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Structural Virology by Mavis Agbandje-McKenna

Over the last ten years, much effort has been devoted to improving the biophysical techniques used in the study of viruses. This has resulted in the visualization of these large macromolecular assemblages at atomic level, thus providing the platform for functional interpretation and therapeutic design. Structural Virology covers a wide range of topics and is split into three sections. The first discusses the vast biophysical methodologies used in structural virology, including sample production and purification, confocal microscopy, mass spectrometry, negative-stain and cryo-electron microscopy, X-ray crystallography and nuclear magnetic resonance spectroscopy. The second discusses the role of virus capsid protein structures in determining the functional roles required for receptor recognition, cellular entry, capsid assembly, genome packaging and mechanisms of host immune system evasion. The last section discusses therapeutic strategies based on virus protein structures, including the design of antiviral drugs and the development of viral capsids as vehicles for foreign gene delivery. Each topic covered will begin with a review of the current literature followed by a more detailed discussion of experimental procedures, a step in the viral life cycle, or strategies for therapeutic development. With contributions from experts in the field of structural biology and virology this exceptional monograph will appeal to biomedical scientists involved in basic and /or applied research on viruses. It also provides up-to-date reference material for students entering the field of structural virology as well as scientists already familiar with the area.

Product Details

ISBN-13: 9780854041718
Publisher: Royal Society of Chemistry, The
Publication date: 12/17/2010
Series: RSC Biomolecular Sciences Series , #21
Pages: 388
Product dimensions: 6.30(w) x 9.30(h) x 1.20(d)

About the Author

Mavis Agbandje-McKenna is currently Director of the Center for Structural Biology and Associate Professor at the University of Florida College of Medicine. She obtained her BSc (Hons) in Human Biology and Chemistry from the University of Hertfordshire and a PhD in Biophysics at the University of London Institute of Cancer Research. Her postdoctoral research was at Purdue University before she was appointed Senior Research Fellow at the University of Warwick, UK. Robert McKenna obtained his BSc (Physics and Biology) and PhD in Crystallography from the University of London. His postdoctoral research was at Purdue University before he accepted the post of Warwick Research Fellow in the Department of Biological Sciences at the University of Warwick. He is currently Associate Professor at the University of Florida College of Medicine.

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Structural Virology

By Mavis Agbandje-McKenna, Robert McKenna

The Royal Society of Chemistry

Copyright © 2011 Royal Society of Chemistry
All rights reserved.
ISBN: 978-0-85404-171-8


Production and Purification of Viruses for Structural Studies


Department of Biochemistry and Molecular Biology, Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USA

1 Introduction

Advances in protein production and purification techniques over the past two decades have allowed the structural study of numerous proteins and macromolecular assemblages that would have otherwise been intractable to the necessary approaches (detailed in the following chapters). This chapter focuses on the production and purification of intact viral capsids (particles) with/ without genome for structure determination. The production and purification of viral proteins for structure determination by X-ray crystallography and NMR spectroscopy are the subjects of Chapters 7 and 8, respectively. Crystallization is often considered a method of purification and a function of purity, often of a protein or virus capsid, and, as such, sample preparation for structure determination by X-ray crystallography places high demands on sample quality. Screening trials to identify the optimal crystallization conditions also require large quantities of sample compared with the majority of other structure determination approaches discussed in the subsequent chapters of this monograph. Virus samples produced for such analyses also have to be both stable and soluble in their storage buffer since degradation and aggregation are detrimental to the crystallization process. Hence this chapter will focus on methodologies to produce and purify virus capsids (Figure 1) in quantities suitable for structure determination by X-ray crystallography, with the premise that such a sample would also be suitable for structural or biophysical analysis using other methodologies.

