a long C-terminal arm. Four loops ema- involved in the association of speci¢c DNA sequences of cel- nate from the L-sheet framework; three of them (B-C, D-E lular and viral genes to the NM. and H-I) form closely interacting structures at the outfacing Results from our and other laboratories [16^21] had sug- side of the L-sandwich that are also exposed at the surface of gested the possibility that VP1, the major capsid protein, the capsomeric structure. The long E-F loop is exposed on the could determine alterations in the host speci¢city of growth side of the L-sandwich core (and on the side of capsomeres). and tumorigenicity. In order to determine the possible mech- anisms that allow the encapsidated polyomaviruses that reach the cell nucleus to associate with the NM, we investigated the *Corresponding author. Fax: (39)-6-4462891. possible role of YY1 in this necessary step of viral function. In E-mail: [email protected] this study we report that YY1 binds in vivo to the capsid 1 Present address: Department of Biological Sciences, Columbia protein VP1. This interaction involves a speci¢c site in the University, New York, NY 10027, USA. D-E loop of VP1 and the activation domain of YY1. The 0014-5793 / 00 / $20.00 ß 2000 Federation of European Biochemical Societies. All rights reserved. PII: S0014-5793(00)01170-4 FEBS 23316 3-2-00 360 Z. Palkova¨ et al./FEBS Letters 467 (2000) 359^364 possible relevance of VP1-YY1 interaction for the biology of the fragment of the 3P part of the VP1 coding sequence from the viral life cycle is discussed. pVL1393-VP1 (11) (PstI/BglII) and (3) the plasmid pGBT9 carrying the GAL4 DNA-binding domain (DNA-BD)) (Clontech; EcoRI/Bam- HI). Production of the fusion protein in yeast was con¢rmed using 2. Materials and methods anti-VP1 antibody. pGBT9-VP1144 carries the fragment StuI^NdeIin pGBT9-VP1 replaced with the corresponding fragment of mutated 2.1. Antibodies VP1-M17 gene containing a SmaI site in position 432 (144 aa) from Rabbit polyclonal anti-VP1 (I58) and anti-D-E loop (against pSVL-VP1-M17 (Amati et al., manuscript in preparation); resulting VP1-Leu137^Glu159 peptide) antibodies were kindly provided by R. pGBT9-VP1-M17 was subsequently cleaved by SmaI and SalI, blunt- Garcea (University of Colorado, CO, USA); mouse monoclonal ended and religated. pGBT9-VP1140 and pGBT9-VP1136 : pGBT9- anti-VP1 and anti-VP2/3B have been already described [22]; anti- VP1-M17 was linearized by SmaI, treated with BAL31 nuclease, YY1 (C-20), anti-Gal4-TA (768), anti-p27 (C-19) and anti-Grb-2 cleaved by SalI, blunt-ended, religated and electroporated into E. (C-23) rabbit polyclonal antibodies were obtained from Santa Cruz coli DH5K [24]; the extent of deletions was analyzed by sequencing Biotechnology, CA, USA. Secondary, Px-conjugated anti-mouse and (kindly performed by CEINGE, Napoli, Italy). pGBT9-DE118170 : the 118 170 anti-rabbit IgG antibodies were from Bio-Rad Laboratories, Hercules, DNA fragment of VP1 (Leu ^Gly ) containing the VP1-D-E loop 137 159 CA, USA. (Leu ^Glu ) was ampli¢ed using synthetic primers (EcoRI/PstI), fused with sequences encoding Gal4bd (Gal4-DNA binding domain) 2.2. Py infection and immunoprecipitation in pGBT9 (EcoRI/PstI); production of fusion protein in yeast was Mouse 3T6 ¢broblast cells were infected with wild type (wt) Py [8] con¢rmed using the anti-D-E loop antibody. pGBT9-EF172248 : con- structed as described for pGBT9-DE , using ampli¢ed fragment at a multiplicity of 50 pfu. At di¡erent times post-infection (p.i.) cells 118170 of VP1 encoding amino acids 172^248. pGAD424-YY1: YY1 gene were lysed and lysates were subjected to ultrasonic treatment and subsequently clari¢ed by centrifugation at 4³C following Pelicci et from pGEM-4Z.DELTA (EcoRI) was fused with sequences encoding al. [23]. After classical immunoprecipitation, immunocomplexes were GAL4 AD (GAL4-activation domain) in pGAD424 (Clontech; EcoRI), and the production of the fusion protein in yeast was con- washed three times with cold lysis bu¡er and once with RIPA, eluted ¢rmed using anti-YY1 antibody. YY1 deletions were derived from and denatured in Laemmli bu¡er (5 min). Proteins were resolved in 10% SDS^PAGE and transferred onto a nitrocellulose ¢lter. Immu- pGAD424-YY1 by cleavage with XmaI (pGAD424-YY1200), PstI nocomplexes were detected using either anti-VP1 or anti-YY1 anti- (pGAD424-YY189) and EagI (pGAD424-YY1157414), respectively, and subsequent religation. bodies and Px-conjugated secondary antibodies [24]. In some experi- ments secondary non-speci¢c anti-rabbit antibodies (NSA) from SEVA, a.f. (Prague, Czech Republic) in control immunoprecipitations 2.6. Yeast strains and methods were used. S. cerevisiae HF7c (MATa, ura3^52, his3^200, ade2^101, lys2^801, trp1^901, leu2^3,112, gal4^542, gal80^538, LYS2: :GAL1-HIS3, UR- A3: :(GAL17-mers) -Cyc1-lacZ and S. serevisiae SFY526 (MATa, 2.3. VP1, VP3 and YY1 over-production in mammalian and insect cells, 3 ura3^52, his3^200, ade2^101, lys2^801, trp1^901, leu2^3,112, canr, and immunoprecipitation gal4^542, gal80^538, URA: :GAL1-lacZ) strains were previously de- COS cells were co-transfected using the calcium-phosphate tech- scribed by Bartel et al.
