Chapter 6: Viruses—Basic Concepts

A virus is a set of genes, composed of either DNA or RNA, packaged in a protein-containing coat called a capsid. Some viruses also have an outer lipid bilayer membrane external to the coat or capsid called an envelope. The resulting complete virus particle is called a virion. Viruses have an obligate requirement for intracellular growth and a heavy dependence on host cell structural and metabolic components. Therefore, viruses are also referred to as obligate intracellular parasites. Viruses do not have a nucleus, cytoplasm, mitochondria, or other cell organelles. Viruses that infect humans are called human viruses, but are considered along with the general class of animal viruses; viruses that infect bacteria are referred to as bacteriophages (phages for short), and viruses that infect plants are called plant viruses.

Virus reproduction requires that a virus particle infect an appropriate host cell and program the cellular machinery to synthesize the viral components required for the assembly of new virions, generally termed progeny virions or daughter viruses. The infected host cell may produce hundreds to hundreds of thousands of new virions, usually accompanied by cell death. Tissue damage as a result of cell death accounts for the pathology of many viral diseases in humans. Many of these viruses cause acute viral infection followed by viral clearance. In some cases, the infected cells survive, resulting in persistent virus production, either a chronic or latent infection that can remain asymptomatic, produce a chronic disease state, or lead to relapse of an infection.

In some circumstances, a virus fails to reproduce itself and, instead, enters a latent state (called lysogeny in the case of bacteriophages), from which there is the potential for reactivation at a later time. A possible consequence of the presence of viral genome in a latent state is a new genotype for the cell. Some determinants of bacterial virulence and some malignancies of animal cells are examples of the genetic effects of latent viruses. Apparently, vertebrates have had to coexist with viruses for a long time because they have evolved the special nonspecific interferon system, which operates in conjunction with the highly specific immune system to combat virus infections.

Two classes of infectious agents exist that are structurally simpler than viruses, namely, viroids and prions. Viroids are infectious circular RNA molecules that lack protein shells; they are responsible for a variety of plant diseases. Prions, which apparently lack any genes and are composed only of protein, are agents that appear to be responsible for some transmissible and inherited spongiform encephalopathies, such as scrapie in sheep; bovine spongiform encephalopathy in cattle; and kuru, Creutzfeldt-Jakob disease, and Gerstmann-Sträussler-Scheinker syndrome in humans.

Viruses are approximately 100- to 1000-fold smaller than the cells they infect. The smallest viruses, virion size (parvoviruses), are approximately 20 nm in diameter (1 nm = 10^–9 m), whereas the largest human viruses (poxviruses) have a diameter of approximately 300 nm (Figure 6–1) and overlap the size of the smallest bacterial cells (Chlamydia and Mycoplasma). Therefore, viruses generally pass through filters designed to trap bacteria, and this property can, in principle, be used as evidence of a viral etiology. Viruses were initially described as filterable agents.

The basic structure of all viruses places the nucleic acid genome (DNA or RNA) on the inside of a protein shell called a capsid. Some human viruses are further packaged into a lipid membrane, or envelope, which is usually acquired from the plasma or cytoplasmic membrane of the infected cell during release from the cell. Viruses that are not enveloped have a defined external capsid and are referred to as naked capsid viruses. The genomes of enveloped viruses form a protein complex and a structure called a nucleocapsid, which is often surrounded by a matrix protein that serves as a bridge between the nucleocapsid and the inside of the viral membrane. Protein or glycoprotein structures called spikes, which often protrude from the surface of virus particles, are involved in the initial contact with receptor on host cells. These basic design features (naked capsid-icosahedral, enveloped-helical nucleocapsid and enveloped-icosahedral capsid) are illustrated schematically in Figure 6–2. Examples of representatives of human/animal viruses electron micrographs are shown in Figure 6–3.

The protein shell forming the capsid or the nucleocapsid assumes one of two basic shapes: cylindrical (helical) or spherical (icosahedral). Examples of these structural categories can be seen in the electron micrographs in Figure 6–3.

The capsid or envelope of viruses functions (1) to protect the nucleic acid genome from damage during the extracellular passage of the virus from one cell to another, (2) to aid in the process of entry into the cell, and (3) in some cases, to package viral enzymes essential for the early steps of the infection process.

In general, the nucleic acid genome of a virus is hundreds of times longer than the longest dimension of the complete virion. It follows that the viral genome must be extensively condensed during the process of virion assembly. For naked capsid viruses, this condensation is achieved by the association of the viral nucleic acid with basic proteins encoded by the virus to form the core of the virus (Figure 6–2). For enveloped viruses, the formation of the nucleocapsid serves to condense the viral nucleic acid genome. The virion may also contain certain virus-encoded essential enzymes and/or accessory/regulatory proteins.

Viral genomes can be made of either RNA or DNA and also can be either single-stranded or double-stranded. The RNA viruses can be either positive sense (indicated by a +) (polarity of mRNA) or negative sense (–) (complementary to or antisense of mRNA), double-stranded (one strand + and the second strand –) or ambisense (both + and – polarity on the same strand). Although the RNA genomes of most viruses are linear, some RNA viruses may also have segmented genomes (several segments or pieces of RNA), with each segment responsible for encoding a protein.

The DNA genome of viruses can be both linear and circular genomes. Most viruses contain a single copy of their genome, except that retroviruses carry two identical copies of its genome and are, therefore, diploid. A few viral genomes (picornaviruses, hepatitis B virus, and adenoviruses) contain covalently attached protein on the ends of the RNA or DNA chains that are remnants of the replication process. Structural diversity among the viruses is most obvious when the makeup of viral genomes is considered.

Subunit Structure of Capsids

The capsids or nucleocapsids are virus-encoded specific proteins that protect the genome and confer shapes to viruses. The capsids of all viruses are composed of many copies of one or, at most, several different kinds of protein subunits. This fact follows from two fundamental considerations. First, all viruses code for their own capsid proteins, and even if the entire coding capacity of the genome were to be used to specify a single giant capsid protein, the protein would not be large enough to enclose the nucleic acid genome. Thus, multiple protein copies are needed, and, in fact, the simplest spherical virus contains 60 identical protein subunits. Second, viruses are such highly symmetrical structures that it is not uncommon to visualize naked capsid viruses in the electron microscope as a crystalline array (eg, simian virus 40 in Figure 6–3B).

The presence of many identical protein subunits in viral capsids or the existence of many identical spikes in the membrane of enveloped viruses has important implications for adsorption, hemagglutination, and recognition of viruses by neutralizing antibodies. Two main architectures are cylindrical (helical symmetry) and spherical (icosahedral or cubic symmetry).

Cylindrical (Helical) Architecture

A cylindrical or helical shape is the simplest structure for a capsid or a nucleocapsid. The first virus to be crystallized and studied in detail was a plant virus called tobacco mosaic virus (TMV). The capsid of TMV is shaped like a rod or a cylinder, with the RNA genome wound in a helix inside it. The capsid is composed of multiple copies of a single kind of protein subunit arranged in a close-packed helix, which places every subunit in the same microenvironment. Because of the helical arrangement of the subunits, viruses that have this type of design are often said to have helical symmetry. The architecture of human viruses with helical symmetry is likely to follow the same general pattern as that of TMV. Thus, the nucleocapsids of influenza virus, parainfluenza virus, measles virus, mumps virus, coronavirus, ebola virus, and rabies virus are likely constructed with a helical arrangement (Figure 6–2, middle) of protein subunits in close association with the nucleic acid genome.

Spherical (Icosahedral) Architecture

The construction of a spherically (icosahedral) shaped virus similarly involves the packing together of many identical subunits, but, in this case, the subunits are placed on the surface of a geometric solid called an icosahedron. An icosahedron has 12 vertices, 30 sides, and 20 triangular faces (Figure 6–4). Because the icosahedron belongs to the symmetry group that crystallographers refer to as cubic (not the cube shape), spherically shaped viruses are said to have cubic symmetry, generally known as icosahedral capsid.

