Viruses: From Understanding to Investigation PDF

Viruses From Understanding to Investigation Susan Payne Department of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, United States Table of Contents Cover image Title page Copyright About the Author Preface Acknowledgments Chapter 1. Introduction to Animal Viruses Abstract What Is a Virus? Diversity in the World of Viruses Are Viruses Alive? Basic Steps in the Virus Replication-Cycle Growing Viruses Categorizing Viruses (Taxonomy) Outcomes of Viral Infection Introduction to Viral Pathogenesis Introduction to Virus Transmission Chapter 2. Virus Structure Abstract Anatomy of a Virus Capsid Structure and Function Capsids Are Built From Many Copies of One or a Few Types of Protein Simple Icosahedral Capsids Larger Icosahedral Capsids Viral Envelopes Other Membrane Proteins Matrix Proteins Nucleocapsid Structure Chapter 3. Virus Interactions With the Cell Abstract Virus Interactions with the Cell Cytoskeleton Virus Assembly Virion Release Virion Maturation Amplification of Viral Proteins and Nucleic Acids in the Context of the Infected Cell Chapter 4. Methods to Study Viruses Abstract Growing Viruses Purifying Viruses Visualizing Viruses Counting Viruses Basic Principles of Diagnostic Virology References Chapter 5. Virus Transmission and Epidemiology Abstract Methods to Count Viral Infections and Disease Infection Versus Disease Developing Models of Virus Transmission Incubation, Latent, and Infectious Periods Virus Transmission Chapter 6. Immunity and Resistance to Viruses Abstract Innate Immunity Intrinsic Immunity Specific Immunity Viruses Fight Back Chapter 7. Viral Vaccines Abstract Classical versus Engineered Vaccines Vaccines Provide a Source of Antigens Replicating versus Inactivated Vaccines Killed, Inactivated, and Subunit Vaccines Viral Vaccines that contain Infectious Agents Antigen Recognition Correlates of Protection Vaccine Development A Final Word about Safety Chapter 8. Virus Evolution and Genetics Abstract Virus Evolution Virus Genetics Chapter 9. Viral Pathogenesis Abstract Natural Barriers to Infection Primary Replication Movement to Secondary Replication Sites Genesis of Disease Immunopathogenesis Virulence Approaches to Study Viral Pathogenesis Chapter 10. Introduction to RNA Viruses Abstract Definition and Basic Properties of RNA Viruses Subgroups of RNA Viruses Structural Features of RNA Virus Genomes Priming Viral RNA Synthesis Mechanisms to Generate Capped mRNAs Mechanisms to Generate Polyadenylated mRNAs Mechanisms to Regulate Synthesis of Genomes and Transcripts RNA Viruses and Quasispecies References Chapter 11. Family Picornaviridae Abstract Genome Organization Virion Structure Picornavirus Replication (Poliovirus Model) Picornaviruses and Disease Chapter 12. Family Caliciviridae Abstract Genome Organization Virion Structure General Replication Cycle Diseases Chapter 13. Family Hepeviridae Abstract Genome Organization Virion Morphology General Replication Cycle of HEV Disease Chapter 14. Family Astroviridae Abstract Genome Organization Virion Morphology Diseases Caused by Astroviruses References Chapter 15. Family Flaviviridae Abstract Genome Structure/Organization Virion Structure General Overview of Replication Protein Products Diseases References Chapter 16. Family Togaviridae Abstract Genome Structure Virion Structure Togavirus Replication Transmission Alphaviruses Associated With Rash and Arthritis Alphaviruses of Veterinary Importance References Chapter 17. Family Coronaviridae Abstract Coronavirus Genome Organization Virion Structure Coronavirus Structural Proteins Coronavirus Replication Cycle Diseases Caused by Coronaviruses References Chapter 18. Family Arteriviridae Abstract Genome Organization Virion Structure Replication Cycle Diseases Caused by Arteriviruses References Chapter 19. Family Rhabdoviridae Abstract Genome Organization Virus Structure Overview of Replication Diseases Chapter 20. Families Paramyxoviridae and Pneumoviridae Abstract Genome Structure/Organization Virion Structure Overview of Replication Diseases Chapter 21. Family Filoviridae Abstract Genome Organization Virion Structure Overview of Replication Disease Chapter 22. Family Bornaviridae Abstract Genome Organization Virion Structure Overview of Replication Diseases Chapter 23. Family Orthomyxoviridae Abstract Genome Structure/Organization Virion Structure Classification and Nomenclature of Orthomyxoviruses Overview of Replication Antigenic Drift and Antigenic Shift Disease Antiviral Drugs Vaccines References Chapter 24. Family Bunyaviridae Abstract Genome Organization Virion Structure General Replication Strategy Diseases Chapter 25. Family Arenaviridae Abstract Genome Organization Virion Structure Overview of Replication Diseases Caused by Arenaviruses Chapter 26. Family Reoviridae Abstract Genome Organization Virus Structure Overview of Replication Diseases Chapter 27. Family Birnaviridae Abstract Genome Structure Virion Structure Replication Cycle Disease Chapter 28. Introduction to DNA Viruses Abstract Definition and Basic Properties of DNA Viruses Viral DNA Replication DNA Viruses and Cell Cycle Oncogenesis Chapter 29. Family Parvoviridae Abstract Genome Organization Virion Structure Overview of Replication Diseases Chapter 30. Other Small DNA Viruses Abstract Family Circoviridae Diseases Family Anelloviridae References Chapter 31. Family Polyomaviridae Abstract Genome Structure Virion Structure Replication Transformation Human Polyomaviruses and Disease Chapter 32. Family Papillomaviridae Abstract Genome Structure Virion Structure Replication Cycle Disease Animal Papillomaviruses Chapter 33. Family Adenoviridae Abstract Genome Organization Virion Structure Adenovirus Replication Cycle Adenoviruses and Human Disease Adenoviruses and Transformation Oncolytic Adenoviruses Adenoviral Vectors Chapter 34. Family Herpesviridae Abstract Genome Organization Virus Structure Replication Cycle Diseases Caused by Human Herpesviruses Antiherpesviral Drugs References Chapter 35. Family Poxviridae Abstract Genome Organization Virion Structure Replication Diseases Rabbit Myxoma Virus References Chapter 36. Family Retroviridae Abstract Virion Structure Genome Organization Overview of the Retroviral Replication Cycle The Process of Reverse Transcription Integration Transcription of Retroviral mRNA Structural Proteins Assembly, Release, and Maturation Mechanisms of Retroviral Oncogenesis Chapter 37. Replication and Pathogenesis of Human Immunodeﬁciency Virus Abstract History of HIV and Acquired Immunodeficiency Syndrome HIV is a Lentivirus HIV Proteins TAT REV Other HIV-1 Proteins Summary of HIV Replication Overview of HIV Pathogenesis HIV Transmission Disease Course Antiretroviral Drugs Vaccines Chapter 38. Family Hepadnaviridae Abstract Genome Organization Virion Structure Replication Cycle Hepatitis B Virus and Disease Index Copyright Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1800, San Diego, CA 92101-4495, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2017 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmi ed in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. 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British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-803109-4 For Information on all Academic Press publications visit our website at h ps://www.elsevier.com/books-and-journals Publisher: Mica Haley Acquisitions Editor: Linda Versteeg-Buschman Editorial Project Manager: Fenton Coulthurst Production Project Manager: Chris Wortley Designer: Mark Rogers Typeset by MPS Limited, Chennai, India Illustrations: Illustrations for this book were provided by Marcy Edelstein, to whom the publisher would like to extend their thanks. About the Author Dr. Susan Payne is an associate professor in the Department of Veterinary Pathobiology at the Texas A&M University, United States. During her career, she has mentored graduate and undergraduate students at three universities and has taught virology to undergraduate, graduate, medical, and veterinary students. Those courses are the basis for this textbook. She has also had an active research career and has wri en over 40 peer reviewed research and review articles. She serves as an ad hoc reviewer for several virology journals. She currently lives in Caldwell, Texas with her husband, mom, ﬁve cats, one dog, nine goats, one donkey, eight chickens (if the dog has not eaten one recently), and eight guinea fowls. She is most easily available in email at [email protected]. Preface This book, Viruses: From Understanding to Investigation, was inspired by a long career of teaching and research. My students have included undergraduate, graduate, medical, and veterinary students. As regards the book title, my intent is to lead students of virology from a basic understanding to an interest in the investigations that have provided the information contained herein. The focus of this textbook is on animal and human viruses, only because these have been the focus of my research and teaching for many years. The viruses of plants, fungi, bacteria, and single-celled organisms are certainly no less interesting. There is a huge