technique (such as electron scattering by membrane-associated protein arrays and cryo-electron microscopy)may help to provide more information in the future, but it is unlikely that these variations will solve this problem completely.One further limitation is that some of the largest virus particles,such as poxviruses, contain hundreds of different proteins and are at present too complex to be analysed using these techniques. Nuclear magnetic resonance (NMR)is increasingly being used to determine the atomic structure of all kinds of molecules,including proteins and nucleic acids. The limitation of this method is that only relatively small molecules can be analysed before the signals obtained become so confusing that they are impossible to deci- pher with current technology.At present,the upper size limit for this technique restricts its use to molecules with a molecular weight of less than about 30.000 to 40,000,considerably less than even the smallest virus particles.Nevertheless,this method may well prove to be of value in the future,certainly for examining iso- lated virus proteins if not for intact virions. Chemical investigation can be used to determine not only the overall compo sition of viruses and the nature of the nucleic acid that comprises the virus genome but also the construction of the particle and the way in which individ- ual components relate to each other in the capsid.Many classic studies of virus structure have been based on the gradual,stepwise disruption of particles by slow alteration of pH or the gradual addition of protein-denaturing agents such as urea phenol,or detergents.Under these conditions,valuable information can sometimes be obtained from relatively simple experiments.For example,as urea is gradually added to preparations of purified adenovirus particles,they break down in an ordered,stepwise fashion which releases subvirus protein assemblies,revealing the composition of the particles.In the case of TMV.similar studies of capsid organi- zation have been performed by renaturation of the capsid protein under variou conditions(Figure 1.5).In simple terms,the reagents used to denature virus capsids can indicate the basis of the stable interactions between its components.Proteins bound together by electrostatic interactions can be eluted by addition of ionic salt or alteration of pH;those bound by nonionic,hydrophobic interactions can be eluted by reagents such as urea;and proteins that interact with lipid components can be eluted by nonionic detergents or organic solvents. In addition to revealing fundamental structure,progressive denaturation can also be used to observe alteration or loss of antigenic sites on the surface of par ticles,and in this way a picture of the physical state of the particle can be devel- oped.Proteins exposed on the surface of viruses can be labelled with variou compounds(e.g.,iodine)to indicate which parts of the protein are exposed and which are protected inside the particle or by lipid membranes.Cross-linking reagents such as psoralens or newer synthetic reagents with side-arms of specifi lengths are used to determine the spatial relationship of proteins and nucleic acids in intact viruses
technique (such as electron scattering by membrane-associated protein arrays and cryo-electron microscopy) may help to provide more information in the future, but it is unlikely that these variations will solve this problem completely. One further limitation is that some of the largest virus particles, such as poxviruses, contain hundreds of different proteins and are at present too complex to be analysed using these techniques. Nuclear magnetic resonance (NMR) is increasingly being used to determine the atomic structure of all kinds of molecules, including proteins and nucleic acids. The limitation of this method is that only relatively small molecules can be analysed before the signals obtained become so confusing that they are impossible to decipher with current technology. At present, the upper size limit for this technique restricts its use to molecules with a molecular weight of less than about 30,000 to 40,000, considerably less than even the smallest virus particles. Nevertheless, this method may well prove to be of value in the future, certainly for examining isolated virus proteins if not for intact virions. Chemical investigation can be used to determine not only the overall composition of viruses and the nature of the nucleic acid that comprises the virus genome but also the construction of the particle and the way in which individual components relate to each other in the capsid. Many classic studies of virus structure have been based on the gradual, stepwise disruption of particles by slow alteration of pH or the gradual addition of protein-denaturing agents such as urea, phenol, or detergents. Under these conditions, valuable information can sometimes be obtained from relatively simple experiments. For example, as urea is gradually added to preparations of purified adenovirus particles, they break down in an ordered, stepwise fashion which releases subvirus protein assemblies, revealing the composition