2 Expression Systems

Most viruses are considered hazardous material in their wild-type (wt) infectious form (for information on safe handling and containment of infectious microorganisms and hazardous biological materials, see http://www.cdc.gov/ biosafety) and are therefore often studied in a recombinant form. Significant effort has been extended into the development of heterologous expression systems to produce recombinant viral proteins which will assemble into viral capsids. The system selected for use is often dependent on the properties of the viral genes and the environmental requirements of the final product. However, the most important factor to consider is the capacity of the host cells to translate the RNA transcript, to ensure proper folding of the gene product and to sustain the protein(s) expressed in an intact and functional state. Protein expression systems contain at least four general components: (1) the genetic elements necessary for transcription/translation and selection; (2) in vector- based systems, a suitable replicon: plasmid, virus genes, etc.; (3) a host strain containing the appropriate genetic traits needed to function with the specific expression signals and selection scheme; and (4) the culturing conditions for the transformed cells or organisms.

Eukaryotic Systems

Mammalian Cells

Since most viruses currently studied are of human or animal origin, mammalian tissue culture is an ideal source to generate viral capsids for structural studies which are generally aimed at functional annotation. In this system, proper folding is achieved and modifications such as complex glycosylation, phosphorylation, acylation, acetylation and γ-carboxylation are obtained. However, yields can be low, depending on gene product(s), ranging from 0.1 to 100 mg L-1 of culture volume. For some of the structural approaches discussed in Section 1 of this monograph, low yields may not be a problem since small amounts of sample are adequate. However, low yields can become problematic in crystallization, especially with a virus that does not have an established crystallization condition. In such a situation, numerous preparation steps may be required to obtain the quantities needed to screen crystallization conditions efficiently. Supplies and reagents can then become expensive, depending on individual cell line requirements. In addition, considerable time and resources can be spent on the construction of a suitable expression system and equally on optimization for suitable yields. In such situations, it is always advisable to seek the expertise of an established molecular biologist before designing new constructs.

Established cell lines and protocols exists for many different tissue systems and, although most of these cell lines are derived from human or mouse tissues, other mammalian cell culture lines are available, such as monkey, raccoon, horse, pig and rabbit. The American Type Culture Collection (ATCC) has over 3400 cell lines from 80 different species, including over 950 cancer cell lines (http://www.atcc.org/). Other cell suppliers include the Health Protection Agency Culture Collections (HPACC; http://www.hpacultures.org.uk/), the German Research Center for Biological Material (DSMZ; http://www.dsmz.de/) and the Riken BioResource Center Cell Bank (Riken; http://www. brc.riken.jp). It is strongly recommended that investigators purchase cell lines from recognized centers such as these listed above to ensure pure, authentic and quality controlled cell lines. The decision to use cells directly from an organism, i.e. primary cells or an immortalized cell line, should be based upon requirements of the virus system and available current protocols. As discussed below, there are three main approaches for virus production in mammalian cell lines: (i) infection of permissive cell lines with wt virus, (ii) transfection of cells with plasmid constructs containing viral genome sequences and (iii) viral vector systems which expression heterologous viral genes.

Although the majority of viruses currently studied are obtained from recombinant expression systems (see below), direct infection of cell lines with wt virus can be used to generate suitable quantities of sample for structural studies under certain conditions and for well-characterized viral systems. For example, the human rhinovirus 3 (HRV3) virion particles used for determining its structure were purified from virus-infected HeLa cells (immortalized human cancer cells). The atomic structure of HRV3 was initially determined to 3 Å, and later refined to 2.15 Å. It was reported that 10–12 L of HeLa cells (at 6–8×105 cells mL-1) were used to generate the amount of virus necessary to carry out crystallization and structure determination. Echovirus-1, also of the Picornaviridae family, was also successfully produced in HeLa cells for its structure determination to ~3.55 Å resolution.

In the use of plasmid constructs, one or more plasmids usually containing capsid proteins alone and, if needed, replication factors, are used to transfect cells, which results in the assembly of virus-like particles (VLPs). Often, another plasmid is added when a packaged gene is desired, e.g. reporter gene, or if genome is needed to produce stable virions. Recovered virus can either be purified for structural studies or, if infectious, used to infect permissive cells for continual propagation of virions. As an example, molecular clones containing the capsid sequence of canine parvovirus was used for the transfection of Norden Laboratories feline kidney cells (NLFK) to produce particles for X-ray crystallographic structural studies to 3.2 Å resolution. For the crystallographic structure determination of the immunosuppressive strain of minute virus of mice (MVMi), infectious virions were harvested from plasmid transfected cell lines and subsequently propagated in a permissive cell line to produce virus for crystallization.