Load more

Recommended publications
  • Cryptic Inoviruses Revealed As Pervasive in Bacteria and Archaea Across Earth’S Biomes
    ARTICLES https://doi.org/10.1038/s41564-019-0510-x Corrected: Author Correction Cryptic inoviruses revealed as pervasive in bacteria and archaea across Earth’s biomes Simon Roux 1*, Mart Krupovic 2, Rebecca A. Daly3, Adair L. Borges4, Stephen Nayfach1, Frederik Schulz 1, Allison Sharrar5, Paula B. Matheus Carnevali 5, Jan-Fang Cheng1, Natalia N. Ivanova 1, Joseph Bondy-Denomy4,6, Kelly C. Wrighton3, Tanja Woyke 1, Axel Visel 1, Nikos C. Kyrpides1 and Emiley A. Eloe-Fadrosh 1* Bacteriophages from the Inoviridae family (inoviruses) are characterized by their unique morphology, genome content and infection cycle. One of the most striking features of inoviruses is their ability to establish a chronic infection whereby the viral genome resides within the cell in either an exclusively episomal state or integrated into the host chromosome and virions are continuously released without killing the host. To date, a relatively small number of inovirus isolates have been extensively studied, either for biotechnological applications, such as phage display, or because of their effect on the toxicity of known bacterial pathogens including Vibrio cholerae and Neisseria meningitidis. Here, we show that the current 56 members of the Inoviridae family represent a minute fraction of a highly diverse group of inoviruses. Using a machine learning approach lever- aging a combination of marker gene and genome features, we identified 10,295 inovirus-like sequences from microbial genomes and metagenomes. Collectively, our results call for reclassification of the current Inoviridae family into a viral order including six distinct proposed families associated with nearly all bacterial phyla across virtually every ecosystem.
    [Show full text]
  • Opportunistic Intruders: How Viruses Orchestrate ER Functions to Infect Cells
    REVIEWS Opportunistic intruders: how viruses orchestrate ER functions to infect cells Madhu Sudhan Ravindran*, Parikshit Bagchi*, Corey Nathaniel Cunningham and Billy Tsai Abstract | Viruses subvert the functions of their host cells to replicate and form new viral progeny. The endoplasmic reticulum (ER) has been identified as a central organelle that governs the intracellular interplay between viruses and hosts. In this Review, we analyse how viruses from vastly different families converge on this unique intracellular organelle during infection, co‑opting some of the endogenous functions of the ER to promote distinct steps of the viral life cycle from entry and replication to assembly and egress. The ER can act as the common denominator during infection for diverse virus families, thereby providing a shared principle that underlies the apparent complexity of relationships between viruses and host cells. As a plethora of information illuminating the molecular and cellular basis of virus–ER interactions has become available, these insights may lead to the development of crucial therapeutic agents. Morphogenesis Viruses have evolved sophisticated strategies to establish The ER is a membranous system consisting of the The process by which a virus infection. Some viruses bind to cellular receptors and outer nuclear envelope that is contiguous with an intri‑ particle changes its shape and initiate entry, whereas others hijack cellular factors that cate network of tubules and sheets1, which are shaped by structure. disassemble the virus particle to facilitate entry. After resident factors in the ER2–4. The morphology of the ER SEC61 translocation delivering the viral genetic material into the host cell and is highly dynamic and experiences constant structural channel the translation of the viral genes, the resulting proteins rearrangements, enabling the ER to carry out a myriad An endoplasmic reticulum either become part of a new virus particle (or particles) of functions5.