When viewed in the electron microscope, many naked capsid viruses and some nucleocapsids appear as spherical particles with a surface topology that makes it appear that they are constructed of identical ball-shaped subunits (Figure 6–3B and E). These visible structures are referred to as morphologic subunits, or capsomeres. A capsomere is generally composed of either five or six individual protein molecules, each one referred to as a structural subunit, or protomer. In the simplest virus with cubic symmetry, five protomers are placed at each one of the 12 vertices of the icosahedron as shown in Figure 6–4 to form a capsomere called a pentamer. In this case, the capsid is composed of 12 pentamers, or a total of 60 protomers. Note that in the case of helical symmetry, this arrangement places every protomer in the same microenvironment as that of every other protomer.

To accommodate the larger cavity required by viruses with large genomes, the capsids contain many more protomers. These viruses are based on a variation of the basic icosahedron in which the construction involves a mixture of pentamers and hexamers rather than only pentamers. A detailed description of this higher level of virus structure is beyond the scope of this text. Examples of icosahedral capsids are shown in Figure 6–5.

Special Surface Structures

Many viruses have structures that protrude from the surface of the virion generally known as spikes or peplomers. In virtually every case, these structures are important for the two earliest steps of infection—adsorption and penetration. The most dramatic example of such a structure is the tail of some bacteriophages which acts as a channel for the transfer of the genome into the bacterial cell. Other examples of surface structures include the spikes of adenovirus (Figure 6–3E) and the glycoprotein spikes found in the membrane of enveloped viruses (see influenza virus in Figure 6–3D). Even viruses without obvious surface extensions probably contain short projections which, like the more obvious spikes, are involved in the specific binding of the virus to the cell surface.

Envelope Structure

Many human viruses have an outer lipid bilayer membrane that is derived from cellular membranes, mainly the plasma membrane, but also, in some cases, cytoplasmic or nuclear membranes. The viral envelope lipid layer membrane contains virus-encoded glycoproteins called “spikes” or “peplomers” or “viral envelope proteins.” The envelope spikes bind to the receptor on the host cells, help the virus envelope membrane fuse with the cellular membrane of the host cells, and act as principal antigens against which the host mounts immune response for the recognition of the virus. Enveloped viruses have another protein, the matrix protein, which serves as a bridge between nucleocapsid and inner membrane of the envelope (Figure 6–2). Examples of enveloped viruses are shown in Figure 6–6, with both helical (Figure 6–6A and B) and icosahedral or cubic (Figure 6–6C and D) symmetry.

Enveloped viruses are more sensitive to detergents, solvents, ethanol, ether, and heat compared with nonenveloped (naked capsid) viruses whose outer coat is capsid protein. Both envelope glycoproteins, and naked capsid viruses’ spikes become antigens after infection and the host mounts both cell-mediated and humoral immune responses for the elimination of virus-infected cells and cell-free virus, respectively. These antigens determine the viral serotypes that are based on antigenic variation and are type-specific such as poliovirus serotypes 1, 2, and 3. Viral serotypes have cross-reactivity but, often, little cross-protection. Viral serotypes arise because of antigenic variations that allow viruses to escape preexisting immune response.

The classification of viruses has evolved at a slower pace than other microorganisms. The International Committee for Taxonomy of Viruses (ICTV) considered various properties, including virions, genome, proteins, envelope, replication, and physical and biologic properties. Based on these properties, virus families are designated with the suffix, -viridae (as in Herpesviridae), virus subfamilies with suffix virinae (Herpesvirinae), virus genera with suffix virus (Herpesvirus), and virus species designated by a virus type (herpes simplex virus 1). Tables 6–1 and 6–2 present a classification scheme for human RNA and DNA viruses, respectively, which is based solely on their structure. The viruses are arranged in order of increasing virion size. It is important to bear in mind that phylogenetic relationships cannot be inferred from this taxonomic scheme. The tables should not be memorized, but rather used as a reference guide to virus structure. In general, viruses with similar structures exhibit similar replication strategies, as discussed later.

Representative and important bacteriophages are listed along with their properties in Table 6–3. In the chapters that follow, the properties of the well-studied temperate bacteriophage, λ, are described to illustrate the replicative strategies of the more medically important, but less well-studied, β phage of Corynebacterium diphtheriae.

Virus Replication

Virus replication cycle typically consists of six discrete phases: (1) adsorption or attachment to the host cell, (2) penetration or entry, (3) uncoating to release the genome, (4) synthetic or virion component production, (5) assembly, and (6) release from the cell. These phases are shown in a general scheme of virus replication cycle in Figure 6–7.

This series of events, sometimes with slight variations, describes what is called the productive or lytic response; however, this is not the only possible outcome of a virus infection. Some viruses can also enter into a very different kind of relationship with the host cell in which no new virus is produced, the cell survives and divides, and the viral genetic material persists indefinitely in a latent state. This outcome of an infection is referred to as the nonproductive response. The nonproductive response in the case of bacteriophages is called lysogeny and, in several human and animal viruses under some circumstances, may be associated with oncogenic transformation. (This use of the term transformation is to be distinguished from DNA transformation of bacteria discussed in Chapter 21.)

Some viruses can also cause a chronic infection where a low level of the virus is produced with little or no damage to the target tissue. Both latent infection and chronic infection are called persistent infection. Virus replication also depends on virus–host cell interaction such as the type of cells it infects—whether permissive or nonpermissive cells. Permissive cells are those that permit production of progeny virus particles and/or viral transformation. However, nonpermissive cells do not allow virus replication, but may allow virus transformation. Some viruses enter cells that do not support virus replication, but some early viral proteins cause cell death; this infection is termed abortive infection.

The outcome of an infection depends on the particular virus–host combination and on other factors such as the extracellular environment, multiplicity of infection, and physiology and developmental state of the cell. Viruses that can enter only into a productive relationship are called lytic or virulent viruses. Viruses that can establish either a productive or a nonproductive relationship with their host cells are referred to as temperate viruses. Some temperate viruses can be reactivated or “induced” to leave the latent state and enter into the productive response. Whether induction occurs depends on the particular virus–host combination, the physiology of the cell, and the presence of extracellular stimuli.

Viruses are generally propagated in the laboratory by mixing the virus and susceptible cells together and incubating the infected cells until lysis occurs. After lysis, the cells and cell debris are removed by a brief centrifugation, and the resulting supernatant is called a lysate.

The growth of human viruses requires that the host cells be cultivated in the laboratory, mostly in human or animal cell lines (cell derived from tumors or cells transformed by viruses) and, in some cases, in primary cells derived from tissues. To prepare cells for growth in vitro, a tissue is removed from an animal, and the cells are disaggregated using the proteolytic enzyme trypsin. The cell suspension is seeded into a plastic Petri dish in a medium containing a complex mixture of amino acids, vitamins, minerals, and sugars. In addition to these nutritional factors, the growth of animal cells requires components present in animal serum. This method of growing cells is referred to as tissue culture, and the initial cell population is called a primary culture. The cells attach to the bottom of the plastic dish and remain attached as they divide and eventually cover the surface of the dish. When the culture becomes crowded, the cells generally cease dividing and enter a resting state. Propagation can be continued by removing the cells from the primary culture plate using trypsin and reseeding a new plate.

Cells taken from a normal (as opposed to cancerous) tissue cannot usually be propagated in this manner indefinitely. Eventually, most of the cells die; a few may survive, and these survivors often develop into a permanent cell line. Cell lines can also be generated directly from tumors or from virus transformed cells. Such cell lines are very useful as host cells for isolating and assaying viruses in the laboratory, but they rarely bear much resemblance to the tissue from which they originated. When cells are taken from a tumor and cultivated in vitro, they display a very different set of growth properties, including long-term survival, reflecting their tumor phenotype.

When a virus is propagated in tissue culture cells, the cellular changes induced by the virus, which usually culminate in cell death, are often characteristic of a particular virus and are referred to as the cytopathic effect of the virus.

Viruses are quantitated by a method called the plaque assay (see Plaque Assay under Quantitation of Viruses for a detailed description of the method). Briefly, viruses are mixed with cells on a Petri plate so that each infectious particle gives rise to a zone of lysed or dead cells called a plaque. From the number of plaques on the plate, the titer of infectious particles in the lysate is calculated. Virus titers are expressed as the number of plaque-forming units per milliliter (pfu/mL).