of the particles. In the case of TMV, similar studies of capsid organization have been performed by renaturation of the capsid protein under various conditions (Figure 1.5). In simple terms, the reagents used to denature virus capsids can indicate the basis of the stable interactions between its components. Proteins bound together by electrostatic interactions can be eluted by addition of ionic salts or alteration of pH; those bound by nonionic, hydrophobic interactions can be eluted by reagents such as urea; and proteins that interact with lipid components can be eluted by nonionic detergents or organic solvents. In addition to revealing fundamental structure, progressive denaturation can also be used to observe alteration or loss of antigenic sites on the surface of particles, and in this way a picture of the physical state of the particle can be developed. Proteins exposed on the surface of viruses can be labelled with various compounds (e.g., iodine) to indicate which parts of the protein are exposed and which are protected inside the particle or by lipid membranes. Cross-linking reagents such as psoralens or newer synthetic reagents with side-arms of specific lengths are used to determine the spatial relationship of proteins and nucleic acids in intact viruses. Introduction 15
16 Principles of Molecular Virology Disk Monomers 5.0 6.0 7.0 8.0 Figure 1.5 The structure and stability of virus particles can be examined by pro- gressive denaturation or renaturation studies.At any particular ionic strength,the purified capsid protein of tobacco mosaic virus (TMV)spontaneously assembles into different structures,dependent on the pH of the solution.At a pH of around 6.0,the particles formed have a helical structure very similar to infectious virus particles.As the pH is increased to about 7.0,disk-like structures are formed.At even higher pH values,individual capsid monomers fail to assemble into more complex structures. Since the 1930s,electron microscopes have overcome the fundamental limita- tion of light microscopes:the inability to resolve individual virus particles owing to physical constraints caused by the wavelength of visible light illumination and the optics of the instruments.The first electron micrograph of a virus (TMV)was published in 1939.Over subsequent years,techniques were developed that allowed the direct examination of viruses at magnifications of over 100,000 times.The two fundamental types of electron microscope are the transmission electron microscope (TEM)and the scanning electron microscope (SEM)(Figure 1.6).Although beau tiful images with the appearance of three dimensions are produced by the SEM, for practical investigations of virusstructure the higher magnifications achievable with the TEM have proved to be of most value.Two fundamental types of infor mation can be obtained by electron microscopy of viruses:the absolute number of virus particles present in any preparation (total count)and the appearance and struc- ture of the virions (see below).Electron microscopy can provide a rapid method of virus detection and diagnosis but in itself may give misleading information.Many cellular components(for example,ribosomes)can resemble'virus-like particles,par ticularly in crude preparations.This difficulty can be overcome by using antisera specific for particular virus antigens conjugated to electron-dense markers such as the iron-containing protein ferritin or colloidal gold suspensions.This highly
Since the 1930s, electron microscopes have overcome the fundamental limitation of light microscopes: the inability to resolve individual virus particles owing to physical constraints caused by the wavelength of visible light illumination and the optics of the instruments. The first electron micrograph of a virus (TMV) was published in 1939. Over subsequent years, techniques were developed that allowed the direct examination of viruses at magnifications of over 100,000 times.The two fundamental types of electron microscope are the transmission electron microscope (TEM) and the scanning electron microscope (SEM) (Figure 1.6). Although beautiful images with the appearance of three dimensions are produced by the SEM, for practical investigations of virus structure the higher magnifications achievable with the TEM have proved to be of most value. Two fundamental types of information can be obtained by electron microscopy of viruses: the absolute number of virus particles present in any preparation (total count) and the appearance and structure of the virions (see below). Electron microscopy can provide a rapid method of virus detection and diagnosis but in itself may give misleading information. Many cellular components (for example, ribosomes) can resemble ‘virus-like particles,’ particularly in crude preparations. This difficulty can be overcome by using antisera specific for particular virus antigens conjugated to electron-dense markers such as the iron-containing protein ferritin or colloidal gold suspensions. This highly 16 Principles of Molecular Virology Helix Disk Monomers 5.0 6.0 7.0 8.0 Figure 1.5 The structure and stability of virus particles can be examined by progressive denaturation or renaturation studies. At any particular ionic strength, the purified capsid protein of tobacco mosaic virus (TMV) spontaneously assembles into different structures, dependent on the pH of the solution. At a pH of around 6.0, the particles formed have a helical structure very similar to infectious virus particles. As the pH is increased to about 7.0, disk-like structures are formed. At even higher pH values, individual capsid monomers fail to assemble into more complex structures