    [Show full text]
  • Receptor-Binding and Oncogenic Properties of Polyoma Viruses Isolated from Feral Mice
    Receptor-Binding and Oncogenic Properties of Polyoma Viruses Isolated from Feral Mice John Carroll1[, Dilip Dey1[, Lori Kreisman1[, Palanivel Velupillai1, Jean Dahl1, Samuel Telford2, Roderick Bronson1, Thomas Benjamin1* 1 Department of Pathology, Harvard Medical School, Boston, Massachusetts, United States of America, 2 Department of Tropical Public Health, Harvard School of Public Health, Boston, Massachusetts, United States of America Laboratory strains of the mouse polyoma virus differ markedly in their abilities to replicate and induce tumors in newborn mice. Major determinants of pathogenicity lie in the sialic binding pocket of the major capsid protein Vp1 and dictate receptor-binding properties of the virus. Substitutions at two sites in Vp1 define three prototype strains, which vary greatly in pathogenicity. These strains replicate in a limited fashion and induce few or no tumors, cause a disseminated infection leading to the development of multiple solid tumors, or replicate and spread acutely causing early death. This investigation was undertaken to determine the Vp1 type(s) of new virus isolates from naturally infected mice. Compared with laboratory strains, truly wild-type viruses are constrained with respect to their selectivity and avidity of binding to cell receptors. Fifteen of 15 new isolates carried the Vp1 type identical to that of highly tumorigenic laboratory strains. Upon injection into newborn laboratory mice, the new isolates induced a broad spectrum of tumors, including ones of epithelial as well as mesenchymal origin. Though invariant in their Vp1 coding sequences, these isolates showed considerable variation in their regulatory sequences. The common Vp1 type has two essential features: 1) failure to recognize ‘‘pseudoreceptors’’ with branched chain sialic acids binding to which would attenuate virus spread, and 2) maintenance of a hydrophobic contact with true receptors bearing a single sialic acid, which retards virus spread and avoids acute and potentially lethal infection of the host.
    [Show full text]
  • REVIEW Hepatitis C Virus
    DOI:http://dx.doi.org/10.7314/APJCP.2012.13.12.5917 Hepatitis C Virus -Proteins, Diagnosis, Treatment and New Approaches for Vaccine Development REVIEW Hepatitis C Virus - Proteins, Diagnosis, Treatment and New Approaches for Vaccine Development Hossein Keyvani1, Mehdi Fazlalipour2*, Seyed Hamid Reza Monavari1, Hamid Reza Mollaie2 Abstract Background: Hepatitis C virus (HCV) causes acute and chronic human hepatitis infection and as such is an important global health problem. The virus was discovered in the USA in 1989 and it is now known that three to four million people are infected every year, WHO estimating that 3 percent of the 7 billion people worldwide being chronically infected. Humans are the natural hosts of HCV and this virus can eventually lead to permanent liver damage and carcinoma. HCV is a member of the Flaviviridae family and Hepacivirus genus. The diameter of the virus is about 50-60 nm and the virion contains a single-stranded positive RNA approximately 10,000 nucleotides in length and consisting of one ORF which is encapsulated by an external lipid envelope and icosahedral capsid. HCV is a heterogeneous virus, classified into 6 genotypes and more than 50 subtypes. Because of the genome variability, nucleotide sequences of genotypes differ by approximately 31-34%, and by 20-23% among subtypes. Quasi-species of mixed virus populations provide a survival advantage for the virus to create multiple variant genomes and a high rate of generation of variants to allow rapid selection of mutants for new environmental conditions. Direct contact with infected blood and blood products, sexual relationships and availability of injectable drugs have had remarkable effects on HCV epidemiology.