The purpose of a one-step growth experiment is to understand various stages of viral infection. To describe an infection in temporal and quantitative terms, it is useful to perform a one-step growth experiment (Figure 6–8). The objective in such an experiment is to infect every cell in a culture so that the whole population proceeds through the infection process in a synchronous fashion. The ratio of infecting plaque-forming units to cells is called the multiplicity of infection (MOI). By infecting at a high MOI (eg, 10, as in Figure 6–8), one can be certain that every cell is infected.

The time course and efficiency of adsorption can be followed by the loss of infectious virus from the medium after removal of the cells (blue line in Figure 6–8). In the example shown, adsorption takes approximately half an hour, and all but 1% of the virus is adsorbed. If samples of the culture containing the infected cells are treated so as to break open the cells before assaying for virus (red line in Figure 6–8), it can be observed that infectious virus initially disappears, because no infectious particles are detectable above the background of unadsorbed virus. The period of infection in which no infectious viruses are found inside the cell is called the eclipse phase and emphasizes that the original virions lose their infectivity soon after entry. Infectivity is lost because, as is discussed later, the virus particles are dismantled as a prelude to their reproduction. Later, infectious virus particles rapidly reappear in increasing numbers and are detected inside the cell prior to their release into the environment (Figure 6–8). The length of time from the beginning of infection until progeny virionsare found outside the cells is referred to as the latent period. Latent periods range from 20 minutes to hours for bacteriophages and from a few hours to many days for human viruses.

The time in the infection at which genome replication begins is typically used to divide the infection operationally into early and late phases. Early viral gene expression is largely restricted to the production of the proteins required for genome replication; later, the proteins synthesized are, primarily, those necessary for construction of the new virus particles.

The average number of plaque-forming units released per infected cell is called the burst size for the infection. In the example shown, the burst size is approximately 1000. Burst sizes range from less than 10 for some relatively inefficient infections to millions for some highly virulent viruses.

Adsorption or Attachment

The first step in every viral infection is the attachment or adsorption of the infecting virus particle to the surface of the host cell. A prerequisite for this interaction is a collision between the virion and the cell. Viruses do not have any capacity for locomotion and, therefore, the collision event is simply a random process determined by diffusion. Therefore, similar to any bimolecular reaction, the rate of adsorption is determined by the concentrations of both the virions and the cells.

Only a small percentage of the collisions between a virus and its host cell lead to a successful infection because adsorption is a highly specific reaction that involves protein molecules on the surface of the virion called virion attachment proteins or spikes and certain molecules on the surface of the cell that are called receptors. Typically, 10⁴ to 10⁵ receptors are on the cell surface. Receptors for some bacteriophages are found on pili of bacteria, although most adsorb to receptors found on the bacterial cell wall. Receptors for human viruses are usually glycoproteins located in the plasma membrane of the cell. Table 6–4 lists some of the receptors that have been identified for medically important viruses. It appears that viruses have evolved to make use of a wide variety of surface molecules as receptors, which are normally signaling devices or immune system components. Any attempts to design agents that block viral infections by binding to the receptors for a long time must consider the possibility that the loss of the normal cellular function associated with the receptors would have serious consequences for the host organism.

For some viruses, two different surface molecules, called coreceptors, are involved in adsorption. Although CD4 was originally thought to be the sole receptor for human immunodeficiency virus type 1 (HIV-1), the discovery of a family of coreceptors that normally function as chemokine receptors (CCR5 and CXCR4) may explain why natural resistance against the virus is found in some individuals with variant forms (Δ32CCR5) of these signaling molecules (discussed in Chapter 18). Although receptors for some human viruses such as influenza viruses are present on lungs cells, these receptors are also found on red blood cells of certain species that are responsible for the phenomena of hemagglutination and hemadsorption discussed later.

Virion attachment proteins are often associated with conspicuous features on the surface of the virion. For example, the virion attachment proteins for the bacteriophages with tails are located at the very end of the tails or the tail fibers. Similarly, the spikes found on adenoviruses (Figure 6–3E) and on virtually all the enveloped human viruses (Figure 6-3D) contain the virion attachment proteins.

In some cases, a region of the capsid protein serves the function of the attachment protein. For polioviruses, rhinoviruses, and probably other picornaviruses, the region on the capsid that binds to the receptor is found at the bottom of a cleft, trough or canyon that is too narrow to allow access to antibodies. This particular arrangement is clearly advantageous to the virus because it precludes the production of antibodies that might directly block receptor recognition.

The repeating subunit structure of capsids and the multiplicity of spikes on enveloped viruses are probably important in determining the strength of the binding of the virus to the cell. The binding between a single virion attachment protein and a single receptor protein is relatively weak, but the combination of many such interactions lead to a strong association between the virion and the cell. The fluid nature of the human cell membrane may facilitate the movement of receptor proteins to allow the clustering that is necessary for these multiple interactions.

A particular kind of virus is capable of infecting only a limited spectrum of cell types called its host range. Thus, although a few viruses can infect cells from different species, most viruses are limited to a single species. For example, dogs do not contract measles, and humans do not contract distemper. In many cases, human viruses infect only a particular subset of the cells found in their host organism. This kind of tissue tropism is clearly an important determinant of viral pathogenesis. In most cases studied, the specific host range of a virus and its associated tissue tropism are determined at the level of the binding between the cell receptors and virion attachment proteins. Thus, these two protein components must possess complementary surfaces that fit together in much the same way as a substrate fits into the active site of an enzyme. It follows that adsorption occurs only in that percentage of collisions that leads to successful binding between receptors and attachment proteins, and that the inability of a virus to infect a cell type is usually due to the absence of the appropriate receptors on the cell. A few cases are known in which the host range of a virus is determined at a step after adsorption and penetration, but these are the exceptions rather than the rule.

When a virus particle has penetrated to the inside of a cell, it is essentially hidden from the host immune system. Thus, if protection from a virus infection is to be accomplished at the level of antibody binding to the virions, it must occur before adsorption and prevent the virus from attaching to and penetrating the cell. It is, therefore, not surprising that most neutralizing antibodies—whether elicited as a result of natural infection or vaccination—are specific for virion attachment proteins.

The disappearance of infectious virus during the eclipse phase is a direct consequence of the fact that viruses are dismantled before being replicated. As discussed later in the text, the uncoating step may be simultaneous with entry or may occur in a series of steps. Ultimately, the nucleocapsid or core structure must be transported to the site or compartment in the cell where transcription and replication will occur.

The Bacteriophage Strategy

The processes of penetration and uncoating are simultaneous for all bacteriophages. Thus, the viral capsids are shed at the surface, and only the nucleic acid genome enters the cell. In some cases, a small number of virion proteins may accompany the genome into the cell, but these are probably tightly associated with the nucleic acid or are essential enzymes needed to initiate the infection.

Bacteriophages with tails are responsible for the attachment of the virion to the bacterial cell wall to facilitate the entry of the genome into the cell. The DNA of the bacteriophage is injected from the head directly into the cell through the hollow tail structure. The process has been likened to the action of a syringe.

Enveloped Human Viruses

There are two basic mechanisms for the entry of an enveloped human virus into the cell. Both mechanisms involve fusion of the viral envelope with a cellular membrane, and the end result in both cases is the release of the free nucleocapsid into the cytoplasm. What distinguishes the two mechanisms is the nature of the cellular membrane that fuses with the viral envelope.

Paramyxoviruses (eg, measles), some retroviruses (eg, HIV-1), and herpesviruses enter by a process called direct fusion (Figure 6–9). The envelopes of these viruses contain protein spikes that promote fusion of the viral membrane with the plasma membrane of the cell, releasing the nucleocapsid directly into the cytoplasm. Because the viral envelope becomes incorporated into the plasma membrane of the infected cell and still possesses its fusion proteins, infected cells have a tendency to fuse with other uninfected cells. Cell-to-cell fusion is a hallmark of infections by paramyxoviruses and HIV-1, and can be important in the pathology of diseases such as measles, respiratory syncytial virus (RSV)-induced bronchiolitis, and acquired immunodeficiency syndrome (AIDS).