SEN Electron /Electron source Electron source gun 不 ■Anode Control grid- Anode■ ◆ ☐Condenser lens First Specimen 0 冂arae len Final image Figure 1.6 Working principles of transmission and scanning electron microscopes specific technique,known as immunoelectron microscopy,is gaining ground as a rapid method for diagnosis. Developments in electron microscopy have allowed investigation of the stru ture of fragile viruses that cannot be determined by x-ray crystallography.These include cryo-electron microscopy,in which the virus particles are maintained at very low temperatures on cooled specimen stages;examination of particles embed- ded in vitreous ice,which does not disrupt the particles by the formation of ice crystals;low-irradiation electron microscopy,which reduces the destructive bom- bardment of the specimen with electrons;and sophisticated image-analysis and image-reconstruction techniques that permit accurate,three-dimensional images to be formed from multiple images that individually would appear as very poor quality.Conventional electron microscopy can resolve structures down to 50 to 70 A in size (a typical atomic diameter is 2-3 A:a protein a-helix,10 A:a DNA
specific technique, known as immunoelectron microscopy, is gaining ground as a rapid method for diagnosis. Developments in electron microscopy have allowed investigation of the structure of fragile viruses that cannot be determined by x-ray crystallography. These include cryo-electron microscopy, in which the virus particles are maintained at very low temperatures on cooled specimen stages; examination of particles embedded in vitreous ice, which does not disrupt the particles by the formation of ice crystals; low-irradiation electron microscopy, which reduces the destructive bombardment of the specimen with electrons; and sophisticated image-analysis and image-reconstruction techniques that permit accurate, three-dimensional images to be formed from multiple images that individually would appear as very poor quality. Conventional electron microscopy can resolve structures down to 50 to 70 Å in size (a typical atomic diameter is 2–3 Å; a protein a-helix, 10 Å; a DNA Introduction 17 Transmission electron microscope (TEM) Scanning electron microscope (SEM) Electron gun Electron source Electron source Anode Anode Electron gun Condenser lens Objective lens Intermediate lens Projector lens Specimen Final Specimen image Control grid First condenser lens Scanning coils Second condenser lens Scanning generator Cathode ray tube (image) Amplifier Electron collector Figure 1.6 Working principles of transmission and scanning electron microscopes
13Principles of Molecular Virology double helix,20 A).Using these newer techniques it is possible to resolve struc- tures of 25 to 30A. In the late 1950s,Sydney Brenner and Robert Horne (among others)devel- oped sophisticated techniques that enabled them to use electron microscopy to reveal many of the fine details of the structure of virus particles.One of the most valuable techniques proved to be the use of electron-dense dyes such as phospho- tungstic acid or uranyl acetate to examine virus particles by negative staining.The small metal ions in such dyes are able to penetrate the minute crevices between the protein subunits in a virus capsid to reveal the fine structure of the particle. Using such data,Francis Crick and James Watson (1956)were the first to suggest that virus capsids are composed of numerous identical protein subunits arranged either in helical or cubic (icosahedral)symmetry.In 1962,Donald Caspar and Aaron Klug extended these observations and elucidated the fundamental principles of symmetry,which allow repeated protomers to form virus capsids,based on the principle of quasi-equivalence (see Chapter 2).This combined theoretical and practical approach has resulted in our current understanding of the structure of virus particles. MOLECULAR BIOLOGY' All of the above techniques of investigation are themselves 'molecular biology'in the original sense of the term;however,the termmolecuar biologyhas taken on the new and different meaning of'genetic engineering'or'genetic manipulation. These techniques for manipulating nucleic acids in vitro (that is,outside living cells or organisms)do not comprise a new discipline but are an outgrowth of earlie developments in biochemistry and cell biology over the previous 50 years.This powerfiul new technology has revolutionized virology and,toa large extent,has shifted the focus of attention away from the virus particle onto the virus genome. Again,this book is not the place to discuss in