amount of information about viruses available online, in journals, books, websites, and blogs. So why the need for another virology textbook? My intent was to organize and present a thoughtful, understandable, and up-to-date summary of the volumes of information available for consumption elsewhere. While every textbook, including this one, contains many facts, I have tried to emphasize general concepts. With 38 chapters, this book contains more than enough material for a semester long course in introductory virology. The book is geared toward students with some background in cell biology, microbiology, immunology, and/or biochemistry, and I hope that it will be useful for both undergraduate and beginning graduate students. I also hope that no instructor will try to cover all of the material contained herein during a single semester. The book is organized into two parts, the ﬁrst nine chapters cover topics including an introduction to viruses (containing information on replication cycle, diversity, taxonomy, and outcomes of virus infection), structure, interactions with the host cell, methods for studying viruses, immunity to viruses, and introductions to viral epidemiology, evolution, and pathogenesis. There are also chapters that serve as introductions to RNA and DNA viruses. I imagine that this will be more than enough information for many instructors and students. The remaining chapters present viruses by family, with information about structure, genome organization, replication strategies, and disease. I have tried to be up-to-date and include virus families that are relatively new (hence these chapters are short). While each chapter includes basic information about a particular virus family, I am fond of narratives that tie the molecular basis of virus replication to pathogenesis, and have provided examples from a variety of animals, including human animals. The inclusion of “animal diseases” speciﬁcally serves as a reminder that companion and food animals play integral roles in human health and well-being. (As do plant and bacterial viruses, but those are subjects for other authors to address.) I encourage instructors to review the material on virus families and choose a handful of these chapters to use in their courses. Positive-strand RNA viruses are presented ﬁrst followed by negative and dsRNA viruses. The DNA viruses are presented from the smallest to the largest. Last, but certainly not least, are chapters covering the reverse transcribing retroviruses and hepadnaviruses. I have included some taxonomic information in each chapter, sometimes more, sometimes less. I imagine this to be a reference resource and starting point for students who wish to know more. (And I ask my colleagues not to make these boxes a giant exercise in memorization.) Throughout the book, I have included brief discussions of both a historical nature (for example, oncogenic retroviruses and an account of the discovery of hepatitis B virus) and current issues such as the recent initiative of the World Health Organization and the World Organization for Animal Health (OIE) to collaborate to reduce human deaths by rabies virus in underdeveloped countries. In the mix are also topics relevant to basic research such as use of p vesicular stomatitis virus G protein for pseudotyping and lymphocytic choriomeningitis virus (LCMV) as a model for pathogenesis. For instructors and colleagues, a ﬁnal word. You will ﬁnd the depth of coverage somewhat mixed throughout, and I may have neglected your favorite virus or disease. I am also quite sure that I have presented ideas with which you disagree. Share these with your students, start a conversation, and call me out if necessary. My research manuscripts have always been improved by thoughtful criticism, and if this book is to have a life beyond the ﬁrst edition, I expect that the same will be true in this case. Acknowledgments I cannot overstate the contributions of my illustrator, and wonderful sister, Marcy Edelstein. As I expected, she went “above and beyond” in assisting with this project. In addition to creating illustrations, she learned virology and gently pushed this project to completion with constant help and advice. Many thanks are also owed to my husband, Ross Payne, and to my mom for their patience and understanding during this project. I also wish to thank Texas A&M University, United States, for providing an incredible work and learning environment and my virology colleagues at the College of Veterinary Medicine and Biomedical Sciences and the Health Science Center for their support and inspiration. Finally, many thanks to my mentors over the years and to the dedicated and imaginative researchers who work to unravel the complex and beautiful world of viruses. C H AP T E R 1 Introduction to Animal Viruses Abstract Viruses are infectious agents that are not cellular in nature. They consist of a nucleic acid genome packaged within a protein shell. Although relatively simple, viruses exhibit signiﬁcant diversity in terms of size, genome organization, and capsid architecture. All viruses are obligate intracellular parasites as they must obtain energy and building blocks from the cell. They subvert many host cell processes for their own replication, and studying virus replication has provided detailed information about the basic workings of cells. At the cellular level, possible outcomes of infection range from production of virus particles without damage to the cell, to cell death, or occasionally cell transformation. In humans and animals the outcomes of infection range from inapparent (no disease) to considerable disease and death. Some viruses cause acute infections lasting for days or a few weeks while others infect their hosts for a lifetime. Viruses can evolve and rapidly adapt to changing conditions. Some viruses are easily replicated in cultured cells, while others require speciﬁc conditions only found in specialized cells within a human or animal. While this text focuses on viruses of humans and other animals, viruses infect organisms of all types, from bacteria to fungi to plants. Viruses are most often classiﬁed based on groups of genome and virion characteristics. Genome sequence comparisons provide an unbiased method for grouping and categorizing viruses. Keywords Virus; infectious agent; capsid; intracellular parasite; virion; host range; transmission; eclipse phase; one-step growth curve OUTLINE What Is a Virus? 1 Diversity in the World of Viruses 2 Are Viruses Alive? 3 Basic Steps in the Virus Replication-Cycle 5 Growing Viruses 5 Categorizing Viruses (Taxonomy) 6 Outcomes of Viral Infection 8 Introduction to Viral Pathogenesis 10 Introduction to Virus Transmission 10 After studying this chapter, you should be able to: Provide a meaningful deﬁnition of a virus. Explain diﬀerence between cell division and virus replication. Explain the correct usage of “virion” versus “virus.” Describe the basic steps in a virus replication-cycle. Draw, label, and describe each part of a “one-step” growth curve. List possible outcomes of a virus infection (1) at the level of the individual cell and (2) at the level of the host animal. Deﬁne the term “host range” as regards viruses. What Is a Virus? Most of us are familiar with the term virus and know viruses as disease causing agents, transmi ed from one person or animal to another. We are familiar with “cold” and “ﬂu” viruses; we fear a worldwide pandemic of Ebola. We may even be aware that viruses are used to deliver genes to cells for the purposes of gene therapy or genetic engineering. But what are viruses? Viruses are infectious agents that are not cellular in nature. Viruses must enter a living host cell in order to replicate, thus all viruses are obligate intracellular parasites. Synthesis of the proteins and nucleic acids (DNA and RNA) for assembly into new virus particles (virions) requires an energy source (ATP), building materials (amino acids and nucleotides), and protein synthesis machinery (ribosomes) supplied by the host cell. The cell also provides scaﬀolds (microtubules, ﬁlaments, membranes) on which virus particles replicate their genomes and assemble. Thus the cell is a factory providing working machinery and raw materials. The infected cell may or may not continue normal cellular processes (host cell mRNA and protein synthesis) during a viral infection. Viruses have nucleic acid genomes that are surrounded by and protected by protein coats called capsids. Capsids protect genomes from environmental hazards and are needed for eﬃcient delivery of viral genomes into new host cells. Some viruses have lipid membranes, called envelopes that surround the capsid (Fig. 1.1). Viruses are structurally much simpler than cells. Some viruses can be crystalized. Viruses do not increase in number by cell division; instead they assemble from newly synthesized protein and nucleic acid parts (building blocks). As viruses are not cells, they have none the organelles associated with cells. A sample of puriﬁed virions has no metabolic activity. Viruses are packages designed to deliver nucleic acids to cells; they are excellent examples of “selﬁsh genes.” FIGURE 1.1 