The mechanism for the entry of most of the remaining enveloped human viruses, such as orthomyxoviruses (eg, influenza viruses), togaviruses (eg, rubella virus), rhabdoviruses (eg, rabies), and coronaviruses, is shown in Figure 6–10. After adsorption, the virus particles are taken up by a cellular mechanism called receptor-mediated endocytosis, which is normally responsible for internalizing growth factors, hormones, and some nutrients. When it involves viruses, the process is referred to as viropexis.

In viropexis, the adsorbed virions become surrounded by the plasma membrane in a reaction that is probably facilitated by the multiplicity of virion attachment proteins on the surface of the particle. Pinching off of the cellular membrane by fusion encloses the virion in a cytoplasmic vesicle termed the endosomal vesicle. The nucleocapsid is now surrounded by two membranes: the original viral envelope and the newly acquired endosomal membrane. The surface receptors are subsequently recycled back to the plasma membrane, and the endosomal vesicle is acidified by a normal cellular process. The low pH of the endosome leads to a conformational change in a viral spike protein, which results in the fusion of the two membranes and release of the nucleocapsid into the cytoplasm. In some cases, the contents of the endosomal vesicle may be transferred to a lysosome before the fusion step that releases the nucleocapsid.

Naked Capsid Human Viruses

Naked capsid human viruses, such as poliovirus, reovirus, and adenovirus, also appear to enter the cell by viropexis (Figure 6–10). However, in this case, the virus cannot escape the endosomal vesicle by membrane fusion as described earlier for some enveloped viruses. For poliovirus, it appears that the viral capsid proteins in the low-pH environment of the endosome expose hydrophobic domains. This process results in the binding of the virions to the membrane and release of the nucleic acid genome into the cytoplasm. In other cases, the virions may escape into the cytoplasm by simply promoting the lysis of the vesicle. This step is a potential target of antiviral chemotherapy, and some drugs have been developed that bind to the capsids of picornaviruses and prevent the release of the virus particles from the endosome.

Reovirus is unusual in that, before release into the cytoplasm, the contents of the endosome are transferred to a lysosome where the lysosomal proteases strip away part of the capsid proteins and activate virion-associated enzymes required for transcription.

Synthetic or virion production is the most important step in the viral replication cycle because the virus must make mRNAs, proteins, and genomes for the assembly of progeny or daughter viruses. In the case of bacteriophages, there is evidence that the entering nucleic acid must be directed to a particular locus in the bacterial cell to initiate the infection process. Pilot proteins accompany the bacteriophage genome to a specific site into the bacterial cell where transcription and replication occur.

For human viruses, the ultimate fate of internalized virus particles depends on the particular virus and on the cellular compartment where replication occurs. Most RNA viruses replicate in the cytoplasm—the immediate site of entry with the exception of influenza viruses and the retroviruses that replicate in the nucleus. All DNA viruses must move from the cytoplasm to the nucleus to replicate, except the poxviruses that replicate in the cytoplasm. The larger DNA viruses, such as herpesviruses and adenoviruses, must uncoat to the level of cores before entry into the nucleus. The smaller DNA viruses, such as the parvoviruses and the papilloma/polyoma viruses, enter the nucleus intact through the nuclear pores and subsequently uncoat inside. The largest of the human viruses, the poxviruses, carry out their entire replicative cycle in the cytoplasm of the infected cell because they make both RNA and DNA polymerases.

From Genome to mRNA

An essential step in every virus infection is the production of virus-specific mRNAs that program the cellular ribosomes to synthesize viral proteins. Besides the structural proteins of the virion, viruses must direct the synthesis of enzymes and other specialized proteins required for genome replication, gene expression, and virus assembly and release. The production of the first viral mRNAs at the beginning of the infection is a crucial step in the takeover of the cell by the virus.

For some viruses, the presentation of mRNA to the cellular ribosomes poses no problems. Thus, the genomes of most DNA viruses are transcribed by the host DNA-dependent RNA polymerase (RNA polymerase II) in the nucleus to yield the viral mRNAs which are exported to the cytoplasm for translation. The (+) strand RNA viruses, such as the picornaviruses, the togaviruses, the flaviviruses, the coronaviruses, the caliciviruses, and the herpesviruses (hepatitis E virus) possess genomes that can be used directly as mRNAs and are translated (at least partially, as discussed later) immediately on entry into the cytoplasm of the cell. One of these viral proteins is RNA-dependent RNA polymerase (also known as viral RNA polymerase or RNA transcriptase) required to synthesize new mRNAs and genomic RNA.

However, for many viruses, the production of mRNA starting from the genome is not so straightforward. The fact that DNA virus such as poxvirus replicates in the cytoplasm means that the cellular RNA polymerase is not available to transcribe the viral DNA genome. Moreover, no cellular machinery exists that can use either single- or double-stranded RNA as a template to synthesize mRNA. Therefore, the poxviruses and viruses that use an RNA template, especially the (–) strand RNA viruses such as the rhabdoviruses, orthomyxoviruses, paramyxoviruses, filoviruses, to make mRNAs must provide their own transcription machinery to produce the viral mRNAs at the beginning of the infection process. This feat is accomplished by synthesizing the polymerases or transcriptases in the later stages of viral development in the previous host cell and packaging the enzymes into the virions, where they remain associated with the genome as the virus enters the new cell and uncoats. In general, the presence of a polymerase or transcriptase in virions is indicative that the host cell is unable to use the viral genome as mRNA or as a template to synthesize mRNA. At later times in the infection, any special enzymatic machinery required by the virus and not initially present in the cell can be supplied among the proteins translated from the first mRNA molecules.

The pathways for the synthesis of mRNA by the major virus groups are summarized in Figure 6–11 and related to the structure of viral genomes. The polarity of mRNA is designated as (+) and the polarity of antisense or complementary to mRNA as (–). The black arrows denote synthetic steps for which host cells provide the required enzymes, whereas the colored arrows indicate synthetic steps that must be carried out by virus-encoded enzymes. Several additional points should be emphasized. The parvoviruses and some bacteriophages have single-stranded DNA genomes. Although the RNA polymerase of the cell requires double-stranded DNA as a template, these viruses need not to carry special enzymes in their virions because host cell DNA polymerases can convert the single-stranded DNA genomes into double-stranded DNA. Note that the production of more mRNA by the picornaviruses and similar (+)-strand RNA viruses requires the synthesis of an intermediate (–)-strand RNA template. The enzyme required for this process is produced by translation of the genome RNA early in infection called RNA-dependent RNA polymerase.

The retroviruses are a special class of (+)-strand RNA viruses. Although their genomes are the same polarity as mRNA and could, in principle, serve as mRNAs early after infection, their replication scheme apparently precludes this. Instead, the RNA genomes of these viruses are copied into (–) DNA strands by an enzyme carried within the virion called reverse transcriptase (RNA dependent DNA polymerase). The (–) DNA strands are subsequently converted by the same enzyme to double-stranded DNA in a reaction that requires the degradation of the original genomic RNA by the RNase H activity of the reverse transcriptase enzyme. The viral DNA product of reverse transcription is integrated into the host cell DNA and ultimately transcribed by the host RNA polymerase to complete the replication cycle as well as produce viral mRNA. The replication of the hepatitis B virus DNA genome is mechanistically similar to that of a retrovirus. Thus, the hepatitis B viral DNA is transcribed to produce a single-stranded RNA by the host RNA polymerase, which in turn is reverse transcribed to produce the progeny viral DNA that is encapsidated into virions.

The Monocistronic mRNA Rule in Human Cells

The ribosome requires input of information in the form of mRNA. For a viral mRNA to be recognized by the ribosome, its production must conform to the rules of structure that govern the synthesis of the cellular mRNAs. Prokaryotic or bacterial mRNA is relatively simple and can be polycistronic, which means it can contain the information for several proteins. Each cistron or coding region is translated independently beginning from its own ribosome binding site.