The development of heterologous surrogate expression systems for virus capsid production has enabled researchers to overcome the lack of efficient expression in homologous systems for several viruses of interest. As an example, for hepatitis C virus (HCV), a herpes simplex virus-1 (HSV-1)-based amplicon vector system that expresses HCV capsid proteins and the two envelope proteins, E1 and E2, under the HSV-1 IE4 promoter was developed. This system has several advantages; (i) the ability to infect a wide range of cells, without the limitation of transfection efficiency, including primary cells in a quiescent state, (iii) the simplicity of cloning desired genes into amplicons, (iii) the high capacity of incorporation of exogenous sequences in the vector genome and the transfer of high copy numbers of the exogenous gene and (iv) the potential for using amplicons in vaccine design and development. A mini-review has covered HSV amplicons from genomes to engineering. Norovirus is another example of a non-cultivable virus that remained refractory to structural studies due to the lack of a reverse genetics system and a permissive cell line until recent advances. A novel expression strategy, which combined the use of a two baculovirus trans-activation system to deliver viral cDNA and an inducible DNA polymerase (pol) II promoter, led to the ability to grow this virus in several cell lines, including HepG2, BHK-21, COS-7 and HEK293T cells.

Yeast Cells

Among the microbial eukaryotic host systems, yeasts can combine the advantages of unicellular organisms (e.g. ease of genetic manipulation and growth) with the capabilities of a protein processing typical of eukaryotic organisms (e.g., protein folding, assembly and posttranslational modifications). The majority of recombinant proteins produced in yeast have been expressed using Saccharomyces cerevisiae. More commonly referred to as baker's or budding yeast, S. cerevisiae was the first eukaryote to have its entire genome sequenced and is still today considered a model organism. A scientific database has been established for S. cerevisiae and is available at http:// www.yeastgenome.org/. With its biochemistry, basic genetics and cellular biology already well established, this simple eukaryote has become a major tool in answering questions of fundamental biological importance and is a central player in post-genomics research.

Appealing aspects of the yeast expression system are its rapid cell growth (with a doubling time of ~90 min), simple growth media, secretion of recombinant proteins to the medium and glycosylation capability. N-linked glycosylation is minimal with high mannose, but O-linked modifications appear similar to mammalian cells. Phosphorylation, acetylation and acylation are also present. Protein yields are comparable with the baculovirus system (see below) at ~10–200 mg L-1 depending on recombinant gene properties. Issues in large-scale protein production involving S. cerevisiae appear to be hyperglycosylation and retention in the periplasmic space. This ultimately leads to a loss of final protein due to retention and degradation. The search for alternative hosts has led to the use of 'non-conventional' yeasts in expression protocols. The most established examples include Hansenula polymorpha, Pichia pastoris, Kluyveromyces lactis, Yarrowia lipolytica, Pichia methanolica, Pichia stipitis, Zygosaccharomyces rouxii, Zygosaccharomyces bailaii, Candida boidinii and Schwanniomyces (Debaryomyces) occidentalis. These systems are broken down even further into two categories: methyltrophic, e.g. P. pastoris, and non-methyltrophic, e.g. S. cerevisiae. These categories are based on the fermentation processes involved and generally dictate the promoter that should be used in the experimental design. The choice of yeast host is one of the most important determinants of the success of the entire project, and many reviews debating the subject can be found in the current literature. Generally, the expression of foreign proteins in yeasts consists of (i) cloning of a foreign protein-coding DNA sequence within an expression cassette containing a yeast promoter and transcriptional termination sequences and (ii) transformation and stable maintenance of this DNA in the fusion host. The transformation process is highly dependent on the yeast strain and detailed studies should be conducted in order to achieve high-efficiency transformation.