Basic features of virions. Panel A. On left, simple diagram of an unenveloped virus with icosahedral symmetry; on right, electron micrograph of calicivirus (Chapter 12: Family Caliciviridae). Panel B. On left, simple diagram of enveloped virus with a helical nucleocapsid; on right, electron micrograph of measles virus, a paramyxovirus (Chapter 20: Families Paramyxoviridae and Pneumoviridae). Panel C. On left a simple diagram of an enveloped virus with an icosahedral nucleocapsid; on right, electron micrograph of hepadnavirus (Chapter 38: Family Hepadnaviridae). The preceding description might suggest uninteresting, inanimate particles, but examining virus replication strategies and interactions with host cells provides a diverse and dynamic view into cellular and molecular processes. Viruses are not a homogenous group. They are an extremely diverse group of infectious agents. It is highly unlikely that they arose from a single common ancestor (Box 1.1). Box 1.1 W h a t is in a D e fin it io n ? By the late 19th century, the term “virus” was used to describe infectious agents that could pass through ﬁlters designed to remove bacteria from liquids. Thus viruses were “smaller than bacteria” and size became an important part of the deﬁnition of a virus. Today we know of a few viruses that are larger than many bacteria, so the trend has been to drop “smallness” from the deﬁnition. Another part of the deﬁnition of a virus is that they are all obligate intracellular parasites. This is certainly true of all viruses, but there are also bacteria and protozoan parasites that are obligate intracellular parasites. When the biochemical nature of viruses was discovered, it became clear that viruses lack many of the complex structures common to cells. This resulted in deﬁnitions of viruses based on comparisons to cells. While these comparisons emphasize the many ways that viruses are diﬀerent from cells, they do not help us understand these unique infectious agents. So, what are viruses? Very simply, they are genes packaged within a protein coat. Their replication process begins when the virion delivers its genome to a cell. The viral genome encodes proteins required for the synthesis of new viral genomes. New viral proteins plus new viral genomes assemble to form new particles or virions, and so the cycle continues. Diversity in the World of Viruses All viruses have nucleic acid genomes, but some utilize DNA as genetic material, while others have RNA genomes. Viral genomes are not always double-stranded molecules; there are single-stranded viral RNA and DNA genomes. There are viral genomes that consist of a single molecule of nucleic acid, but some genomes are segmented. For example, reoviruses (Chapter 26: Family Reoviridae) package 11–12 diﬀerent pieces of double- stranded RNA and each genome segment encodes a diﬀerent gene. Some viruses have lipid envelopes in addition to a genome and protein coat. Viral envelopes are not homogenous. Diﬀerent types of host membranes may be utilized, and their speciﬁc lipid and protein components can diﬀer. Viruses range in size from 10 to 1000 nm is size (Fig. 1.2). Viral genomes range in size from 3000 nucleotides (nt) to over 1,000,000 base pairs. Outcomes of viral infections are diverse. Infection does not always result in cell or host death. Some host genes are derived from viruses and have played key roles in evolution. (Some plant viruses are beneﬁcial in extreme environments.) Some viruses complete their replication cycles in minutes while others take days. Some viruses are transiently associated with an infected host (days or weeks) while others (for example, herpesviruses) are life-long residents. Where did viruses come from? Three general scenarios for virus evolution have been proposed: Retrograde evolution: Intracellular parasites lost the ability for independent metabolism keeping only those genes necessary for replication. Poxviruses are very large complex viruses that may have evolved in this manner. Origins from cellular DNA and RNA components: Some DNA genomes resemble plasmids or episomes. Did these DNAs acquire protein coats and the ability to be transferred from cell to cell eﬃciently? Descendants of primitive precellular life forms: Viruses originated and evolved along with primitive, self-replicating molecules. This is the likeliest origin of the RNA viruses described in this text. FIGURE 1.2 Relative sizes of an animal cells and virions. For the most part, names of speciﬁc viruses have been omi ed in this section, to emphasize the general subject of viral diversity. Throughout this text, the details will be forthcoming. But I hope that now, when reading about any virus, you will want to learn its place in the complex world of viruses. (Big? small? friend? foe? transient visitor? life-long partner?) Are Viruses Alive? Viruses parasitize every known form of life on this planet and they have both short-term and long-term impacts on their hosts. But are viruses alive? This question is the subject of ongoing debate, but the answer does not change the nature of the virus. As we discuss and describe viruses it is easy to assume that they are alive. They replicate to increase in number and the terms “virus replication- cycle” and “virus life-cycle” are often used interchangeably. Viruses also evolve (change their genomes), sometimes very rapidly. In this manner they adapt to new hosts and environments. In contrast, the virion (the physical package that we view with an electron microscope) has no metabolic activity. Some viruses can be assembled simply by mixing puriﬁed genomes and proteins in a test tube. The genomes may have been synthesized by machine and the viral proteins may have been produced in bacteria. If those component parts combine under suitable conditions, a fully infectious virion can be produced. To avoid the question of living versus nonliving, the term “infectious agent” is both appropriate and descriptive. We can then speak of infectious virions that are capable of entering a cell and initiating a replication-cycle, or inactivated virions that cannot complete a replication-cycle. As we will see in later chapters, the diﬀerence between an infectious and a noninfectious virion may be as small as the cleavage of a single peptide bond. Basic Steps in the Virus Replication-Cycle The ﬁrst step in a virus life-cycle is a achment (or binding) to the host cell (Figs. 1.3 and 1.4). A achment results from very speciﬁc interactions between viral proteins and molecules on the surface of the host cell. The interactions are usually hydrophobic and ionic, rather than covalent bonds. Thus a achment is inﬂuenced by environmental conditions such as pH and salt concentration. A achment becomes stronger as many copies of a viral surface protein interact with multiple copies of the host cell receptor molecules. FIGURE 1.3 Simple schematic of a eukaryotic cell identifying some major organelles. FIGURE 1.4 The basic virus life-cycle is shown in a generic cells. (For simplicity no cell organelles are shown but the processes of virus replication are intimately associated with cell organelles and structures.) The basic virus life-cycle begins with: (1) Attachment of the virion to receptors on a cell. (2) The genome is delivered into cytoplasm (penetration). (3) Viral proteins and nucleic acids are synthesized (amplification). (4) Genomes and proteins assemble to form new virions. (5) Virions are released from the cell. The next step in the virus life-cycle is penetration of the viral genome into the host cell cytoplasm or nucleoplasm. After penetration, there may be a further rearrangement of viral proteins to release the viral genome, a process called uncoating. Penetration and uncoating are two distinct steps for some viruses while for others the viral genome is uncoated during the process of penetration. The processes of penetration and uncoating are irreversible, the infecting virion cannot reassemble. The next phase in the virus life-cycle is synthesis of the new viral proteins and genomes. This is a complex process that requires transcription (synthesis of mRNA), translation (protein synthesis), and genome replication to generate the parts that will assemble into new virions. Synthesis of viral proteins and genomes occurs in close association with, and depends upon, many host cell proteins and structures. The great diversity among viruses will be evident as we examine processes that regulate transcription, translation, genome replication and the speciﬁc virus–host cell interactions that shape these processes. The next step in the virus replication-cycle is assembly of new virions. New particles assemble from the genome and protein components that accumulate in the infected cell. Viruses are assembled at diﬀerent sites in host cells; sometime large areas of the cell become virus factories, concentrated regions of viral proteins and genomes from which host cell organelles are excluded. The ﬁnal step(s) in the virus replication-cycle are release from the host cell and maturation of the released virions. Virion release may occur upon cell rupture or lysis. Enveloped viruses must acquire their envelopes