The Molecular Biology

The Molecular Biology of Poliovirus Friedrich Koch and Gebhard Koch Springer-~rlag Wien New York

Dr. Friedrich Koch Prof. Dr. Gebhard Koch Abteilung Molekularbiologie Universitat Hamburg, Federal Republic of Germany This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machine or similar means, and storage in data banks. 1985 by Springer-VerlaglWien Softcover reprint of the hardcover 1st edition 1985 With 122 Figures Library of Congress Cataloging in Publication Data. Koch, Friedrich, 1957-. The molecular biology of poliovirus. Includes index.!. Poliovirus. 2. Molecular biology. I. Koch, Gebhard. II. Title. QR201. P73K58. 1985. 576'.6484. 85-4757. ISBN-13:978-3-7091-7467-8 e-isbn-13:978-3-7091-7000-7 DOl: 10.1007/978-3-7091-7000-7

Preface Years ago when we were asked to write a book on the present-day knowledge of the molecular biology of poliovirus, we did not expect that such an apparently simple task could involve so much time and effort. Our writing was hampered by the fact that both of us are full time "workers", so that this monograph is mainly a spare time expedience. The main attention of this book focuses on a detailed review of the molecular biology of poliovirus and especially on the advances of the last decade; medical and environmental aspects are only briefly mentioned. Observations from older studies are considered in view of more recent information. Some of the older observations provided fundamental insights and paved the way for present day research; too often such data has been neglected or independently rediscovered. Today, poliovirus research has again attracted considerable interest. High points gained within the last few years were the elucidation of the complete nucleotide sequences of the RNAs of the three poliovinls serotypes and the corresponding vaccine strains, the demonstration of genome evolution during transmission of poliovirus in an epidemic, further characterization of the antigenic sites on the virus particle and of the antigenic drift, characterization of alternate conformational states of the virion capsid, the development of monoclonal antibodies against some of the virus proteins, observations on the role of the plasma membrane, cytoskeleton, and cytoplasmic membranes as mediators in the virusinduced redirection of the synthetic machinery of the host cell, and characterization of proteins involved in RNA replication. The review is certainly not allinclusive. We are grateful for any comments, corrections, additions, and criticism. We wish to thank Kurt Bienz, John Bilello, Richard Crowell, Keith Dunker, Denise Egger, John Mapoles, Paolo La Colla, Roland Rueckert, and Douglas Scraba for many fruitful discussions and helpful comments. We also thank all those who provided us with material for illustrations. The personal proceeds of this book will be donated to a preventive health care project in Nicaragua. As part of this project, the Nicaraguan health ministry has initiated mass vaccination programs against poliomyelitis, which will for the first time succeed in reaching most of the children in this country. Hamburg, March 1985 F. Koch and G. Koch

Part I: The Poliovirus. 1. History..... 3 I. Introduction. 3 II. Early History-Occasional Nonepidemic Cases of Poliomyelitis 4 III. The 18th and 19th Centuries: Polioepidemics, Poliomyelitis Is Described as a Clinical Entity............ 6 IV. Early 20th Century: Research on Polio Begins, Virology Is still a Clinical Discipline................ 6 V. The Development of Vaccines Against the Poliomyelitis Virus 9 VI. The Fifties and the Advent of Molecular Biology... 10 VII. Advances in Polio Research During the Past Two Decades 11 2. Classification........ 15 I. General Considerations.. 15 A. Nature of the Genome. 15 B. The Cryptogram... 19 II. The Distinguishing Features of Picornaviruses. 19 III. Comparison of Different Picornavirus Genera. 24 A. Disease Aspects 25 B. Serotypes.......... 25 C. Physical Properties...... 25 D. Relative Relatedness to Polioviruses 26 3. Composition and Structure of the Virion 28 I. Introduction... 28 II. Composition and Physical Properties of the Virion. 30 III. Overall Architecture of the Viral Capsid 38 A. Building Blocks........ 38 1. Functional Requirements.... 38 2. Biological Arguments of Building Block Economy and Efficiency of Assembly............ 38 3. Thermodynamic Forces and Structural Consequences 38 B. The Geometric Design: The Icosahedron 39 1. Helical Tubes Versus Spherical Shells...... 39