detail the technical aspects of these methods,and readers are referred to one of the many relevant texts,such as those given at the end of this chapter. Virus infection has long been used to probe the working of 'normal'(i.e.,un- infected)cells -for example,to look at macromolecular synthesis.This is true,for example,of the applications of bacteriophages in bacterial genetics and in many instances where the study of eukaryotic viruses has revealed fundamental informa tion about the cell biology and genomic organization of higher organisms.In 1970, John Kates first observed that vaccinia virus mRNAs were polyadenylated at their 3'ends.In the same year,Howard Temin and David Baltimore jointly identified the enzyme reverse transcriptase(RNA-dependent DNA polymerase)in retrovirus- infected cells.This finding shattered the so-called'central dogma'of biology that there is a one-way flow of information from DNA through RNA into protein and
double helix, 20 Å). Using these newer techniques it is possible to resolve structures of 25 to 30 Å. In the late 1950s, Sydney Brenner and Robert Horne (among others) developed sophisticated techniques that enabled them to use electron microscopy to reveal many of the fine details of the structure of virus particles. One of the most valuable techniques proved to be the use of electron-dense dyes such as phosphotungstic acid or uranyl acetate to examine virus particles by negative staining. The small metal ions in such dyes are able to penetrate the minute crevices between the protein subunits in a virus capsid to reveal the fine structure of the particle. Using such data, Francis Crick and James Watson (1956) were the first to suggest that virus capsids are composed of numerous identical protein subunits arranged either in helical or cubic (icosahedral) symmetry. In 1962, Donald Caspar and Aaron Klug extended these observations and elucidated the fundamental principles of symmetry, which allow repeated protomers to form virus capsids, based on the principle of quasi-equivalence (see Chapter 2). This combined theoretical and practical approach has resulted in our current understanding of the structure of virus particles. ‘MOLECULAR BIOLOGY’ All of the above techniques of investigation are themselves ‘molecular biology’ in the original sense of the term; however, the term ‘molecular biology’ has taken on the new and different meaning of ‘genetic engineering’ or ‘genetic manipulation.’ These techniques for manipulating nucleic acids in vitro (that is, outside living cells or organisms) do not comprise a new discipline but are an outgrowth of earlier developments in biochemistry and cell biology over the previous 50 years. This powerful new technology has revolutionized virology and, to a large extent, has shifted the focus of attention away from the virus particle onto the virus genome. Again, this book is not the place to discuss in detail the technical aspects of these methods, and readers are referred to one of the many relevant texts, such as those given at the end of this chapter. Virus infection has long been used to probe the working of ‘normal’ (i.e., uninfected) cells—for example, to look at macromolecular synthesis. This is true, for example, of the applications of bacteriophages in bacterial genetics and in many instances where the study of eukaryotic viruses has revealed fundamental information about the cell biology and genomic organization of higher organisms. In 1970, John Kates first observed that vaccinia virus mRNAs were polyadenylated at their 3¢ ends. In the same year, Howard Temin and David Baltimore jointly identified the enzyme reverse transcriptase (RNA-dependent DNA polymerase) in retrovirusinfected cells. This finding shattered the so-called ‘central dogma’ of biology that there is a one-way flow of information from DNA through RNA into protein and 18 Principles of Molecular Virology
revealed the plasticity of the eukaryote genome.Subsequently,the purification of this enzyme from retrovirus particles permitted cDNA cloning,which greatly accelerated the study of viruses with RNA genomes- a good illustration of the catalytic nature of scientific advances.In 1977,Richard Roberts and,independ- ently,Phillip Sharp recognized that adenovirus mRNAs were spliced to remove intervening sequences,indicating the similarities between virus and cellular genomes. Initially at least,the effect of this new technology was to shift the emphasis of investigation from proteins to nucleic acids.As the power of the techniques devel oped,it quickly became possible to determine the nucleotide sequences of entire virus genomes,beginning with the smallest bacteriophages in the mid-1970 and working up to the largest of all virus genomes,those of the herpesviruses and poxviruses,many of which have now been determined. This nucleic acid-centred technology,in addition to its ultimate achievement of nucleotide sequencing and the artificial manipulation of virus genomes,also offered significant advances in detection of viruses and virus infections involving nucleic acid hybridization techniques.There are many variants of this basic