from cellular membranes in a process called budding. Some enveloped viruses bud through the plasma membrane, but budding can occur at other, intracellular membranes. The budding process can, but does not always, kill the host cell. Other viruses obtain their lipid enveloped by budding into cellular vesicles. These vesicles then fuse with the plasma membrane to release the virions; this is process called exocytosis. Maturation is the term used to describe changes in virus structure that occur after a virus is released from the host cell. Maturation may be required before a virus is able to infect a new cell; maturation may involve cleavage or rearrangement of viral proteins. Viruses assemble in the cell (under conditions of favorable energy) but when the released virions encounter new cells they must be able to disassemble (uncoating). Maturation events that occur after virus release set the stage for a productive g p encounter with the next cell. Maturation processes are well understood for several important animal viruses and examples will be presented in future chapters. It is important to stress that each step in the virus replication- cycle requires speciﬁc interactions between viral proteins and host cell proteins. Some viruses can infect many diﬀerent cell types and organisms because they interact with proteins found on, and in, many cell types. These viruses are said to have a broad host range. Other viruses have a very narrow host range due to their need to interact with speciﬁc cellular proteins that are expressed only in a few cell types. Factors that impact virus replication include the presence or absence of receptors, the metabolic state of the cell, the presence or absence of any number of intracellular proteins required to complete the virus replication-cycle. Another way to view the replication-cycle of a virus is the one- step growth curve (Fig. 1.5). This graph illustrates the concept that penetration of a virus into the host cell is not reversible. During the so-called eclipse phase infectious virions cannot be detected, even if cells are broken open (lysed), there are no infectious particles to be found! FIGURE 1.5 One-step virus growth curve. The red curve represents infectious virions released from the infected cells. The blue curve represents infectious virions released if the cells are lysed. Key to understanding the one-step growth curve is to note that after attachment, the number of virions detected in media and within cells decreases. These virions have penetrated cells and their genomes have uncoated, thus they are no longer “infectious.” New virions are detected only after amplification and assembly. Growing Viruses Viruses are obligate intracellular parasites; they replicate only within living cells. Thus in the laboratory, susceptible cells or organisms are required to study virus replication. For the virologist, ideal host cells are easily grown and maintained in the laboratory. Animal virologists often use cell and (less often) organ cultures. To culture animal cells, tissues or organs are harvested and disrupted (using mechanical and enzymatic methods) to obtain individual cells. Often cells are derived from tumors that grow robustly in culture. Cells circulating in the blood, such as lymphocytes, can be obtained directly from animal blood samples. If cells are provided with the appropriate environment (growth media, temperature, pH, and CO2), they will remain metabolically active and may undergo cell divisions. Cell cultures will be described in more detail in a later chapter (Box 1.2). Box 1.2 Pe rm issiv e o r N o t ? Some viruses replicate very poorly when ﬁrst introduced into cultured cells. There may be no visible signs of virus infection, but upon prolonged incubation or “blind passage” (often over a period of weeks or months) the virus will adapt to the new environment. This “cell culture adapted” virus now grows well in cultured cells. Therefore the initial virus infection was permissive, although very poorly so. After becoming adapted to cell culture conditions, the virus may be a enuated (replicate poorly or become incapable of causing disease) in the animal host. Often the best-studied viruses are those that have been adapted for robust growth in a culture system. However, cell or organ cultures may be very diﬀerent from the natural environment of the human or animal host. The biggest diﬀerence is that the cultured cells lack the many antiviral defenses encountered in an organism. Thus it is not uncommon for a virus highly adapted to cell cultures to perform poorly when used to infect an animal. In fact, propagation in culture is a common method for producing a enuated (weakened) live viral vaccines. A enuated viruses replicate in a host, but do not cause disease. When considering experiments with viruses, it is very important to understand both the host system and the origins of the virus being studied. Categorizing Viruses (Taxonomy) The most widely accepted method to group viruses is by the type of nucleic acid (RNA or DNA) that serves as the viral genome. Within this scheme, there are three overarching groups of viruses: DNA viruses: Package DNA genomes synthesized by a DNA- dependent DNA polymerase. RNA viruses: Package RNA genomes synthesized by an RNA- dependent RNA polymerase (RdRp). The third group of viruses uses the enzyme reverse transcriptase (RT) during the replication-cycle. RT is an RNA-dependent DNA polymerase as it synthesizes a DNA copy of an RNA molecule. Reverse transcribing viruses (examples are the retroviruses and hepadnaviruses) use both RNA and DNA versions of their genomes (at diﬀerent times) during their replication cycles. The DNA and RNA viruses are further diﬀerentiated by the physical makeup of their genomes (single stranded, double stranded, unsegmented, segmented, linear, circular). The importance of genome type, and how it inﬂuences virus replication will be covered in upcoming chapters. In addition to genome type, other physical traits are used to subdivide viruses into smaller groups. Some viruses have lipid envelopes (enveloped viruses) while others do not (naked viruses). Capsids also come in diﬀerent shapes and sizes. The goal of viral taxonomy is to categorize viruses using groups of traits. Borrowing nomenclature from the Linnaean classiﬁcation system, viruses are grouped into orders, families, genera, and species (Fig. 1.6). Orders contain two or more related families, and families can be subdivided into multiple genera. A genus is further subdivided into species (or strains). The family is often called the fundamental unit of viral taxonomy. Viruses in the same family are considerably more closely related than viruses from diﬀerent families. Placement of viruses into families is accomplished by examining shared characteristics such as genome type, presence or absence of an envelope, shape of the capsid, arrangement of genes on the viral genome, etc. All viruses within a family share a core set of properties. Thus, if one knows the major characteristics of any single member of the family Picornaviridae (for example, poliovirus), one know the genome type, general genome p g yp g g organization, approximate size, and shape of all picornaviruses. One needs only to learn the characteristics of a handful of virus families, rather than thousands of individual viruses. FIGURE 1.6 Viral taxonomy is based on groups of characteristics such as genome type, genome organization, capsid structure, presence/absence of an envelope. The virus family is often considered the focal point of virus taxonomy. Viruses in a family share genome type, overall genome organization, size, and shape. Related families can be group into orders. Families are also subdivided into smaller groups of more closely related viruses (genera) within the family. A genus can contain a number of different species or strains. These may differ by up to 10% at the nucleotide sequence level. Closely related strains may sometimes be quite phenotypically distinct. Viral taxonomy is determined by groups of expert virologists from around the world who volunteer to serve on the International Commi ee on the Taxonomy of Viruses (ICTV). Visit the ICTV website at h p://ictvonline.org/virusTaxonomy.asp to ﬁnd the most recent virus classiﬁcation schemes. The site also provides a helpful history of virus names. Before it was possible to generate genome sequences quickly and cheaply, classifying viruses was often done using phenotypic traits such as host range, or tissue tropism. Now it is standard practice to use genome sequences to categorize or classify viruses. Genome sequences provide detailed and objective criteria to subdivide viruses into related groups. Genome sequences from many diﬀerent viruses can be compared to generate phylogenies that provide a visual “map” of relationships among viruses (Fig. 1.7). In some cases, many thousands of viral genome sequences are compared in order to generate detailed phylogenies. Such is the case with human immunodeﬁciency viruses (HIV). FIGURE 1.7 Phylogeny of the family Picornaviridae showing recognized genera. In some cases a genus contains only one virus isolate or strain. Phan, T. G., Kapus ins zky, B., Wang, C., Ros e, R., Lipton, H., E. Delaware. 