VIII 2. Cubic Symmetries, the Platonic Polyhedra 39 3. The Icosahedron.......... 40 a) Advantages of Icosahedral Symmetry. 40 b) Limitations of the Icosahedral Skeleton as a Model for Virus Structure.............. 41 c) Related Regular Polyhedra with Icosahedral Symmetry 42 d) The Bonding Pattern of an Icosahedral Lattice... 42 C. Experimental Evidence that the Poliovirus Capsid Is an Icosahedral Lattice........ 44 1. X-Ray Diffraction Studies......... 45 2. Electron Microscopic Observations..... 45 3. Resolving the 32 or 60 Capsomers Controversy 47 4. Similar Construction Principles for Picornaviruses and the Small Plant Viruses.............. 47 IV. Characterization of the Building Blocks: The Capsid Proteins. 50 A. Separation and Identification.... 50 B. Amino Acid Composition and Sequence....... 53 C. Microheterogeneity............... 59 V. Relative Localization of the Viral Proteins in the Capsid and Bonds Involved...... 59 A. General Reflections............... 59 1. Geometric Restrictions............. 60 2. Structural Principles Borrowed from Plant Picornaviruses 64 3. Implications for the Capsid Features of Poliovirus: The Concept of a Rigid Capsid Backbone and Variable Surface Protrusions............... 69 B. Experimental Results............ 71 1. Specific Chemical Modifications of Capsid Proteins 71 2. Chemical Crosslinking of Capsid Proteins. 75 3. UV Irradiation of Poliovirus....... 78 VI. The Dissociation of Poliovirus........ 79 A. Breakdown During Preparatory Procedures for Electron Microscopy.... 82 B. Alkaline Degradation 84 C. Heat Degradation.. 86 D. Guanidine Degradation. 89 E. Urea Degradation... 89 F. Reassociation of Poliovirus from Products of Urea Degradation................ 93 G. Conclusions.................. 93 VII. Conformational Forms of the Poliovirus Capsid...... 94 A. Poliovirus Capsid Structures During Early Interactions with the Host Cell and upon Experimentally Induced Disruption: Two Conformational States of the Shell 95 B. Viral Structures During Maturation......... 97

IX C. Two Distinct, Reversibly Interconvertible Conformational States of Intact Poliovirions............ 99 D. Conformational Alterations Monitored by Changes in the Intrinsic and Induced Fluorescence of Poliovirus Components................... 103 E. Dense Particles................. 106 F. Possible Functions of the Alternate Conformational States of the Poliovirus Capsid............. 108 VIII. Antigenicity................... 114 A. The Main Poliovirus Antigenic Sites: The D- and C-Antigenic States.................... 118 1. Attempts to IdentifY the C- and D-Antigenic Determinants in Terms of the Constituent Capsid Proteins with Polyclonal Antisera.......... 121 2. Studies with Monoclonal Antibodies.. 126 B. Other, Minor Antigenic Sites on Poliovirus 128 C. Serodifferentiation of Polioviruses. 132 IX. Summary... 132 A. Composition 132 B. Structure.. 133 C. The Capsid Proteins. 133 1. Number of Peptides and Type of Association on the Structure Units 133 2. Relative Localizations.... 134 3. Heterogeneity....... 135 D. Two Conformational States of the Capsid. 135 E. Antigenicity....... 136 4. Structure and Function of the Genome I. Introduction... II. Genome Structure.... A. Characteristic Features. B. Structural Organization C. Secondary Structure.. D. The Genome-Linked Protein VPg. III. Nucleotide Sequences.... A. Oligonucleotide Mapping.... B. Cloning of Poliovirus cdna C. The Consensus Sequence of Poliovirus Type I D. Terminal Sequences. 1. 5' End Sequences 2. 3' End Sequences 3. The Poly (A) Tract E. Features of the Coding Region. 1. Codon Usage...... 2. Cleavage Signals for Proteolytic Processing 138 138 139 139 140 142 143 146 146 150 150 161 161 165 166 167 167 167