Eukaryotic mRNAs are structurally more complex, containing special 5′-cap and 3′-poly(A) attachments. In addition, their synthesis often involves removal of internal sequences (introns) and joining of coding sequences (exons) by a process called splicing. Most important, almost all eukaryotic mRNAs are monocistronic, which means one mRNA encodes one protein. Accordingly, eukaryotic translation is initiated by the binding of a ribosome to the 5′-cap, followed by movement of the ribosome along the RNA until the first AUG initiation codon is encountered. The outcome to this first AUG rule is that eukaryotic ribosomes, unlike prokaryotic ribosomes, generally cannot initiate translation at internal sites on an mRNA. To conform to the monocistronic mRNA, most human viruses produce mRNAs that are translated to yield only a single polypeptide chain (protein) following initiation near the 5′ end of the mRNA.

Because most DNA human viruses replicate in the nucleus, they adhere to the monocistronic mRNA rule either by having a promoter precede each gene or by programming the transcription of precursor RNAs that are processed by nuclear splicing enzymes into monocistronic mRNAs (Figure 6–12A). The virion transcriptase or polymerase of the cytoplasmic poxviruses apparently must synthesize monocistronic mRNAs by initiation of transcription in front of each gene.

RNA human viruses have evolved three strategies to circumvent or conform to the monocistronic mRNA rule. The simplest strategy involves having a segmented RNA genome (Figure 6–12B). For the most part, each genome segment of the orthomyxoviruses and the reoviruses corresponds to a single gene; therefore, the mRNA transcribed from a given segment constitutes a monocistronic mRNA. Unlike most RNA viruses, the orthomyxovirus virus (influenza virus) replicates in the nucleus, and some of its monocistronic mRNAs are produced by splicing of precursor RNAs by host cell enzymes. Moreover, orthomyxoviruses use small 5′ RNA fragments derived from host cell pre-mRNAs, found in the nucleus, to prime the synthesis of their own mRNAs. However, the synthesis of mRNA and genomic RNA is completed by the viral RNA dependent RNA polymerase.

A second solution to the monocistronic mRNA rule mainly seen in negative-strand RNA viruses that carry RNA-dependent RNA polymerase in its virus particle. The negative-strand RNA viruses, including paramyxoviruses, rhabdoviruses, filoviruses, bunyaviruses, and arenaviruses, and some positive-strand RNA viruses, such as togaviruses and coronaviruses, synthesize monocistronic mRNAs by initiating the synthesis of each mRNA at the beginning of a gene. In most cases, the RNA-dependent RNA polymerase terminates mRNA synthesis at the end of the gene such that each message corresponds to a single gene (Figure 6–12C). For coronaviruses and togaviruses, the positive-strand RNA is initially translated to synthesize RNA-dependent RNA polymerase, which transcribes positive-strand RNA into a negative-strand RNA intermediate that is used as a template for RNA synthesis. RNA synthesis is initiated on the negative-strand RNA intermediate template at the beginning of each gene and continues to the end of the genome so that a nested set of mRNAs is produced. However, each mRNA is functionally monocistronic and is translated to produce only the protein encoded near its 5′ end.

The positive-strand RNA viruses such as picornaviruses and flaviviruses have evolved yet a third strategy to deal with the monocistronic mRNA requirement (Figure 6–12D). The (+)-strand genome contains just a single ribosome binding site near the 5′ end. It is translated into one long polypeptide chain called a polyprotein, which is subsequently broken into the final set of protein products by a series of proteolytic cleavages. Most of the required protease activities reside within the polyprotein itself.

Several viruses use more than one of these strategies to conform to the monocistronic mRNA rule. For example, retroviruses, togaviruses, arenaviruses, and bunyaviruses synthesize multiple mRNAs, each one coding for a polyprotein that is subsequently cleaved into the individual protein molecules. For some viruses such as retrovirus (HIV) and flavivirus (HCV), viral protease enzyme that cleaves polyprotein can be inhibited by potent antivirals (protease inhibitors) in infected patients.

DNA Viruses

Host cells contain the enzymes and accessory proteins that are required for the replication of DNA. In bacteria, these proteins are present continuously, whereas in the eukaryotic cells they are present only during the S phase of the cell cycle, and restricted to the nucleus. The extent to which viruses use the cell replication machinery depends on their protein-coding potential and, thus, on the size of their genome.

The smallest of the DNA viruses, the parvoviruses, are so completely dependent on host machinery that they require the infected cells to be dividing to ensure that a normal S phase occurs and replicates the viral DNA together with the cellular DNA. At the other end of the spectrum are the large DNA viruses, which are relatively independent of cellular functions. The largest bacteriophages such as T4 degrade the host (bacterial) cell chromosome early in infection and replace all the host replication machinery with bacteriophage-specified proteins. The largest human viruses, the poxviruses, are similarly independent of the host. Because they replicate in the cytoplasm, they must code for almost all of the enzymes and other proteins required for replicating their DNA.

The remainder of the DNA viruses is only partially dependent on host machinery. For example, bacteriophages ϕX174 and λ code for proteins that direct the initiation of DNA synthesis to the viral origin. However, the actual synthesis of DNA occurs by the complex of cellular enzymes responsible for replication of the Escherichia coli DNA. Similarly, the small DNA human viruses, such as the polyomaviruses and papillomaviruses (formerly known as papovaviruses), code for a protein that is involved in the initiation of synthesis at the origin, but the remainder of the replication process is carried out by host machinery. The somewhat more complex adenoviruses and herpesviruses, in addition to providing origin-specific proteins, also encode for their own DNA polymerases and other accessory proteins required for DNA replication.

The fact that the herpesviruses encode for their own DNA polymerase has important implications for the treatment of infections by these viruses and illustrates a central principle of antiviral chemotherapy. Certain antiviral drugs such as acyclovir (acycloguanosine) preferentially kills herpesvirus-infected cells because the viral thymidine kinase, unlike the cellular counterpart, phosphorylate the nucleoside analog, converting it to a form that inhibits further DNA synthesis when DNA polymerases incorporate it into DNA. The host cell enzyme is more discriminating and fails to phosphorylate the acyclovir analog and inhibit synthesis of cellular DNA; thus, this drug does not kill uninfected cells. Similar principle applies to the chain-terminating drugs such as zidovudine (ZDV or AZT) and dideoxyinosine (ddI) that are phosphorylated by cellular kinase and target not only the HIV-1 reverse transcriptase but also inhibit cellular DNA polymerase to some extent. In principle, any viral process that is distinct from a normal cellular process is a potential target for antiviral drugs such as HIV protease and integrase inhibitors and HCV protease and polymerase inhibitors. As more knowledge becomes available about the details of viral replication, more antiviral drugs will become available that are targeted to these unique viral processes.

As noted earlier, with the exception of the poxviruses, all the DNA human viruses are at least partially dependent on host cell machinery for the replication of their genomes. However, unlike the parvoviruses, the other DNA viruses do not need to infect dividing cells for a productive infection to ensue. Instead, all these viruses code for a protein expressed early in infection that induces an unscheduled cycle of cellular DNA replication (S phase). In this way, these viruses ensure that the infected cell makes all the machinery required for the replication of their own DNA. It is noteworthy that all the DNA viruses except the parvoviruses are capable, in some circumstances, of transforming a normal cell into an abnormal or cancerous cell. This correlation suggests that the unlimited proliferative capacity of the cancer cells may be due to the continual synthesis of the viral protein(s) responsible for inducing the unscheduled S phase in a normal infection. The fact that these DNA viruses can induce oncogenic transformation of cell types that are nonpermissive for viral multiplication may simply be an accident related to the need to induce cellular enzymes required for DNA replication during the lytic infection.

All DNA polymerases, including those encoded by viruses, synthesize DNA chains by the successive addition of nucleotides onto the 3′ end of the new DNA strand. Moreover, all DNA polymerases require a primer terminus containing a free 3′-hydroxyl to initiate the synthesis of a DNA chain. In cellular replication, a temporary primer is provided in the form of a short RNA molecule. This primer (RNA) is synthesized by an RNA polymerase, and after elongation by the DNA polymerase, it is removed. With circular chromosomes, such as those found in bacteria and many viruses, the unidirectional chain growth and primer requirement of the DNA polymerase pose no structural problems for replication. However, as illustrated in Figure 6–13, when a replication fork encounters the end of a linear DNA molecule, one of the new chains (heavy lines) cannot be completed at its 5′ end, because there exists no means of starting the DNA portion of the chain exactly at the end of the template DNA. Thus, after the RNA primer is removed, the new chain is incomplete at its 5′ end. This constraint on the completion of DNA chains on a linear template is called the end problem in DNA replication. Some eukaryotic cells add short repetitive sequences to chromosome ends using an enzyme called telomerase to prevent the shortening of the DNA with each successive round of replication.