2011. The Fecal Flora of Wild Rodents. PLOS Pathogen 7:e1002218. The recent explosion viral in genome sequence data has necessitated extensive taxonomic changes in some virus families. For example, until recently the site of infection (respiratory versus enteric) was used as a criterion to deﬁne genera within the family Picornaviridae. However a phylogeny based on genome sequences does not split the picornaviruses cleanly along these lines. Thus the family Picornaviridae still contains the genus Enterovirus, but there is no longer a genus Rhinovirus, although you will see frequent reference to it in older literature. Alternatives to ICTV taxonomy are sometimes used to group viruses that share common phenotypic characteristics. Hepatitis viruses are so named because they share the phenotype of replicating in the liver. However the hepatitis B virus (HBV) and the hepatitis C virus (HCV) are not related, either structurally or genetically, and vaccines and antiviral treatments developed for HCV are not eﬀective for treating, or preventing, HBV infection. Another common phenotypic grouping is use of the term arbovirus (meaning arthropod-borne virus) to describe viruses that are transmi ed by insects. Members of many diﬀerent virus families can properly be called arboviruses; the term does not imply genetic relatedness among the diverse members of this “group”. You might ask if it is useful to generate or understand, phylogenies of viruses. The answer is a resounding yes. For example, the origins of a disease outbreak can be determined using detailed genetic information. Information from genome sequencing can be used to analyze past outbreaks and track the transmission of viruses from one person or animal to another in order to determine the best methods to curb virus transmission during an epidemic. Outcomes of Viral Infection Virus infection impacts individual cells, and these cellular changes may or may not noticeably inﬂuence the health and ﬁtness of the organism. There are four general outcomes when a virus encounters a cell: Productive or permissive infection. Viral proteins and nucleic acids are synthesized and virions are assembled and released. Nonpermissive infection. The cell is completely resistant to infection. Abortive or nonproductive infection. The virus enters the cell, but replication becomes irreversibly blocked at some step before particles are produced. Latent infection. Describes a situation where a viral genome is present in the cell, but no or only a few viral proteins are produced. Latency implies that the virus can productively replicate given the right conditions (Box 1.3). Box 1.3 L a t e n t Ve rsu s C h ro n ic In fe ct io n s: W h e re Is t h e Bo u n d a ry ? A latent infection is one in which viral genomes are present in cells but virions are not produced. The term chronic infection describes one where virions can be routinely detected. Thus the sensitivity of the assays used for virus detection becomes an important factor in the distinction. As virus detection methods become more sensitive, the distinction between latency and chronic infection has become blurred. Consider genital herpes, caused by human herpesviruses 1 and 2 (HHV1 and 2). These viruses are abundant in visible lesions but also can be transmi ed when there are no visible lesions. So is the infection latent or is it chronic? How often are the HHVs found on the skin in the absence of lesions? How often must a latent virus reactivate before it is considered chronic? From a public health standpoint calling genital herpes, a chronic infection might be er convey the fact that herpesvirus can be transmi ed in the absence of lesions. Both productive and nonproductive infections can impact the cell. The eﬀects of infection can range from no apparent change, to cell death, to transformation (immortalization). Productive infection often results in cell death (lytic or cytopathic infection), but this is not always the case. Some viruses can replicate without damaging the cell, resulting in an inapparent infection. Viruses that cause inapparent infections are often produced in small amounts for the life of the cell. Sometimes an inapparent infection results from latency. A much less frequent outcome of infection is transformation or immortalization that allows the cell to divide without restriction. Immortalized cells may be productively infected (virus is released) or the condition may result from a nonproductive infection. In the preceding paragraphs we learned that cells can be inapparently infected by a virus. Inapparent infection also occurs at the level of the animal host. Some viruses replicate in hosts without causing disease. After all, the “job” of a virus is replicate and infect another host; disease is not a required side eﬀect. Until very recently it was hard to ﬁnd viruses that caused inapparent infections. But many inapparent infections are now being identiﬁed through large-scale sequencing of host nucleic acids. (Methods for virus detection and discovery will be discussed in a later chapter). Disease is the result of damage to tissues or organs. Many viral infections cause disease, and diseases can be described as acute, chronic, or latent (Fig. 1.8). Acute disease has a rapid onset, lasts from days to months, and the virus is either controlled or cleared, or causes death of the host. There are many examples of acute viral infections, the common cold being one. From a public health standpoint, it is important to know that virus replication and spread may begin well before symptoms develop and virus may be shed for days or weeks after symptoms have resolved. The peak of clinical signs and symptoms may or may not correspond to peak virus titers, or the time of maximum transmissibility. FIGURE 1.8 Outcomes of viral infection at the level of the animal host are quite variable. The red areas under the curves depict periods of clinical disease. In the examples depicted here, virus shedding begins before the onset of symptoms and ends after symptoms have resolved. Note that periods of virus shedding vary. Shedding may begin at the time of onset of clinical symptoms and may end prior to the resolution of disease. During latent infection, there may be intermittent virus shedding without clinical symptoms. Chronic viral infections have a slower progression and the time to resolution is years to a lifetime. These viral infections may, but do not always, lead to death of the host. Chronic infections are also called persistent infections. Virus is produced and shed continuously (albeit sometimes at very low levels). Examples of viruses that may cause chronic or persistent infections of humans are hepatitis C virus (HCV), hepatitis B virus (HBV), and human immunodeﬁciency virus (HIV). It should be noted that a chronic viral infection can be without symptoms (inapparent) for years. Latent infection describes the maintenance of a viral genome without the production of detectable virus. Herpesviruses are a good example of viruses that cause latent infections. The chickenpox/shingles virus, formerly known as varicella-zoster virus, but recently renamed human herpesvirus 3 (HHV3) is an instructive example. Prior to 1995 chickenpox was a common childhood infection in the United States. Chickenpox infection is usually mild, characterized by blister-like pustules that resolve in about a week. However, HHV3 remains in the body long after the pustules have disappeared. HHV3 genomes are silently maintained in neurons, for decades. Shingles, a very painful and debilitating disease of adults, occurs when HHV3 exits latency and travels down neurons to the skin to produce blister-like lesions. These lesions contain infectious virus, thus a person with shingles can transmit chickenpox to a nonimmune person. HHV3 reactivates (breaks out of latency) when the host’s immune system is impaired (by advancing age or stress, for example). Shingles vaccines boost immune responses to HHV3, reducing the likelihood of virus reactivation. Introduction to Viral Pathogenesis Viral pathogenesis is deﬁned as the mechanism by which viruses causes disease. A simple view of viral pathogenesis is that viruses replicate and kill cells, thus causing disease. For example, death of liver cells (hepatocytes) causes hepatitis, death of enterocytes may cause diarrhea, death of respiratory epithelial cells may cause severe respiratory tract disease. However loss of cell function, without death, can also produce disease. During HIV infection, immunodeﬁciency is not simply caused by cell death; the virus also alters the function of some cells needed to maintain a healthy immune system. Signs and symptoms of disease can also result from tissue damage caused by host immune responses. Inﬂammation, killing of virus-infected cells by the immune system, or deposition of immune complexes are examples. Of course, like any biological event, disease is often a complex combination of direct damage by virus in concert with host immune responses. Understanding viral pathogenesis, the mechanism by which disease develops, is