x IV. Genome Products and Gene Order. A. Mapping of the Genome Products on the Polioviral Genome.... 1. Relatedness of Viral Proteins: Tryptic Peptide Analysis 2. Biochemical Mapping............ 3. The Genome Map as Deduced from Genetic Studies B. Function of the Viral Proteins.......... 1. The P-1 Proteins: Coat-Proteins, Proteinkinase, Shut-Off of Protein Synthesis............... 2. The P-2 Proteins: Guanidine Sensitivity, VPO Protease. 3. The P-3 Proteins: glu/gly Protease, VPg Replicase, Cytophathic Effect...... V. Genomic Variation of Polioviruses. VI. Genetics... A. Mutations........ B. Mutant Types....... 1. Temperature Sensitive Mutants. 2. Structural Markers.. 3. Non Structural Markers. C. Genetic Recombination D. Mechanism of Recombination VII. Summary........ 171 175 176 177 178 178 180 180 181 182 188 188 191 191 192 195 195 199 200 Part II: The Replication of Poliovirus 203 5. Introduction......... I. The "Life-Cycle" of Poliovirus II. Timecourse of Poliovirus Replication III. The Host Cell..... A. Constituents of the Cell B. The Nucleus.... C. The Plasma Membrane. D. The Ionic Environment E. The Cytoskeleton... F. Intracellular Membranes G. The Cell Cycle H. Employment of the Metabolic Machinery of the Host Cell by the Virus.............. IV. Some Speculations on Abortive Infections of Poliovirus... 203 203 208 212 213 215 215 216 218 220 220 221 222 6. Morphological Alterations of the Host Cell as an Essential Basis for Poliovirus Replication..... I. Introduction... II. Microscopic Oberservations A. Light Microscopy.. B. Electron Microscopy.. 226 226 227 228 230

XI III. IV. 1. The Nucleus 230 2. Ribosomes. 235 3. "Viroplasm". 235 4. Alterations of Intracellular Membranes. 237 a) Nuclear "Extrusions"...... 237 b) Membraneous Cisternae..... 240 c) Biochemical Aspects of Membrane Formation 245 5. Changes in the Cytoskeletal Framework. 250 6. Assembly and Release of Progeny Virions. 250 7. Lysosomes and Autophagic Vesicles... 252 8. Inhibitors of Morphological Alterations 253 Speculations on the Function of Compartmentalization in Virion RNA Synthesis and Assembly............ 256 A. Concerning the Mode of Vesicle Formation...... 256 B. Concerning the Relative Localization of RNA Synthesis and Virion Assembly with Respect to the Vesicle Membrane 258 Poliovirus induced Alterations in Functions of the Plasma Membrane, in the Intracellular Ionic Environment, and in Cell Size...................... 262 A. Membrane Changes Accompanying Adsorption and Penetration of Poliovirus................ 262 B. Membrane Alterations Accompanying Virus Replication at the Maximal Rate 264 V. Summary... 265 7. Early Interactions of Virus and Host Cell 267 I. Introduction... 267 A. Overview and Definition of Terms 267 B. The Superposition of Abortive and Productive Pathways in Infection...... 270 II. Adsorption and Attachment 271 A. The Reaction Partners. 272 1. The Virus Particle 272 2. The Virus Receptor Complex on the Host Cell 275 a) Properties of the Poliovirus Receptor.. 276 b) Other Functions and Components of the Receptor Complex............. 278 c) Number of Attachment Sites/Cell.... 278 d) Specificity and Genetics of Virus Receptors.... 279 B. The Interaction of Poliovirions with the Host Cell Membrane.... 280 1. Adsorption. 282 2. Attachment. 285