Several viruses are faced with the end problem during replication of their linear genomes, but none uses the cellular telomerase to synthesize DNA ends. It is beyond the scope of this book to detail all of the strategies that viruses have evolved to deal with the end problem, but it is worth mentioning some of the structural features found in linear viral genomes whose presence is related to solutions of the end problem. These structures are diagrammed schematically in Figure 6–14. The linear double-stranded genome of bacteriophage λ possesses 12-bp single-stranded extensions that are complementary in sequence to each other and, thus, called cohesive ends. Very early after entry into the cell, the two ends pair up to convert the linear genome into a circular molecule to avoid the end problem in replication. The linear double-stranded adenovirus genome contains a protein molecule covalently attached to the 5′ end of both strands. These proteins provide the primers required to initiate the synthesis of the DNA chains during replication, circumventing the need for RNA primers and, thus, solving the end problem in replication. The single-stranded parvovirus genome contains a self-complementary sequence at the 3′ end, which causes the molecule to fold into a hairpin and make it self-priming for DNA replication. The poxviruses contain linear double-stranded genomes in which the ends are continuous. With the parvovirus and poxvirus genomes, the solutions to the end problem create additional problems that must be solved to produce replication products that are identical to the starting genomes.

RNA Viruses

Because nuclear functions are primarily designed for DNA metabolism, RNA viruses mostly replicate in the cytoplasm. Moreover, cells do not have RNA polymerases that can copy RNA templates (RNA-based RNA transcription or replication). Therefore, RNA viruses not only need to encode for transcriptases or polymerases (required for transcription), as discussed earlier, but also must provide the replicases or polymerases required to duplicate the RNA genome into daughter RNA genomes. Furthermore, except in the cases of the picornaviruses, in which transcription and replication are synonymous, the RNA viruses must temporally and functionally separate transcription from replication. This requirement is especially apparent for the rhabdoviruses, paramyxoviruses, togaviruses, and coronaviruses, in which a complete genome, or complementary copy of the genome, is transcribed into a set of small monocistronic mRNAs early in infection. After replication begins, these same templates are used to synthesize full-length strands for replication.

Two mechanisms exist to separate the process of transcription from replication. First, in some cases, transcription is restricted to subviral particles and involves a transcriptase transported into the cell within the virion. Second, in other cases, the replication process either involves a functionally distinct RNA polymerase or depends on the presence of some other viral-specific accessory protein that directs the synthesis of full-length copies of the template rather than the shorter monocistronic mRNAs. In reoviruses, the switch from transcription to replication appears to involve the synthesis of a replicase that converts the (+) mRNAs synthesized early in infection to the double-stranded genome segments.

Viral RNA polymerases, similar to DNA polymerases, synthesize chains in only one direction; however, in general, RNA polymerases can initiate the synthesis of new chains without primers. Thus, there is no obvious end problem in RNA replication. There is one exception to this general rule. The picornaviruses contain a protein that is covalently attached to the 5′ end of the genome, called VPg. This protein is present on the viral RNA because it is involved in the priming of new RNA viral genomes during the infection, similar to the process described earlier for adenoviruses.

The process of enclosing the viral genome in a protein capsid is called assembly or encapsidation. Four general principles govern the construction of capsids and nucleocapsids. First, the process generally involves self-assembly of the component parts. Second, assembly is stepwise and ordered. Third, individual protein structural subunits or protomers are usually preformed into capsomeres in preparation for the final assembly process. Fourth, assembly often initiates at a particular locus on the genome called a packaging site.

Viruses with Helical Symmetry

The assembly of the helical or cylindrically nucelocapsids has been extensively studied in the TMV. In helical symmetry, doughnut-shaped disks containing a number of individual structural subunits are preformed and added stepwise to the growing structure. Elongation occurs in both directions from a specific packaging site on the single-stranded viral RNA. The addition of each disk involves an interaction between the protein subunits of the disk and the genome RNA. The nature of this interaction is such that the assembly process ceases when the ends of the RNA are reached. The structural subunits as well as the RNA trace out a helical path in the final virus particle. The individual protein subunits are intimately associated with the RNA and that the nucleoprotein complexes are assembled by the stepwise addition of protein subunits or complexes of subunits.

For influenza and other helical viruses with segmented genomes, the various genome segments are assembled into nucleocapsids independently and then brought together during virion assembly by a mechanism that is as yet poorly understood. It is notable that virtually all of the human RNA viruses with helical symmetry are enveloped.

Viruses with Icosahedral or Cubic Symmetry

For both human viruses and bacteriophages, icosahedral capsids are generally preassembled and the nucleic acid genomes, usually complexed with condensing proteins, are threaded into the empty structures. Construction of the hollow capsids appears to occur by a self-assembly process, sometimes aided by other proteins. The stepwise assembly of components involves the initial aggregation of structural subunits into pentamers and hexamers, followed by the condensation of these capsomeres to form the empty capsid. In some cases, it appears that a small complex of capsid proteins associates specifically with the viral genome and nucleates the assembly of the complete capsid around the genome.

Bacteriophages

Most bacteriophages escape from the infected cell by coding for one or more enzymes synthesized late in the latent phase, which causes the lysis of the cell. The enzymes are either lysozymes or peptidases that weaken the cell wall by cleaving specific bonds in the peptidoglycan layer. The damaged cells burst as a result of osmotic pressure.

CELL DEATH

Nearly all productively infected cells die (see further for exceptions), presumably because the viral genetic program is dominant and precludes the continuation of normal cell functions required for survival. In many cases, direct viral interference with normal cellular metabolic processes leads to cell death. For example, picornaviruses shut off host protein synthesis soon after infection, and many DNA human viruses interfere with normal cell-cycle controls. In many cases, the end result of such insults is a triggering of a cellular stress response called programmed cell death or apoptosis. Some viruses are known to code for proteins that block or delay apoptosis, probably to stave off cell death until the virus replication cycle has been completed. Ultimately, the cell lysis that accompanies cell death is responsible for the release of naked capsid viruses into the environment.

BUDDING

Most enveloped human viruses acquire their membrane by budding either through the plasma membrane or, in the case of herpesviruses, through the nuclear membrane; however, in some other viruses such as coronaviruses and poxviruses, budding occurs through cytoplasmic membranes. Thus, for these viruses, release from the cell is coupled to the final stage of virion assembly. The herpesviruses ultimately escape from the cell when the membrane of the exocytic vesicle fuses with the plasma membrane. The poxviruses appear to program the formation of membrane structures and acquire membrane from Golgi apparatus that is lost upon the release of extracellular enveloped virions.

The membrane changes that accompany budding appear to be just the reverse of the entry process described before for those viruses that enter by direct fusion (compare Figure 6–9 and Figure 6–15). The region of the cellular membrane where budding is to occur acquires a cluster of viral glycoprotein spikes. These proteins are synthesized by the pathway that normally delivers cellular membrane proteins to the surface of the cell by way of the Golgi apparatus. At the site of the glycoprotein cluster, the inside of the membrane becomes coated with a virion structural protein called the matrix or M protein. The accumulation of the matrix protein at the proper location is probably facilitated by the presence of a binding site for the matrix protein on the cytoplasmic side of the transmembrane glycoprotein spike. The matrix protein attracts the completed nucleocapsid that triggers the envelopment process leading to the release of the completed particle to the outside (Figure 6–15).

For viruses that bud, it is important to note that the plasma membrane of the infected cell contains virus-specific glycoproteins that represent foreign (viral) antigens. This means that infected cells become targets for the immune system. In fact, cytotoxic T lymphocytes that recognize these antigens can be a significant factor in combating a virus infection.