    [Show full text]
  • Structural Basis and Evolution of Glycan Receptor Specificities Within the Polyomavirus Family
    RESEARCH ARTICLE Host-Microbe Biology crossm Structural Basis and Evolution of Glycan Receptor Specificities within the Polyomavirus Family Luisa J. Ströh,a* Nils H. Rustmeier,a Bärbel S. Blaum,a Josephine Botsch,a Philip Rößler,a Florian Wedekink,a W. Ian Lipkin,b Nischay Mishra,b Thilo Stehlea,c Downloaded from aInterfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany bCenter for Infection and Immunity, Columbia University, New York, New York, USA cDepartment of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee, USA ABSTRACT Asymptomatic infections with polyomaviruses in humans are common, but these small viruses can cause severe diseases in immunocompromised hosts. New Jersey polyomavirus (NJPyV) was identified via a muscle biopsy in an organ transplant recipient with systemic vasculitis, myositis, and retinal blindness, and hu- http://mbio.asm.org/ man polyomavirus 12 (HPyV12) was detected in human liver tissue. The evolutionary origins and potential diseases are not well understood for either virus. In order to define their receptor engagement strategies, we first used nuclear magnetic reso- nance (NMR) spectroscopy to establish that the major capsid proteins (VP1) of both viruses bind to sialic acid in solution. We then solved crystal structures of NJPyV and HPyV12 VP1 alone and in complex with sialylated glycans. NJPyV employs a novel binding site for a ␣2,3-linked sialic acid, whereas HPyV12 engages terminal ␣2,3- or ␣2,6-linked sialic acids in an exposed site similar to that found in Trichodysplasia on October 12, 2020 by guest spinulosa-associated polyomavirus (TSPyV). Gangliosides or glycoproteins, featuring in mammals usually terminal sialic acids, are therefore receptor candidates for both viruses.
    [Show full text]
  • Protein-Mediated Viral Latency Is a Novel Mechanism for Merkel Cell
    Protein-mediated viral latency is a novel mechanism PNAS PLUS for Merkel cell polyomavirus persistence Hyun Jin Kwuna, Yuan Changa,1,2, and Patrick S. Moorea,1,2 aCancer Virology Program, University of Pittsburgh, Pittsburgh, PA 15213 Contributed by Patrick S. Moore, April 10, 2017 (sent for review March 8, 2017; reviewed by Daniel DiMaio and Peter M. Howley) Viral latency, in which a virus genome does not replicate in- binding. Expression of LT is known to be autoinhibited in two ways: dependently of the host cell genome and produces no infectious LT inhibits its own promoter in a negative feedback loop (5, 6), and particles, is required for long-term virus persistence. There is no a virus-encoded miRNA transcribed during early viral gene ex- known latency mechanism for chronic small DNA virus infections. pression inhibits LT mRNA expression (7–10). Merkel cell polyomavirus (MCV) causes an aggressive skin cancer Merkel cell polyomavirus (MCV) is a human polyomavirus that after prolonged infection and requires an active large T (LT) phos- causes ∼80% of human Merkel cell carcinomas (MCCs), which phoprotein helicase to replicate. We show that evolutionarily are among the most severe skin cancers (11, 12). It was the first conserved MCV LT phosphorylation sites are constitutively recog- human pathogen discovered through nondirected transcriptome nized by cellular Fbw7, βTrCP, and Skp2 Skp-F-box-cullin (SCF) sequencing using an approach called digital transcriptome sub- E3 ubiquitin ligases, which degrade and suppress steady-state LT traction. MCV infection is nearly ubiquitous among human adults, protein levels. Knockdown of each of these E3 ligases enhances LT and MCC tumors arise as rare biological accidents after prolonged stability and promotes MCV genome replication.