XII III. 3. Response of the Plasma Membrane..... 286 a) Changes in Membrane Fluidity and Capping of Viruses............. 286 b) Changes in Membrane Permeability and Membrane Potential........... 288 c) Interaction with Modifying and Stabilizing Membrane Components............. 288 Penetration of Virus Particles into the Cell: Insertion and Phagocytosis............. 291 IV. Uncoating: A Multistep Process..... 297 A. Possible Steps and Sites of Uncoating. 297 B. The Fate of the Parental Capsid Proteins 300 V. Infection of Cells Lacking Receptors. 301 A. Introduction........ 301 B. Adsorption of Viral RNA to Cells 302 C. Penetration of Isolated Viral RNA into Cells. 304 1. RNA-Penetration by Passive Influx of RNA 304 2. Stimulation of Active Uptake of Viral RNA. 304 3. Entrance of Poliovirus RNA into Cells Via Lipid Vesicles (Liposomes).............. 305 D. Cellular Competence for Infection by Viral RNA.... 305 1. Optimal Conditions for the Use of Polycations.... 306 2. Relationship Between RNA Concentration and Yield of Infections Centers.............. 307 3. The Combined Effect of Dimethylsulfoxide and DEAE Dextran on the Competence of Cells for Infection by Viral RNA................. 308 4. Competence of HeLa Cells for Infection by Viral RNA at Different Stages in a Cell Growth Cycle 309 E. Conclusions. 309 VI. Summary....... 310 8. Translation of the Viral Genome. 313 I. Mammalian Protein Synthesis.... A. The Protein Synthesizing Machinery. 1. Ribosomes........ a) Structure and Composition.. b) Monosomes and Polysomes.. c) Free and Membrane Bound Polysomes. 2. mrna.... a) The Cap............ b) The 5' Terminal Untranslated Region. c) The 3' Terminal Untranslated Region and the A Tract............... Poly 313 313 314 314 314 315 315 315 317 319

XIII d) Monocistronic mrnas and Potential Internal Initiation Sites............. 320 e) mrnps.............. 320 3. Initiation Factors and the Process of Initiation. 322 4. Elongation and Termination of Translation 325 5. Cotranslational Processing and Membrane Insertion of Nascent Polypeptide Chains... 326 B. The Regulation of Protein Synthesis. 327 1. The Role of Culture Conditions. 328 2. Competition Between mrnas.. 331 a) The Role of mrna Concentration 331 b) Relative Translational Efficiencies of mrnas 331 c) The Role of Limiting Initiation Components 332 3. Alteration or Inactivation of the Cap Binding Protein and Other Initiation Factors.. 333 4. Modification of Ribosomes........... 334 5. The Role of Uncharged trna.......... 334 6. Transfer of mrnas Between Untranslatable and Translatable Pools... 335 7. Control of Free and Membrane Bound Pools of Ribosomes.......... 336 II. Translation of the Poliovirus Genome...... 337 A. Overview and Introduction......... 337 B. Translation of Poliovirus RNA in Cell Free Extracts 342 1. General Comments....... 342 2. Initiation of in vitro Translation...... 343 3. In vitro Elongation and Termination.... 344 C. The Additional Complexity of in vivo Translation During Infection................... 344 1. The Shut-Off Phenomenon........... 345 a) The Activation of an Inherent Host-Cell Regulatory Mechanism................ 346 b) Competition Between Viral and Host Cell mrnas. 348 c) The Role of Ionic Disturbances and Membrane Leakiness................ 350 d) Alterations of Initiation Factors or Ribosomes... 351 e) The Role of Virus-Specific Factors in Mediating the Shut-Off................ 353 f) In Summary, a Concert of Mechanisms with a Purpose................ 355 2. Non-Uniform Synthesis of Viral Proteins..... 356 3. The Role of Interaction Between Poliovirus Proteins and Intracellular Membranes.. 358 4. Distribution of Viral Proteins 359 5. Protein Processing...362 a) The Role of Cleavage.. 362