The process of initial viral budding usually does not lead directly to cell death because the plasma membrane can be repaired after budding. It is likely that cell death for most enveloped viruses, as for naked capsid viruses, is related to the loss of normal cellular functions required for survival or as a result of apoptosis. Unlike most retroviruses that do not kill the host cell, HIV-1 is cytopathic. Although the mechanism of HIV-1 cell killing is not entirely understood, factors such as the accumulation of viral DNA in the cytoplasm, the toxic effects of certain viral proteins, alterations in plasma membrane permeability, apoptosis, and cell–cell fusion are believed to contribute to the cytopathic potential of the virus.

CELL SURVIVAL

For retroviruses (except HIV-1 and other lentiviruses) and the filamentous bacteriophages, virus reproduction and cell survival are compatible. Retroviruses convert their RNA genome into double-stranded DNA, which integrates into a host cell chromosome and is transcribed just like any other cellular gene (see Chapter 18). Thus, the impact on cellular metabolism is minimal. Moreover, these retroviruses bud through the plasma membrane without any permanent damage to the cell (except HIV). How the cell escapes permanent damage in this case is unknown. As with the retroviruses, the infected cell continues to produce virus indefinitely.

QUANTITATION OF VIRUSES

Hemagglutination Assay

For some human viruses such as influenza viruses, red blood cells from one or more human species contain receptors for the virion attachment proteins. Because the receptors and attachment proteins are present in multiple copies on the cells and virions, respectively, an excess of virus particles coats the cells and causes them to aggregate. This aggregation phenomenon was first discovered with influenza virus and is called hemagglutination. The virion attachment protein on the influenza virion is appropriately called the hemagglutinin. Furthermore, the presence of the hemagglutinin in the plasma membrane of the infected cell means that the cells as well as the virions bind the red blood cells. This reaction, called hemadsorption, is a useful indicator of infection by certain viruses.

Hemagglutination can be used to estimate the titer of virus particles in a virus-containing sample. Serially diluted samples of the virus preparation are mixed with a constant amount of red blood cells, and the mixture is allowed to settle in a test tube. Agglutinated red blood cells settle to the bottom to form a thin, dispersed layer. If there is insufficient virus to agglutinate the red blood cells, they will settle to the bottom of the tube and form a tight pellet. The difference is easily scored visually, and the endpoint of the agglutination is used as a relative measure of the virus concentration in the sample. Furthermore, hemagglutination can be inhibited by virus attachment protein-specific antibodies known as hemagglutination inhibition (HI), which can be used to determine titer of the antibody.

Plaque Assay

The plaque assay is a method for determining the titer of infectious virions in a virus preparation or lysate. The sample is diluted serially, and an aliquot of each dilution is added to a vast excess of susceptible host cells. For a human virus, the host cells are usually attached to the bottom of a plastic Petri dish; for bacterial cells, adsorption is typically carried out in a cell suspension. In both cases, the cells are then immersed in a semisolid medium such as agar, which prevents the released virions from spreading throughout the entire cell population. Thus, the virus released from the initial and subsequent rounds of infection can invade only the cells in the immediate vicinity of the initial infected cell on the plate. The end result is an easily visible clearing of dead cells at each of the sites on the plate where one of the original infected cells was located. The clearing is called a plaque (Figure 6–16). Visualization in the case of human cells usually requires staining the cells. By counting the number of plaques and correcting for the dilution factor, the virus titer in the original sample can be calculated. The titer is usually expressed as the number of plaque-forming units per milliliter (pfu/mL).

Immunologic Assay

Viral antigen can be quantified by using antigen–antibody specificity, as measured by enzyme immunoassay (EIA), enzyme linked immunosorbent assay (ELISA), and immunofluoresence assay (IFA). Similar to other assays, in immunologic assays the antigen–antibody specificity and conditions should be worked out. For most viruses, commercial antibodies are available and can be used to detect or quantify the antigen of viruses in culture and body fluids, tissue biopsies, serum, plasma, and cerebrospinal fluid (CSF). The most common example is the detection and sometimes quantification of RSV by IFA in which RSV antigens are be measured in nasopharyngeal and throat washing, sputum, or bronchoalveolar lavage. In addition, viral antigens can be detected and quantified in blood (plasma or serum), which can then provide information on the amount of virus present in the blood. For example, HIV can be quantified by the levels of p24 (capsid) antigen in the culture fluid or blood. On the other hand, these immunoassays can also be used to detect antibodies produced during infection and are very powerful tools to diagnose infection. In this scenario, commercial antigens-coated plates are available that can be used to detect antibodies in patients’ samples for diagnosis of infection and monitoring the effectiveness of vaccines.

Molecular Assay

Viral genomes, both RNA and DNA, can be quantified to determine the amount of virus (viral load) in blood (serum or plasma) or any given samples. The RNA genomes of the viruses are first reversely transcribed to cDNA by reverse transcriptase enzyme and then amplified by polymerase chain reaction (PCR). However, viral DNA genomes can be directly amplified by PCR to quantify the viral genomes. On the basis of the number of copies of the viral genomes, the amount of virus in any sample can be determined. This is the most sensitive and specific method to detect and quantify viral genomes. PCR is routinely used to determine viral load in HIV, hepatitis C virus, and other viral and microbial infections.

VIRAL GENETICS

Viruses generally use two mechanisms—mutation and recombination—by which viral genomes change during infection and there are virologic, immunologic, and clinical consequences of some of these changes. Typically, the majority of the virus particles derived from a cell infected with a human virus are noninfectious in other cells as determined by a plaque assay. Although some of this discrepancy may be attributable to inefficiencies in the assay procedures, it is clear that many defective particles are being produced. In part, this production of defective particles arises because the mutation rates for human viruses are unusually high and many infections occur at high multiplicities, where defective genomes are complemented by nondefective viruses and therefore propagated.

Mutation

Many DNA viruses use the host DNA synthesis machinery for replicating their genomes. Therefore, they benefit from the built-in proofreading and other error-correcting mechanisms used by the host cell. However, the large human viruses (adenoviruses, herpesviruses, and poxviruses) code for their own DNA polymerases, and these enzymes are not as effective at proofreading as the cellular polymerases. The resulting higher error rates in DNA replication endow the viruses with the potential for a high rate of mutation, but they are also partially responsible for the high frequency of defective viral particles.

The replication of RNA viruses is characterized by even higher error rates of mutation because viral RNA polymerases do not possess any proofreading capabilities. The result is that error rates for RNA viruses commonly approach one mistake for every 2500 to 10 000 nucleotides polymerized. Such a high misincorporation rate means that, even for the smallest RNA viruses, virtually every round of replication introduces one or more nucleotide changes somewhere in the genome. If it is assumed that errors are introduced at random, most of the members of a clone (eg, in a plaque) are genetically different from all other members of the clone. The resulting mixture of different genome sequences for a particular RNA virus has been referred to as quasispecies to emphasize that the level of genetic variation is much greater than what normally exists in a species.

Because of the redundancy in the genetic code, some mutations are silent and are not reflected in changes at the protein level, but many occur in essential genes and contribute to the large number of defective particles found for RNA human viruses. The concept of genetic stability takes on a new meaning in view of these considerations, and the RNA virus population as a whole maintains some degree of homogeneity only because of the high degree of fitness exhibited by a subset of the possible genome sequences. Thus, strong selective forces continually operate on a population to eliminate most mutants that fail to compete with the few very successful members of the population. However, any time the environment changes (eg, with the appearance of neutralizing antibodies), a new subset of the population is selected and maintained as long as the selective forces remain constant.

The high mutation rates found for RNA viruses endow them with a genetic plasticity that leads readily to the occurrence of genetic variants and permits rapid adaptation to new environmental conditions. The large number of serotypes of rhinoviruses causing the common cold, for instance, likely reflects the potential to vary by mutation. Although rapid genetic change occurs for most if not all viruses, no medically important RNA virus has exhibited this phenomenon as conspicuously as influenza virus. Point mutations accumulate in the influenza genes coding for the two envelope proteins (hemagglutinin and neuraminidase), resulting in changes in the antigenic structure of the virions. These changes lead to new variants not recognized by the immune system of previously infected individuals. This phenomenon is called antigenic drift (see Chapter 9). Figure 6–17 shows the effect of mutations resulting in antigenic drift. Apparently, the domains of the two envelope proteins that are most important for immune recognition are not essential for virus entry and, as a result, can tolerate amino acid changes leading to antigenic variation. This feature may distinguish influenza from other human RNA viruses that possess the same high mutation rates, but do not exhibit such high rates of antigenic drift. Antigenic drift in epidemic influenza viruses from year to year requires continual updating of the strains used to produce annual influenza vaccines.

