Review of Medical Microbiology and Immunology, 13th Edition

29. Replication

CHAPTER CONTENTS

Introduction

Viral Growth Curve

Specific Events During the Growth Cycle

Attachment, Penetration, & Uncoating

Gene Expression & Genome Replication

Assembly & Release

Lysogeny

Relationship of Lysogeny in Bacteria to Latency in Human Cells

Pearls

Self-Assessment Questions

Practice Questions: USMLE & Course Examinations

INTRODUCTION

The viral replication cycle is described in this chapter in two different ways. The first approach is a growth curve, which shows the amount of virus produced at different times after infection. The second is a stepwise description of the specific events within the cell during virus growth.

VIRAL GROWTH CURVE

The growth curve depicted in Figure 29–1 shows that when one virion (one virus particle) infects a cell, it can replicate in approximately 10 hours to produce hundreds of virions within that cell. This remarkable amplification explains how viruses spread rapidly from cell to cell. Note that the time required for the growth cycle varies; it is minutes for some bacterial viruses and hours for some human viruses.

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FIGURE 29–1 Viral growth curve. The figure shows that one infectious virus particle (virion) entering a cell at the time of infection results in more than 100 infectious virions 10 hours later, a remarkable increase. Note the eclipse period during which no infectious virus is detectable within the infected cells. In this growth curve, the amount of infecting virus is 1 virion/cell (i.e., 1 infectious unit/cell). (Modified and reproduced with permission from Joklik WK et al. Zinsser Microbiology. 20th ed. Originally published by Appleton & Lange. Copyright 1992 by McGraw-Hill.)

The first event shown in Figure 29–1 is quite striking: the virus disappears, as represented by the solid line dropping to the x axis. Although the virus particle, as such, is no longer present, the viral nucleic acid continues to function and begins to accumulate within the cell, as indicated by the dotted line. The time during which no virus is found inside the cell is known as the eclipse period. The eclipse period ends with the appearance of virus (solid line). The latent period, in contrast, is defined as the time from the onset of infection to the appearance of virus extracellularly. Note that infection begins with one virus particle and ends with several hundred virus particles having been produced; this type of reproduction is unique to viruses.

Alterations of cell morphology accompanied by marked derangement of cell function begin toward the end of the latent period. This cytopathic effect (CPE) culminates in the lysis and death of cells. CPE can be seen in the light microscope and, when observed, is an important initial step in the laboratory diagnosis of viral infection. Not all viruses cause CPE; some can replicate while causing little morphologic or functional change in the cell.

SPECIFIC EVENTS DURING THE GROWTH CYCLE

An overview of the events is described in Table 29–1 and presented in diagrammatic fashion in Figure 29–2. The infecting parental virus particle attaches to the cell membrane and then penetrates the host cell. The viral genome is “uncoated” by removing the capsid proteins, and the genome is free to function. Early mRNA and proteins are synthesized; the early proteins are enzymes used to replicate the viral genome. Late mRNA and proteins are then synthesized. These late proteins are the structural, capsid proteins. The progeny virions are assembled from the replicated genetic material, and newly made capsid proteins and are then released from the cell.

TABLE 29–1 Stages of the Viral Growth Cycle

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FIGURE 29–2 Viral growth cycle. The growth cycle of adenovirus, a nonenveloped DNA virus, is shown. (Modified and reproduced with permission from Jawetz E, Melnick JL, Adelberg EA. Review of Medical Microbiology. 16th ed. Originally published by Appleton & Lange. Copyright 1984 by McGraw-Hill.)

Another, more general way to describe the growth cycle is as follows: (1) early events (i.e., attachment, penetration, and uncoating); (2) middle events (i.e., gene expression and genome replication); and (3) late events (i.e., assembly and release). With this sequence in mind, each stage will be described in more detail.

Attachment, Penetration, & Uncoating

The proteins on the surface of the virion attach to specific receptor proteins on the cell surface through weak, noncovalent bonding. The specificity of attachment determines the host range of the virus. Some viruses have a narrow range, whereas others have quite a broad range. For example, poliovirus can enter the cells of only humans and other primates, whereas rabies virus can enter all mammalian cells. The organ specificity of viruses is governed by receptor interaction as well. Those cellular receptors that have been identified are surface proteins that serve various other functions. For example, herpes simplex virus type 1 attaches to the fibroblast growth factor receptor, rabies virus to the acetylcholine receptor, and human immunodeficiency virus (HIV) to the CD4 protein on helper T lymphocytes.

The virus particle penetrates by being engulfed in a pinocytotic vesicle, within which the process of uncoating begins. A low pH within the vesicle favors uncoating. Rupture of the vesicle or fusion of the outer layer of virus with the vesicle membrane deposits the inner core of the virus into the cytoplasm.

The receptors for viruses on the cell surface are proteins that have other functions in the life of the cell. Probably the best known is the CD4 protein that serves as one of the receptors for HIV but whose normal function is the binding of class 2 major histocompatibility complex (MHC) proteins involved in the activation of helper T cells. A few other examples will serve to illustrate the point: rabies virus binds to the acetylcholine receptor, Epstein–Barr virus binds to a complement receptor, and vaccinia virus binds to the receptor for epidermal growth factor,

Certain bacterial viruses (bacteriophages) have a special mechanism for entering bacteria that has no counterpart in either human viruses or those of animals or plants. Some of the T group of bacteriophages infect Escherichia coli by attaching several tail fibers to the cell surface and then using lysozyme from the tail to degrade a portion of the cell wall. At this point, the tail sheath contracts, driving the tip of the core through the cell wall. The viral DNA then enters the cell through the tail core, whereas the capsid proteins remain outside.