This system is extensively used for studying biological processes in higher eukaryotes and also allows replication of eukaryotic viruses. The first eukaryotic virus for which replication and genome encapsidatation was conducted in S. cerevisiae was brome mosaic virus (BMV), a positive strand RNA [(+)RNA] virus that infects plants. The BMV VLPs were subsequently purified for structure-to-function studies using cryo-electron microscopy (cryo-EM) studies. Other (+) RNA viruses that have been successfully replicated in S. cerevisiae include the plant viruses tomato bushy stunt virus and carnation Italian ringspot virus and animal viruses Flock House virus (FHV) and Nodamura virus. Human papillomavirus-16 (HPV-16) VLPs have also been successfully expressed in the yeast system in addition to the bovine papillo-mavirus-1 (BPV-1). The yeast virus L-A was isolated and purified from S. cerevisiae and the structure was solved to 3.4 Å resolution.

Insect Cells

Originally isolated from the alfalfa looper (Autographa californica) insect, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most widely used and best characterized baculovirus for recombinant gene expression (a recent review on baculovirus molecular biology is available). The rather large genome (~134 kbp) can stably accommodate an insertion of ~38kb, making expression of large genes possible. This virus is also known to infect several other insect species including Spodoptera frugiperda. The most commonly used insect host cell lines, Sf9 and Sf21AE, are derived from S. frugiperda pupal ovarian tissue and the BTI-Tn-5B1-4 line, also known as 'High 5 cells', derived from Trichoplusia ni egg cell homogenates. The wt nucleopolyhedrovirus (NPV) produces small inclusion bodies composed of a polyhedron protein which allows for the encapsulation of many virions into a crystalline protein matrix. This protein is expressed in the very late phase of gene expression and is controlled by a very strong promoter, the polydron promoter (a review on baculovirus late expression factors is available). The baculovirus expression vector system (BEVS) takes advantage of this very strong polyhedron promoter to drive foreign protein expression. It has also been shown that the non-structural p10 protein is expressed at similar levels in the same very late phase of expression. Both proteins have been shown to be nonessential in the production of baculovirus particles, making the replacement of their open reading frame (ORF) ideal for use in foreign gene expression.

The coupling of the very strong polyhedron promoter with a foreign gene-coding region results is the production of high levels of recombinant protein (~5–200 mg L-1) in a relatively short amount of time using the BEVS. Since the baculovirus genome is generally considered too large to insert the foreign gene of choice by direct ligation, transfer vectors are used. There are many different vectors available for gene insertion, which are variants of a basic design (a review appeared recently). These offer single gene, multiple genes and fusion gene expression. Multiple copies of the promoter can also be engineered into BEVS for the expression of multiple recombinant proteins concurrently in infected cells, which permits the assembly of structures that are made up of heterologous proteins, such as viruses.


Excerpted from Structural Virology by Mavis Agbandje-McKenna, Robert McKenna. Copyright © 2011 Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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Table of Contents

Introduction: Principles of Virus Structure;Section 1 Methodologies for Structural Analysis of Viruses; Production and Purification of Viruses for Structural Studies; Microscopic (Confocal) Analysis of Viral Entry and Infection; Cryo-electron Microscopy of Virus Infection - Tomography and Asymmetric Structure Determination; Cryo-electron Microscopy of Viruses - 3D of Virus Capsids; X-ray Crystallography of Virus Capsids; Structural Studies of Viral Proteins; Probing Viral Capsid Structures in Solution; Section 2 Structure to Function Correlation for Viruses; Evolution of Viral Capsid Structures - the Three Domains of Life; Mechanisms of Virus Capsid Assembly; Mechanisms of Genome Packaging; Attachment and Viral entry - Receptor Recognition in Viral Pathogenesis; Attachment and Entry - Viral Cell Fusion; Virus Antibody Recognition; Section 3 Therapeutic Strategies Based on Viral Structures; Development of anti-HIV drugs; Design of Influenza Vaccines and Antiviral Agents; Engineering Viral Capsids as Nano Tools; Viral Vectors for Gene Delivery;

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