idea, but,essentially,a hybridization probe,labelled in some fashion to facilitate detec- tion,is allowed to react with a crude mixture of nucleic acids.The specific inter action of the probe sequence with complementary virus-encoded sequences,to which it binds by hydrogen-bond formation between the complementary base pairs,reveals the presence of the virus genetic material (Figure 1.7).This approach has been taken a stage further by the development of various in vitro nucleic acid amplification procedures,such as polymerase chain reaction (PCR),which is an even more sensitive technique,capable of detecting just a single molecule of virus nucleic acid (Figure 1.8). More recently,there has also been renewed interest in virus proteins based on a new biology which is itself dependent on manipulation of nucleic acids in vitro and advances in protein detection arising from immunology.Methods for in vitro synthesis and expression of proteins from molecularly cloned DNA have advanced rapidly,and many new analytical techniques are now available.Studies of protein-nucleic acid interactions are proving to be particularly valuable in under standing virus structure and gene expression.Advances in electrophoresis have made it possible to study simultaneously all of the proteins in a virus-infected cell,called the proteome of the cell (by analogy to the genome). Molecular biologists have one further trick up their sleeves.Because of the repetitive,digitized nature of nucleotide sequences,computers are the ideal means of storing and processing this mass of information.Bioinformatics'is a broad term coined in the 1980s to encompass any application of computers to biology.This can imply anything from artificial intelligence and robotics to genome analysis More specifically,the term applies to computer manipulation of biological sequence data,including protein structural analysis.Bioinformatics permits the inference of
revealed the plasticity of the eukaryote genome. Subsequently, the purification of this enzyme from retrovirus particles permitted cDNA cloning, which greatly accelerated the study of viruses with RNA genomes—a good illustration of the catalytic nature of scientific advances. In 1977, Richard Roberts and, independently, Phillip Sharp recognized that adenovirus mRNAs were spliced to remove intervening sequences, indicating the similarities between virus and cellular genomes. Initially at least, the effect of this new technology was to shift the emphasis of investigation from proteins to nucleic acids. As the power of the techniques developed, it quickly became possible to determine the nucleotide sequences of entire virus genomes, beginning with the smallest bacteriophages in the mid-1970s and working up to the largest of all virus genomes, those of the herpesviruses and poxviruses, many of which have now been determined. This nucleic acid-centred technology, in addition to its ultimate achievement of nucleotide sequencing and the artificial manipulation of virus genomes, also offered significant advances in detection of viruses and virus infections involving nucleic acid hybridization techniques. There are many variants of this basic idea, but, essentially, a hybridization probe, labelled in some fashion to facilitate detection, is allowed to react with a crude mixture of nucleic acids. The specific interaction of the probe sequence with complementary virus-encoded sequences, to which it binds by hydrogen-bond formation between the complementary base pairs, reveals the presence of the virus genetic material (Figure 1.7). This approach has been taken a stage further by the development of various in vitro nucleic acid amplification procedures, such as polymerase chain reaction (PCR), which is an even more sensitive technique, capable of detecting just a single molecule of virus nucleic acid (Figure 1.8). More recently, there has also been renewed interest in virus proteins based on a new biology which is itself dependent on manipulation of nucleic acids in vitro and advances in protein detection arising from immunology. Methods for in vitro synthesis and expression of proteins from molecularly cloned DNA have advanced rapidly, and many new analytical techniques are now available. Studies of protein–nucleic acid interactions are proving to be particularly valuable in understanding virus structure and gene expression.Advances in electrophoresis have made it possible to study simultaneously all of the proteins in a virus-infected cell, called the proteome of the cell (by analogy to the genome). Molecular biologists have one further trick up their sleeves. Because of the repetitive, digitized nature of nucleotide sequences, computers are the ideal means of storing and processing this mass of information. ‘Bioinformatics’ is a broad term coined in the 1980s to encompass any application of computers to biology. This can imply anything from artificial intelligence and robotics to genome analysis. More specifically, the term applies to computer manipulation of biological sequence data, including protein structural analysis. Bioinformatics permits the inference of Introduction 19