an important consideration in developing eﬀective treatments. Introduction to Virus Transmission How are viruses transmi ed from one animal to another? Common routes of infection include: fecal-oral, respiratory droplets, contact with contaminated fomites, exchange of infected bodily ﬂuids, tissues, or organs, airborne, insect vectors. Fecal-oral transmission occurs via ingestion of contaminated food or water. Virus enters the body through epithelial cells or lymphoid in the gastrointestinal tract. Examples include rotaviruses and the Norwalk-like viruses (noroviruses). Noroviruses have caused notable outbreaks on cruise ships, sickening hundreds of guests and crew in a ma er of days. Human hepatitis A virus is also transmi ed by the fecal-oral route via contaminated produce or uncooked shellﬁsh. Fomites (objects contaminated with infectious organisms) can also play a role in fecal-oral transmission. Respiratory transmission occurs when viruses in the respiratory tract are expelled as droplets. The transmission may be directly from one individual to another (please do not cough in my face) or may occur through fomites, hence the advice to wash your hands often! Viruses expelled from the respiratory tract may also be transmi ed by contact with mucosal surfaces such as the eye. Health care providers and infectious disease researchers must remember to keep gloved hands away from their eyes. Examples of viruses that can be spread by the respiratory route are inﬂuenza viruses, rhinoviruses (one of the common cold viruses), and the severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses. Transmission of viruses via exchange of bodily ﬂuids can result from blood transfusions, use of dirty needles, trauma (bleeding), organ or tissue transplantation, sexual contact, or artiﬁcial insemination. The human viruses HIV, HBV, and HCV are all transmi ed via contaminated blood. But these viruses can also be transmi ed through contact with other bodily ﬂuids such as semen or saliva. HIV can be transmi ed via breast milk. Rabies virus is transmi ed by saliva. A few viruses, such as foot-and-mouth disease virus of livestock, can be transmi ed over long distances through the air, a process called airborne transmission. Measles virus is also known for airborne transmission. Simply si ing in a room with a measles- infected individual can lead to infection! It should be noted that airborne transmission is distinct from aerosol transmission. In airborne transmission, particle sizes are very small and remain suspended in the air for long periods. The importance of understanding the distinction between these two types of transmission is exempliﬁed by the 2014 Ebola virus epidemic. Ebola virus is transmi ed through contact with body ﬂuids of an infected individual. Transmission occurs when the patient is clearly symptomatic and virus titers are highest. Ebola can be transmi ed via respiratory droplets but there is no evidence that the virus is transmi ed in the absence of direct contact with respiratory droplets, or other secretions, thus Ebola is not considered to be an “airborne” virus. Many viruses (West Nile virus, the equine encephalitis viruses, dengue virus, chikungunya virus, and zika virus, for example) are transmi ed from one host to another primarily via an insect intermediary. Blood-feeding insects such as mosquitos, ticks, and midges are common vectors. Viruses transmi ed by insect vectors are collectively called arboviruses. It should be emphasized that a virus can be transmi ed by more than one route. The SARS coronavirus, considered primarily a respiratory virus, is also transmi ed by the fecal-oral route. Blood transfusions, dirty needles, and organ transplants may facilitate transmission of viruses usually spread by other routes. Mucosal surfaces, such as the eye, can be entry points for transmission of virus in present in blood or other bodily ﬂuids. Some mosquito- p y q vectored viruses (West Nile, chikungunya, yellow fever, and equine encephalitis viruses) require special precautions to avoid transmission in a research se ing, where these viruses can be transmi ed via aerosols. Finally, a discussion of virus transmission should also include brief mention of virus transmissibility. Transmissibility is the ease of virus spread from one host to another. Measles virus is highly transmissible by the airborne route, and outbreaks can quickly become widespread in nonimmune population. Transmissibility is not related to the ability of a virus to cause disease (virulence). A virus may be relatively diﬃcult to transmit, but highly virulent if transmission does occur. It is easy to overestimate the transmissibility of a highly virulent virus. In this chapter we have learned that: Viruses are infectious agents (but are not cells). Viruses are obligate intracellular parasites that require host cells for their replication. Virions are the packages that contain the viral genome. Virions assemble from viral proteins and genomes synthesized within the infected cell. In the laboratory viruses are cultured or grown in cell or organ culture. Viruses can change or adapt to new growth conditions. Viruses have diﬀerent genome types, capsid types, routes of infection, and diverse interactions with host cells. Virus infection may but does not always lead to cell death or host disease. Virus infections may be relatively short lived (acute infections) or may be life-long (chronic or persistent). C H AP T E R 2 Virus Structure Abstract Simple viruses are genomes packaged in a protein shell called a capsid. Capsids are assembled from many copies of a single, or a few capsid proteins. Capsids are symmetrical structures stabilized by repeated contacts between protein subunits. Capsids of naked (unenveloped) viruses function in a achment and entry. Capsids also have roles in genome packaging and exit from the cell. Capsids accomplish these diverse tasks because they are dynamic structures that change conformation in response to environmental clues. Some viruses surround their capsids with a lipid bilayer called the envelope. Enveloped viruses encode proteins that associated with the lipid bilayer. They are usually glycosylated and contain transmembrane anchoring domains. They often project out from the surface of the envelope forming distinct spikes. Envelope proteins have a achment and fusion functions. Many enveloped viruses have a matrix (MA) protein positioned inside, and associated with the envelope (via direct membrane interactions or through interactions with the cytosolic tails of envelope glycoproteins). The MA protein forms a link between the membrane and the nucleocapsid. The terms nucleocapsid and core refer to the complex of viral nucleic acid and protein found within the envelope. Keywords Capsid; icosahedron; envelope glycoprotein; nucleocapsid; envelope; matrix protein; triangulation number; eight-stranded jelly roll β-barrel motif OUTLINE Anatomy of a Virus 13 Capsid Structure and Function 14 Capsids Are Built From Many Copies of One or a Few Types of Protein 14 Simple Icosahedral Capsids 15 Larger Icosahedral Capsids 16 Polyomavirus and Papillomavirus Capsids 16 Adenovirus Capsids 16 Reoviruses 17 Herpesvirus Capsids 18 Poxvirus Structure 18 Viral Envelopes 18 Glycosylation 19 Other Membrane Proteins 19 Matrix Proteins 20 Nucleocapsid Structure 21 After studying this chapter, you should be able to: Deﬁne “capsid” and explain its functions. Deﬁne “nucleocapsid,” “envelope,” and “envelope protein.” Describe the diﬀerence between “structural” and “nonstructural” proteins. Be able to distinguish between icosahedral and helical capsids. Indicate the twofold, threefold, and ﬁvefold axes of symmetry on an icosahedral capsid. Describe the activities of envelope glycoproteins. Describe the location and a common function of matrix (MA) proteins of enveloped viruses. Anatomy of a Virus The simplest viruses consist of genome packaged in a protein shell or capsid (see Fig. 1.1). Capsids are assembled from many copies of a single, or a few types of capsid proteins. Some viruses surround their capsids with a lipid bilayer called the envelope. Envelopes may be derived from the cell plasma membrane, nuclear membrane, or other intracellular membranes. All enveloped viruses encode proteins that are associated with the lipid bilayer. They are usually glycosylated (thus are envelope glycoproteins) and often contain transmembrane anchoring domains. They often project out from the surface of the envelope forming distinct spikes. Many enveloped viruses have a matrix protein positioned inside, and associated with the envelope (via direct membrane interactions or through interactions with the cytosolic tails of envelope glycoproteins). The matrix protein often forms a link between the membrane and the nucleocapsid. The term nucleocapsid refers to a complex of viral nucleic acid and protein. The term is most often used to refer to the assemblage of protein and nucleic acid within an enveloped virus. If viral envelopes are gently lysed, the nucleocapsids are released. The proteins that assemble to form the virion (the extracellular particle) are called structural proteins. Additional