    [Show full text]
  • High Aspect Ratio Viral Nanoparticles for Cancer Therapy
    HIGH ASPECT RATIO VIRAL NANOPARTICLES FOR CANCER THERAPY By Karin L. Lee Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Dr. Nicole F. Steinmetz Biomedical Engineering CASE WESTERN RESERVE UNIVERSITY August 2016 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Karin L. Lee candidate for the Doctor of Philosophy degree*. (signed) Horst von Recum (chair of the committee) Nicole Steinmetz Ruth Keri David Schiraldi (date) June 29, 2016 *We also certify that written approval has been obtained for any proprietary material contained therein. TABLE OF CONTENTS Table of Contents List of Tables .................................................................................................................... ix List of Figures and Schemes .............................................................................................x Acknowledgements ........................................................................................................ xiv List of Abbreviations .................................................................................................... xvii Abstract......................................................................................................................... xxiii Chapter 1: Introduction ....................................................................................................1 1.1 Cancer statistics...............................................................................................................1
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 7,527,801 B2 Coit Et Al
    USOO7527801B2 (12) United States Patent (10) Patent No.: US 7,527,801 B2 Coit et al. (45) Date of Patent: May 5, 2009 (54) NOROVIRUS AND SAPOVIRUS ANTIGENS Genbank Accession # AAQ63158, ORF3 of Norwalk virus, pub lished Aug. 31, 2003.* (75) Inventors: Doris Coit, Petaluma, CA (US); Glass et al., Two Nonoverlapping Domains on the Norwalk Virus Michael Houghton, Danville, CA (US); Open Reading Frame 3 (ORF3) Protein Are Involved in the Forma Colin McCoin, Castro Valley, CA (US); tion of the Phosphorylated 35K Protein and in ORF3-Capsid Protein Interactions, 2003, Journal of Virology, vol. 77. No. 6, pp. 3569 Angelica Medina-Selby, San Francisco, 3577.: CA (US); Michael Vajdy, Orinda, CA Harrington et al., Systemic, Mucosal, and Heterotypic Immune (US) Induction in Mice Inoculated with Venezuelan Equine Encephalitis Replicons ExpressingNorwalkVirus-Like Particles, 2002, Journal of (73) Assignee: Novartis Vaccines and Diagnostics, Virology, vol. 76, No. 2, pp. 730-742.* Inc., Emeryville, CA (US) Asanaka, et al., “Replication And Packaging Of Norwalk Virus RNA In Cultured Mammalian Cells. PNAS USA 102: 10327-10332 (*) Notice: Subject to any disclaimer, the term of this (2005). patent is extended or adjusted under 35 Belliot, et al., “In Vitro Proteolytic Processing Of The MD 145 U.S.C. 154(b) by 0 days. Norovirus ORF1 Nonstructural Polyprotein Yields Stable Precursors And Products Similar To Those Detected In Calicivirus-Infected (21) Appl. No.: 11/603.913 Cells.”J Virol 77: 10957-10974 (2003). Chakravarty, et al., “Evolutionary Trace Residues. In Noroviruses: (22) Filed: Nov. 22, 2006 Importance In Receptor Binding, Antigenicity, Virion Assembly, And Strain Diversity.”J Virol 79:554-568 (2005).
    [Show full text]
  • Phosphorylation of the Budgerigar Fledgling Disease Virus Major Capsid Protein Vp1t
    JOURNAL OF VIROLOGY, JUIY 1992, p. 4551-4555 Vol. 66, No. 7 0022-538X/92/074551-05$02.00/0 Copyright © 1992, American Society for Microbiology Phosphorylation of the Budgerigar Fledgling Disease Virus Major Capsid Protein VP1t JOHN I. HAYNES II AND RICHARD A. CONSIGLI* Division of Biology, Section of Virology and Oncology, Ackert Hall, Kansas State University, Manhattan, Kansas 66506-4901 Received 19 February 1992/Accepted 30 March 1992 The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyoma- virus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32p; to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein. Budgerigar fledgling disease virus (BFDV) has been iso- murine counterpart revealed 47% identity between the VP1 lated from fledgling budgerigars (Melopsittacus undulatus) proteins and 26.5 and 29.5% identity between the VP2 and showing lesions in numerous tissues, including skin, feather, VP3 proteins, respectively (35).