The retroviruses likewise show high rates of variation because of error-prone reverse transcriptase enzyme that converts retroviral RNA into double-stranded DNA. For example, error rate for HIV-1 reverse transcriptase is approximately four to five errors per reverse transcription of the genome. After the viral DNA has integrated into the chromosome of the host cell, the retroviral DNA is transcribed by the host RNA polymerase II, which is also capable of generating errors. Accordingly, HIV-1 exhibits a high rate of mutation, and this property gives HIV-1 the ability to evolve rapidly in response to changing conditions in the infected host. Genetic variation has resulted in several clades or subtypes of HIV-1 worldwide.

Retroviruses that exhibit high rates of antigenic variation such as HIV-1 pose particularly difficult problems for the development of effective vaccines. Attempts are being made to identify conserved and, therefore, presumably essential domains of the envelope proteins for these viruses, which might be useful in developing a genetically engineered vaccine.

Von Magnus Phenomenon and Defective Interfering Particles

In early studies with influenza virus, it was noted that serial passage of virus stocks at high multiplicities of infection led to a steady decline of infectious titer with each passage. At the same time, the titer of noninfectious particles increased. As discussed later, the noninfectious genomes interfere with the replication of the infectious virus and so are called defective interfering (DI) particles. Later, these observations were extended to include virtually all RNA and DNA human viruses. The phenomenon is now named after von Magnus, who described the initial observations with the influenza virus.

A combination of two separate events leads to von Magnus phenomenon. First, deletion mutations occur at a significant frequency for all viruses. For DNA viruses, the mechanisms are not well understood, but deletions presumably occur as a result of mistakes in replication or by nonhomologous recombination. The basis for the occurrence of deletions in RNA viruses is better understood. All RNA polymerases (replicases) have a tendency to dissociate from the template RNA, but remain bound to the end of the growing RNA chain. By reassociating with the same or a different template at a different location, the replicase “finishes” replication, but, in the process, creates a shorter or longer RNA molecule. A subset of these variants possesses the proper signals for initiating RNA synthesis and continues replicating. Because the deletion variants in the population require less time to complete a replication cycle, they eventually predominate and constitute the DI particles.

Second, as their name implies, the DI particles interfere with the replication of nondefective or normal (wild-type) particles. Interference occurs because the DI particles successfully compete with the nondefective genomes for a limited supply of replication enzymes. The virions released at the end of the infection are therefore enriched for the DI particles. With each successive infection, the DI particles can predominate over the normal particles as long as the multiplicity of infection is high enough that every cell is infected with at least one normal infectious particle. If this condition is satisfied, then the normal virus particle can complement any defects in the DI particles and provide all of the viral proteins required for the infection. This process is called complementation. Eventually, however, as serial passage is continued, the multiplicity of infectious particles drops below one, and the majority of the cells are infected only with DI particles. When this happens, the proportion of DI particles in the progeny virus decreases.

Recombination

Besides mutation, genetic recombination between related viruses is a major source of genomic variation. Bacterial cells as well as the nuclei of human cells contain the enzymes necessary for homologous recombination of DNA. Thus, it is not surprising that recombinants arise from mixed infections involving two different strains of the same type of DNA virus. The larger bacteriophages such as λ and T4 code for their own recombination enzymes, a fact that attests to the importance of recombination in the life cycles and possibly the evolution of these viruses. The fact that recombination has also been observed for cytoplasmic poxviruses suggests that they too code for their own recombination enzymes.

As far as is known, cells do not possess the machinery to recombine RNA molecules. However, recombination among at least some RNA viruses has been observed by two different mechanisms. The first, which is unique to the viruses with segmented genomes (orthomyxoviruses and reoviruses), involves reassortment of segments during a mixed infection involving two different viral strains. Recombinant progeny viruses that differ from either parent can be accounted for by the formation of new combinations of the genomic segments that are free to mix with each other at some time during the infection. Reassortment of this type occurring during infections of the same cell by human and certain animal influenza viruses is believed to account for the occasional drastic change in the antigenicity of the human influenza A virus. These dramatic changes, called antigenic shifts (Figure 6–18), produce strains to which much of the human population lacks immunity and, thus, can have enormous epidemiologic and clinical consequences (see Chapter 9).

The second mechanism of RNA virus recombination is exemplified by the genetic recombination between different forms of poliovirus. Because the poliovirus RNA genome is not segmented, reassortment cannot be invoked as the basis for the observed recombinants. In this case, it appears that recombination occurs during replication by a “copy choice” type of mechanism. During RNA synthesis, the replicase (polymerase) dissociates from one template and resumes copying a second template at the exact place where it left off on the first. The end result is a progeny RNA genome containing information from two different input RNA molecules. Strand switching during replication, therefore, generates a recombinant virus. Although this is not frequently observed, it is likely that most of the RNA human viruses are capable of this type of recombination.

A “copy choice” mechanism has also been invoked to explain a high rate of recombination observed with retroviruses. Early after infection, the reverse transcriptase within the virion synthesizes a DNA copy of the RNA genome by a process called reverse transcription. In the course of reverse transcription, the enzyme is required to “jump” between two sites on the RNA genome (see Chapter 18). This propensity to switch templates apparently explains how the enzyme generates recombinant viruses. Because reverse transcription takes place in subviral particles, free mixing of RNA templates brought into the cell in different virus particles is not permitted. However, retroviruses are diploid, because each particle carries two copies of the genome. This arrangement appears to be a situation readymade for template switching during DNA synthesis, and most likely accounts for retroviral recombination.

Occasionally, animal retroviruses package a cellular mRNA into the virion rather than a second RNA genome. This arrangement can lead to copy choice recombination between the viral genome and a cellular mRNA. The end result is, sometimes, the incorporation of a cellular gene into the viral genome. This mechanism is believed to account for the production of highly oncogenic retroviruses containing modified cellular genes (see later).

Phenotype Mixing

Mixing of two closely related viruses, A and B, may result in generation of a hybrid virus with genome of virus A and outer surface protein of B. Upon infection of the new appropriate cells, the surface protein determines the tropism (means binds to the receptor on the host cells). However, the progeny viruses produced from this hybrid virus will be of type A virus because the genome in this hybrid virus belonged to type A virus.

THE LATENT STATE

Temperate viruses, which can establish both productive and nonproductive responses, can infect a cell and enter a latent state that is characterized by little or no virus production. The viral DNA genome is replicated and segregated along with the cellular DNA when the cell divides. There exist two possible states for the latent viral genome. It can exist extrachromosomally (herpesviruses) like a bacterial plasmid, or it can become integrated into the chromosome (retroviruses) like the bacterial F factor in the formation of a high-frequency recombination (HFR) strain (see Chapter 21). Because the latent genome is usually capable of reactivation and entry into the lytic cycle, it is called a provirus or, in the case of bacteriophages, a prophage. In many cases, viral latency goes undetected; however, limited expression of proviral genes can occasionally endow the cell with a new set of properties. For example, the latent herpes simplex virus infection is characterized by the presence of viral DNA (extrachromosomal) in the nerve ganglion without any production of infectious virus particles as well as no symptoms (clinical latency) in infected individuals. However, this latent state can be reactivated characterized by the presence of infectious virus particles and symptoms. For instance, lysogeny (latent state) can lead to the production of virulence-determining toxins in some bacteria (lysogenic conversion— details in Chapter 21) and latency by a human virus may produce oncogenic transformation.

The significance of lysogeny and lysogenic conversion is described in Chapter 21. Diphtheria, scarlet fever, and botulism all are caused by toxins produced by bacteria that have been “converted” by a temperate bacteriophage. In each case, the gene that codes for the toxin protein resides in the phage DNA and is expressed together with the repressor gene in the lysogenic state.