proteins may be encoded by a virus, but are not present in the virion. These so- called nonstructural proteins have a variety of functions in the virus replication cycle. For example, nonstructural proteins may modulate cell and host antiviral responses; others are enzymes such as proteases or polymerases. It is important to note: nonstructural does not mean nonfunctional, or unimportant. Most nonstructural proteins are in fact essential for virus replication. Capsid Structure and Function In the simplest terms, viral capsids are protein packages that protect the genome. However capsids should not be considered static boxes, as they are dynamic structures that have other important functions. In addition to simply providing “packaging,” the capsids of unenveloped viruses mediate a achment to, and penetration into, the host cell. Capsids must also be able to assemble, speciﬁcally package viral genomes and direct budding or release from cells. Capsids come in two basic shapes: helical (rod shaped) or icosahedral (spherical). The simplest capsids are assemblies of many copies of a single protein (often called the capsid protein). As capsids assemble, they are stabilized by the repeated interactions (largely electrostatic) of the capsid protein building blocks. It should come as no surprise that there are few covalent bonds between these building blocks, because the genome must be released from the capsid at a later time! The repeated occurrence of similar protein–protein interfaces leads to construction of a symmetrical capsid. The simple helical capsid of tobacco mosaic virus (TMV), a plant virus, is assembled from many copies of a single capsid protein. Each capsid protein forms identical same side-to-side and top to bo om interactions with its neighbors, as indicated in Fig. 2.1. In addition to interacting with neighboring proteins, each TMV capsid protein interacts with three nucleotides of the viral (RNA) genome. The capsid proteins are tightly packed around the RNA and form a rigid rod whose length is determined by the length of the genome. Not all helical capsids are rigid rods, many enveloped animal viruses have very ﬂexible, helical nucleocapsids surrounded by an envelope. FIGURE 2.1 Capsids come in two basic shapes helices (rods shown on left) and spherical particles with icosahedral symmetry (shown on right). (A) The red and blue bars on the helical virus indicate sites of repeating contacts between subunits. Red bars indicate side-to-side contacts. Blue bars indicated top to bottom contacts. (B) The two, three and five fold axes symmetry are indicated for this simple icosahedral capsid. Viruses with spherical capsids have icosahedral symmetry. An icosahedron is a closed cube with twofold, threefold, and ﬁvefold axes of symmetry (Fig. 2.1). The simplest icosahedron can be assembled from 20 equilateral triangles. More complex, and larger, icosahedra can be built by assembling more than 20 triangular subunits. Capsids that vary from the deﬁnitions of a helix or an icosahedron are sometimes called “complex.” For example, there are viruses of bacteria (bacteriophage) with icosahedral “heads,” rod like “tails” and long “ﬁbers” extending from the tails. Capsids Are Built From Many Copies of One or a Few Types of Protein Biological constraints require that capsids be assembled from multiple copies of one, or a few, small (usually in the range of 20– 60 kDa) proteins. A key consideration of capsid assembly is the relative size of an amino acid and the triplet codon for that amino acid. The average size of an amino acid is 110 daltons (Da) while the average size of a triplet codon is ~330 Da×3, or nearly 1000 Da. The codon is considerably larger than the amino acid, so it requires many copies of any single amino acid to package it. A single polypeptide is always physically smaller than the gene that encodes it. Luckily, translation produces many copies of a protein from each mRNA. Another biological constraint to the size of a capsid protein is its ability to fold. Small polypeptides can fold tightly while larger ones are unable to fold without leaving gaps in the structure; gaps could render the genome susceptible to environmental damage. Larger proteins also need chaperones to help them fold correctly. Indeed, there are some viral capsids that assemble with the help of chaperones. Fidelity of protein synthesis is another issue that constrains the size of a capsid protein. The observed error rate of protein synthesis is ~10−4, or 1 mistake per 1000 amino acids polymerized. Thus on average, a 1000 amino acid long protein would contain one mistake. In contrast, synthesis of 10 copies of a 100 amino acid protein would produce, on average, nine perfect proteins and one with a mistake. Simple Icosahedral Capsids In the previous section the icosahedron was described as an assembly of equilateral triangles. However, capsids are not assembled from “triangular” or even symmetrical, proteins. Among animal viruses, the most common type of capsid protein structure is called an eight-stranded jelly roll β-barrel motif. This structure consists of four pairs of antiparallel β sheets (Fig. 2.2). FIGURE 2.2 (A) The icosahedral capsids of most animal viruses are assembled from proteins that have a common shape: an eight- stranded jelly roll β-barrel motif. (B) Each triangular ‘face’ of the capsid is assembled from three copies of an eight-stranded jelly roll β-barrel motif. The most common icosahdral capsids can be envisioned as an arrangement of three capsid proteins into a triangle. Thus, as shown in Fig. 2.2, the simplest icosahedral capsid would contain 60 capsid proteins (20 triangular faces, each having three capsid proteins). This structure is called a T=1 capsid, and T is the triangulation number (Box 2.1). Without reviewing the mathematics involved, a T=1 icosahedron is an assembly of 20 triangular faces, a T=3 icosahedron has 3×20 or 60 triangular faces, a T=4 icosahedron has 4×20 or 80 triangular faces and so on. The T number can be used to determine the theoretical number of capsid proteins required to assemble a shell, if each triangular face is constructed from three capsid proteins. A T=1 virus (for example, a parvovirus) has a shell assembled from 60 capsid proteins. Caliciviruses have T=3 capsids, assembled from 180 (3×60) molecules of a capsid protein. Togaviruses have T=4 capsids that are assembled from 240 (3×80) molecules of capsid protein (Table 2.1). Thus larger capsids are assembled by using more building blocks (Fig. 2.3). Box 2.1 Tria n g u la t io n N u m b e rs a n d S t ru ct u ra l S u b u n it s The T number was ﬁrst described by Caspar and Klug in 1962 to explain the structural basis for icosahedral capsids of diﬀerent sizes. In their models, the T number predicted the number of capsid proteins in a structure, and their predictions proved true for many small viruses. However, as more icosahedral capsids were analyzed it became apparent that T numbers more often predicted numbers of “structural subunits” rather than numbers of polypeptides in a capsid. The most common structural subunits of icosahedral animal virus capsids are β-barrels (eight- stranded jelly roll β-barrel motifs). This common structure is shown in Fig. 2.3. Capsid proteins of diﬀerent viruses do not have conserved amino acid sequences; however, they do assume this conserved structure. The distinction between a structural subunit and a polypeptide in building capsids can be demonstrated by comparing the capsids of caliciviruses, picornaviruses, and cowpea mosaic virus. All three virus groups have T=3 capsid architecture. The calicivirus capsid is assembled from 180 copies of a single capsid (C) protein while picornaviruses are assembled from 60 copies each, of three capsid proteins (VP1, VP2, and VP3). The three picornavirus capsid proteins have diﬀerent primary amino acid sequences but fold to assume similar structures. Thus the capsids of picornaviruses are assembled from 180 equivalent structural subunits. The T=3 capsids of picornaviruses are sometimes called pseudo T=3 or P=3 capsids as they do not strictly adhere to the original predictions put forth by Caspar and Klug. In the case of cowpea mosaic virus the capsid is assembled from two diﬀerent polypeptides, 60 copies each of a large (L) and a small (S) capsid protein. However, a close look at capsid architecture shows that L folds into two independent β-barrels connected by a hinge region. Thus the cowpea mosaic virus capsid is assembled from 180 “structural” domains, where the structural unit is the β-barrel. Table 2.1 Construction of Simple Icosahedral Capsids FIGURE 2.3 Examples of large and small icosahedral capsids. (A) T=1 icosahedral capsid; (B) T=3 icosahedral capsid; (C) T=4 icosahedral capsid; (D) T=7d polyomavirus capsid; (E) T=25 adenovirus capsid; (F) Triple layer particle of rotavirus contains a T=1 core surrounded by T=13 shells. (G) HIV capsid. Elongated icosahedral capsids are formed by adding rows of hexamers to the middle of the structure. How do capsid proteins assemble to form a simple icosahedral shell? The process is stepwise and sometimes the substructures can be