    [Show full text]
  • Nucleotide Substitution Rates of HIV-1
    AIDS Rev 2000; 2: 39-47 Nucleotide Substitution Rates of HIV-1 Yoshiyuki Suzuki1, Yumi Yamaguchi-Kabata2, and Takashi Gojobori1 1Center for Information Biology, National Institute of Genetics, Mishima, Japan 2Laboratory of Viral Pathogenesis, Institute for Virus Research, Kyoto University, Kyoto, Japan Abstract The molecular evolution of human immunodeficiency virus type 1 (HIV-1) is reviewed from the viewpoint of the rate of nucleotide substitution, which is defined as the number of nucleotide substitutions per site per year. The rate of nucleotide substitution is useful not only for estimating the origin and history of HIV-1 epidemics, but also for detecting natural selection operating at the amino acid sequence level. The RNA viruses may be classified into two groups, the rapidly and slowly evolving RNA viruses, according to their rates of nucleotide substitution; 10-3-10-4 and 10-6-10-7 per site per year for the former and the latter, respectively. HIV-1 is a member of the rapidly evolving RNA viruses, with the rate of 10-3 per site per year, which is several million times faster than the eukaryotic and prokaryotic genes. The linear regression analysis of a large number of HIV-1 sequences revealed that HIV-1 has a weak molecular clock. The latest divergence time between human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus (SIV) was estimated as about 30 years ago, and that between HIV-1 and SIV as several hundred years ago. These observations, as well as the inconsistency between the topologies of phylogenetic trees reconstructed for primate lentiviruses and for their host species, indicate that the interspecies transmission occurred during the evolution of primate lentiviruses.
    [Show full text]
  • Structural Presentation of Influenza Epitopes on Modular Virus-Like Particles Doan Thanh Tam Master of Biotechnology
    Structural presentation of influenza epitopes on modular virus-like particles Doan Thanh Tam Master of Biotechnology A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2018 Australian Institute for Bioengineering and Nanotechnology i Abstract Vaccine development has increasingly developed toward subunit vaccine approach which uses highly-purified and well-characterised components, such as antigenic peptides, to elicit an immune response. An antigenic peptide requires a protein carrier or platform e.g. virus-like particle (VLP), to improve its poor immunogenicity. Based on this approach, murine polyomavirus (MuPyV) VLP and its subunit (capsomere) have been employed as vaccine platforms to present heterogeneous epitopes or antigenic elements from targeted pathogens. The design rule for epitope presentation on VLPs still remains unclear. A tandem repeat display strategy, in which antigenic element was repetitively arrayed, led to the induction of a significant antibody titer against its native structure, compared with the use of flanking sequence. Tandem repeat display strategy might retain the epitope conformation that is close to its native structure. This work further investigates the structural presentation of Helix 190 (H190) and Helix A (HA2A), from influenza hemagglutinin, using tandem repeat strategy. Different tools are exploited to characterise the structures of presented epitopes. The major outcomes of this research are: (i) demonstrating the limitation of phage antibody panning as a tool to inform the structural presentation of epitope on modular VLPs; (ii) developing a simple tool for rapid screening of presented epitope conformation using circular dichroism spectroscopy coupled with singular value decomposition algorithms (CD-SVD); (iii) demonstrating the capability of tandem repeat strategy in promoting the helicity of the helical structure; (iv) establishing initial steps in determining atomic structure of presented epitope using X-ray crystallography.
    [Show full text]
  • Resurrection of a Global, Metagenomically Defined Microvirus 1 2 3 4 Paul C. Kirchberger* and Howard Ochman 5 6 7 8 9 Department
    bioRxiv preprint doi: https://doi.org/10.1101/743518; this version posted August 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Resurrection of a Global, Metagenomically Defined Microvirus 2 3 4 5 Paul C. Kirchberger* and Howard Ochman 6 7 8 9 10 Department of Integrative Biology 11 University of Texas at Austin 12 Austin, Texas 78712, USA 13 14 15 16 17 18 *Corresponding author. Email: [email protected] 19 bioRxiv preprint doi: https://doi.org/10.1101/743518; this version posted August 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 20 Abstract 21 The Gokushovirinae (family Microviridae) are a group of single-stranded, circular 22 DNA bacteriophages that have been detected in metagenomic datasets from every 23 ecosystem on the planet. Despite their abundance, little is known about their biology or 24 their bacterial hosts: isolates are exceedingly rare, known only from a very small 25 number of obligate intracellular bacteria. By synthesizing circularized phage genomes 26 from prophages embedded in diverse enteric bacteria, we produced viable 27 gokushovirus phage particles that could reliably infect E. coli, thereby allowing 28 experimental analysis of its life cycle and growth characteristics.
    [Show full text]