XIV b) Types of Cleavages....... 362 c) Types of Proteases....... 363 d) Role of Cleavage in RNA-Replication 366 e) Interference with Protein Processing. 366 f) The Effect of Guanidine on the Processing of Polioviral Proteins 367 III. Summary...... 369 9. Replication of the Viral RNA. 372 I. Introduction... 372 II. Isolation and Characterization of Virus Specific RNAs Isolated from Infected Cells............... 377 A. Preparation and Purification of Poliovirus Specific RNAs 377 B. Properties of Poliovirus Specific RNAs 379 1. The Single-Stranded Viral RNA 379 2. The Replicative Form-RNA... 380 3. The Replicative Intermediate-RNA 382 4. Double Stranded Forms of RNA-Extraction Artefacts?. 386 III. Time Course and Kinetics of Synthesis of Virus Specific RNAs.................. 386 A. The Onset of RNA Synthesis........ 389 B. The Exponential Phase: crna -+ mrna -+ crna. 391 C. The Linear Phase: crna -+ vrna, mrna 392 D. Cessation of RNA Synthesis. 395 IV. The Sites of RNA Synthesis....... 395 V. The Viral RNA-Polymerases....... 397 A. The Crude Replication Complex: Synthesis of Plus Strand RNA.................. 399 B. Soluble Replicase(s): Synthesis of Minus Strand RNA 402 VI. The Effects of Guanidine on Poliovirus Replication 404 VII. Some Thoughts on the Mode of RNA Replication. 408 A. Initiation of Viral RNA Synthesis.. 409 B. Elongation of Viral RNA Replication 412 C. Inhibition of Host Nuclear Functions 414 D. On the Infectivity of RF-RNA.. 415 E. Regulation of Viral RNA Synthesis 416 VIII. Summary... 416 10. Assembly of the Virion 421 I. The Cytoplasmic Sites of Assembly-Virus-Induced Intracellular Membranes.......... 421 A. Electron Microscopic Observations......... 421 B. Biochemical Approaches............. 426 II. Subviral Particles in the Infected Cell-Potential Assembly Intermedia tes. A. Overview.. 427 427

XV B. NCVP1a and the 5S Protomer 430 C. The 14S Pentamer 430 D. The 55S Particle.... 432 E. The 80S Shell..... 433 F. Ribonucleoprotein Particles 436 1. The Slow Sedimenting (80S) RNPs 436 2. The 125S and 150S Provirion(s).. 440 3. Association of RNPs with the Replication Complex in Smooth Membranes.............. 441 III. Assembly Kinetics................. 443 A. Chasing of Radioactive Precursors: Nucleosides and Amino Acids................ 443 B. Assembly of Isolated Subviral Particles in vitro..... 445 1. Self Assembly of Isolated Subunits........ 445 2. Assembly-Enhancing Activity in Extracts of Infected Cells.............. 447 C. Studies with Inhibitors of Assembly.... 448 1. Reversible Inhibition of Assembly by Py-ll 449 2. Studies with the Assembly Inhibitor Guanidine. 450 3. Inhibition of Poliovirus Maturation Under Hypotonic Culture Conditions.... 452 4. Assembly Defective Mutants 452 IV. The Individual Steps of Assembly. 454 A. Principles of Assembly.... 454 B. Formation, Activation, and Assembly of the 5S Protomer. 457 C. Activation and Assembly of the 14S Pentamer 459 D. Encapsidation of the Viral RNA. 462 1. Condensation of the Viral RNA. 463 2. Formation of the RNP..... 463 a) Assembly Around an RNP Core 464 b) Insertion of RNA into a Procapsid 464 3. Stepwise Condensation of the RNPs. 466 4. The Possible Role of Mg++.. 466 E. The Final Morphogenetic Cleavage 466 V S~~ry 4~ 11. Conclusions. Appendix I: Laboratories Engaged in Poliovirus Research Appendix II: Poliovirus Models......... A. A Paper Model of a Prototype Picornavirus. B. The Apple Model of Poliovirus..... 472 476 484 484 486

XVI Appendix III: The Geometry of Isometric Polyhedra. 488 488 488 488 490 492 A. The Platonic Polyhedra a) Models... b) Characteristics.. c) Duality..... d) The Golden Proportion. e) Geometric Restriction of the Maximal Number Subunits....... B. Other Icosahedron-Related Polyhedra... a) Characteristics.......... of 492 497 497 b) The Triangulation-Number-Classification of Icosahedral Lattices....... 499 Appendix IV: Complete Nucleotide and Amino Acid Sequences of Poliovirus Type 1, 2 and 3 502 References.. 509 Subject Index 572