identiﬁed. T=1 viruses assemble from 60 capsid proteins. A general model is that three capsid proteins assemble to form a capsomere. Five capsomers form a pentamer and 12 pentamers assemble to form the closed shell. T=3 and T=4 capsids are built with pentamers and hexamers (hexamers are assemblies of six capsid proteins). T=3 capsids assemble from 12 pentamers and 20 hexamers while T=4 viruses assemble from 12 pentamers and 30 hexamers. The surfaces of icosahedral capsids can be quite variable. Some capsids are relatively smooth, while others have prominent projections and/or deep canyons or pits (Fig. 2.4). Electron micrographs of unenveloped icosahedral viruses are shown in Fig. 2.5. In the case of unenveloped viruses, a achment proteins may project out from the capsid to engage a receptor, while in other cases a achment sites are inside a canyon or pit. FIGURE 2.4 Molecular models of icosahedral viruses. FIGURE 2.5 Examples of unenveloped viruses. Larger Icosahedral Capsids Large icosahedral capsids can also be envisioned as assemblages of triangular faces. For example, one could construct a T=7 icosahedron using 140 equilateral triangles (420 structural subunits) or a T=13 icosahedron from 260 equilateral triangles (780 structural subunits). However cryo-electron microscopic visualization of viruses with larger icosahedral capsids reveals that some of these structures (described below) are not simply larger versions of a T=1 or T=3 virus. Polyomavirus and Papillomavirus Capsids Based on the predictions of Caspar and Klug, these T=7 capsids (~50 nm diameter for polyomavirus and ~60 nm diameter for papillomavirus) would contain 420 structural subunits assembled into 12 pentamers and 80 hexamers, but this is not exactly the case. Instead the capsids of polyomaviruses and papillomaviruses are assembled from 360 structural subunits organized as 72 pentamers (Fig. 2.3). These capsids structures are referred to at T=7d (rather than T=7). Papillomavirus capsids are assembled from two structural proteins, L1 and L2. Each pentamer assembles from ﬁve molecules of L1 and a single molecule of L2. Adenovirus Capsids The large (~95 nm diameter) icosahedral capsids of adenoviruses contain 12 diﬀerent polypeptides. Their capsid structure is described as pseudo T=25. The major capsid protein (MCP) is called the hexon protein and 720 copies of hexon protein assemble to form 240 trimers. Each trimer forms a hexamer subunit. But how can a hexamer be assembled from a trimer of capsid proteins? It turns out that hexon protein is a large capsid protein that contains two structural domains. Adenovirus capsids also contain 12 pentamers or vertices (Fig. 2.3, Panel E) assembled from three additional structural proteins, the penton base protein (polypeptide III), the ﬁber protein (polypeptide IV), and polypeptide IIIa. The long spikes that extend from each vertex are homotrimers of the ﬁber protein. They are anchored in place by the penton protein. Five copies of polypeptide IIIa are arranged in a ring beneath each vertex. Thus pentons and hexons are assembled from diﬀerent types of capsid proteins. Reoviruses Reoviruses have multilayered icosahedral capsids. Viruses in the genus Rotavirus have triple-layered particles (Fig. 2.3, Panel F). The smallest, innermost layer is a T=1 capsid formed from 60 copies of viral protein (VP) 2. The middle layer is a T=13 capsid assembled from the ~45 kDa VP6. The outmost layer of the rotavirus capsid is assembled from two additional structural proteins, VP4 and VP7. Members of the genus Reovirus have two capsids layers, an inner T=1 core surrounded by an outer T=13 shell. Herpesvirus Capsids Herpesviruses are large, enveloped viruses with icosahedral capsids. The major capsid protein or MCP is quite large (~1300 amino acids) and folds to assume a structure that is distinct from the eight-stranded jelly roll β-barrel motif of other animal viruses. MCP assumes a shape very similar to the capsid proteins of double- stranded (ds) DNA bacteriophage such as P22 or HK97. Herpesviral capsids are assembled from a total of 162 capsomers (150 hexons and 12 pentons). MCP forms all of the hexons and 11 of 12 pentons. The 12th penton is formed from a diﬀerent herpesvirus protein called the portal protein to form the portal “complex.” The portal complex is cylindrical and contains a channel. Bacterio phage portal complexes actively transport DNA into and out of bacteriophage capsids. Herpesvirus portals likely serve a similar function. The structure of herpesvirus capsids suggests an evolutionary relationship between herpesviruses and dsDNA phage. Poxvirus Structure Poxviruses are among the largest of the animal viruses. Virions are brick shaped or ovoid. They are enveloped and range in size from 140 to 260 nm in height and 220 to 450 nm in length. The envelope surrounds a dumb bell-shaped core (capsid) and two globular protein structures called lateral bodies. Morphogenesis of poxvirions is described in Chapter 35, Family Poxviridae. Viral Envelopes Many important human and animal pathogens are enveloped viruses. Their helical or icosahedral capsids are enclosed within a lipid bilayer. Enveloped viruses come in a variety of shapes, ranging from the long, ﬁlamentous ebolaviruses (Chapter 21: Family Filoviridae) to the icosahedral togaviruses (Chapter 16: Family Togaviridae). Most enveloped viruses acquire their lipid bilayer by budding through a cellular membrane (for example, plasma membrane, endoplasmic reticulum (ER), Golgi, or nuclear membranes). However some large viruses, such as poxviruses apparently build their membranes from crescent-shaped lipid fragments associated with protein scaﬀolds (Chapter 35: Family Poxviridae). Enveloped viruses (with the exception of poxviruses) have proteins that are anchored into or across the lipid bilayer. Thus they are membrane proteins and can be categorized as type I or type II depending on their orientation in the membrane (Fig. 2.6). Types I and II membrane proteins span the lipid bilayer a single time, via an α-helical TM domain. Type I proteins are inserted into membranes with their amino-terminal domain to the outside and their carboxyl-terminal domain to the inside of the cell or virion. Type II membrane proteins have the opposite polarity with respect to the membrane Envelope proteins often take the form of long spikes. Spikes are usually homo- or heterodimers or trimers and many have distinct globular heads. Examples of viral type I membrane proteins include: inﬂuenza virus HA, rabies virus G protein, paramyxovirus fusion proteins, and hepatitis C virus E1 and E2. Paramyxovirus a achment proteins are type II membrane proteins. FIGURE 2.6 Arrangement of types I and II membrane proteins. Virus envelope proteins must carry out at least two functions: receptor binding and fusion. These activities may be carried out by a single protein (inﬂuenza virus HA, rabies virus G) or by two distinct proteins in the case of the paramyxoviruses. In the case of inﬂuenza virus, the HA glycoprotein is cleaved by cellular proteases to generate a fusion-active version of the protein but the two halves of HA (HA1 and HA2) remain associated via disulﬁde bonds. Rabies virus G protein also has both a achment and fusion activities; however, rabies virus G remains uncleaved. Human immunodeﬁciency virus (HIV), a retrovirus, also encodes a single glycoprotein precursor, but it is cleaved during synthesis to produce two distinct structural proteins. The amino-terminal domain of the envelope precursor is the surface unit (SU) protein, which functions in a achment. The carboxyl-terminal half of the precursor is the transmembrane (TM) protein. As its name implies, it has a membrane-spanning domain, anchoring it into the envelope. TM is the fusion protein. SU and TM are not covalently linked. SU is on the outside of the virion, held thereby noncovalent interactions with ectodomain of TM (Chapter 37: Replication and Pathogenesis of Human Immunodeﬁciency Virus). Viruses in the family Paramyxoviridae (see Chapter 20: Families Paramyxoviridae and Pneumoviridae) produce two envelope glycoproteins using distinct open reading frames. Both are membrane-anchored proteins. The a achment protein is a type II integral membrane protein while the fusion protein is a type I integral membrane protein. Glycosylation The ectodomains of envelope proteins are usually glycosylated (they have polysaccharides linked to the protein backbone). Carbohydrates can be linked to the peptide backbone via the nitrogen atom on an asparagine side chain (N)-linked or to the oxygen atoms of serine or threonine side chains (O-linked). Glycosylation occurs during transit of envelope proteins through the ER and Golgi. The amount of carbohydrate decorating envelope proteins varies. Some viral envelope proteins have one molecule of polysaccharide per polypeptide chain. In the case of the HIV SU, the molecular mass of carbohydrate equals that of the polypeptide backbone. HIV SU protein has an apparent molecular weight of 120 kDa (as determined by SDS-PAGE) but the calculated mass of the amino acid backbone is

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