It is appropriate at this point to describe the phenomenon of infectious nucleic acid, because it provides a transition between the concepts of host specificity described earlier and early genome functioning, which is discussed later. Note that we are discussing whether the purified genome is infectious. All viruses are “infectious” in a person or in cell culture, but not all purified genomes are infectious.

Infectious nucleic acid is purified viral DNA or RNA (without any protein) that can carry out the entire viral growth cycle and result in the production of complete virus particles. This is interesting from three points of view:

(1) The observation that purified nucleic acid is infectious is the definitive proof that nucleic acid, not protein, is the genetic material.

(2) Infectious nucleic acid can bypass the host range specificity provided by the viral protein–cell receptor interaction. For example, although intact poliovirus can grow only in primate cells, purified poliovirus RNA can enter nonprimate cells, go through its usual growth cycle, and produce normal poliovirus. The poliovirus produced in the nonprimate cells can infect only primate cells because it now has its capsid proteins. These observations indicate that the internal functions of the nonprimate cells are capable of supporting viral growth once entry has occurred.

(3) Only certain viruses yield infectious nucleic acid. The reason for this is discussed later. Note that all viruses are infectious, but not all purified viral DNAs or RNAs (genomes) are infectious.

Gene Expression & Genome Replication

The first step in viral gene expression is mRNA synthesis. It is at this point that viruses follow different pathways depending on the nature of their nucleic acid and the part of the cell in which they replicate (Figure 29–3).

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FIGURE 29–3 Synthesis of viral mRNA by medically important viruses. The following information starts at the top of the figure and moves clockwise: Viruses with a double-stranded DNA genome (e.g., papovaviruses such as human papillomavirus) use host cell RNA polymerase to synthesize viral mRNA. Note that hepadnaviruses (e.g., hepatitis B virus) contain a virion DNA polymerase that synthesizes the missing portion of the DNA genome, but the viral mRNA is synthesized by host cell RNA polymerase. Parvoviruses use host cell DNA polymerase to synthesize viral double-stranded DNA and host cell RNA polymerase to synthesize viral mRNA. Viruses with a single-stranded, negative-polarity RNA genome (e.g., orthomyxoviruses such as influenza virus) use a virion RNA polymerase to synthesize viral mRNA. Viruses with a double-stranded RNA genome (e.g., reoviruses) use a virion RNA polymerase to synthesize viral mRNA. Some viruses with a single-stranded, positive-polarity RNA genome (e.g., retroviruses) use a virion DNA polymerase to synthesize a DNA copy of the RNA genome but a host cell RNA polymerase to synthesize the viral mRNA. Some viruses with a single-stranded, positive-polarity RNA genome (e.g., picornaviruses) use the virion genome RNA itself as their mRNA. (Modified and reproduced with permission from Ryan K et al. Sherris Medical Microbiology. 3rd ed. Originally published by Appleton & Lange. Copyright 1994 by McGraw-Hill.)

DNA viruses, with one exception, replicate in the nucleus and use the host cell DNA-dependent RNA polymerase to synthesize their mRNA. The poxviruses are the exception because they replicate in the cytoplasm, where they do not have access to the host cell RNA polymerase. They therefore carry their own polymerase within the virus particle. The genome of all DNA viruses consists of double-stranded DNA, except for the parvoviruses, which have a single-stranded DNA genome (Table 29–2).

Most RNA viruses undergo their entire replicative cycle in the cytoplasm. The two principal exceptions are retroviruses and influenza viruses, both of which have an important replicative step in the nucleus. Retroviruses integrate a DNA copy of their genome into the host cell DNA, and influenza viruses synthesize their progeny genomes in the nucleus. In addition, the mRNA of hepatitis delta virus is also synthesized in the nucleus of hepatocytes.

TABLE 29–2 Important Features of DNA Viruses

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The genome of all RNA viruses consists of single-stranded RNA, except for members of the reovirus family, which have a double-stranded RNA genome. Rotavirus is the important human pathogen in the reovirus family.

RNA viruses fall into four groups with quite different strategies for synthesizing mRNA (Table 29–3).

TABLE 29–3 Important Features of RNA Viruses

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(1) The simplest strategy is illustrated by poliovirus, which has single-stranded RNA of positive polarity1 as its genetic material. These viruses use their RNA genome directly as mRNA.

(2) The second group has single-stranded RNA of negative polarity as its genetic material. An mRNA must be transcribed by using the negative strand as a template. Because the cell does not have an RNA polymerase capable of using RNA as a template, the virus carries its own RNA-dependent RNA polymerase. There are two subcategories of negative-polarity RNA viruses: those that have a single piece of RNA (e.g., measles virus [a paramyxovirus] or rabies virus [a rhabdovirus]) and those that have multiple pieces of RNA (e.g., influenza virus [a myxovirus]).

Certain viruses, such as arenaviruses and some bunyaviruses, have a segmented RNA genome, most of which is negative stranded, but there are some positive strand regions as well. RNA segments that contain both positive polarity and negative polarity regions are called “ambisense.”

(3) The third group has double-stranded RNA as its genetic material. Because the cell has no enzyme capable of transcribing this RNA into mRNA, the virus carries its own polymerase. Note that plus strand in double-stranded RNA cannot be used as mRNA because it is hydrogen-bonded to the negative strand. Rotavirus, an important cause of diarrhea in children, has 11 segments of double-stranded RNA.

(4) The fourth group, exemplified by retroviruses, has single-stranded RNA of positive polarity that is transcribed into double-stranded DNA by the RNA-dependent DNA polymerase (reverse transcriptase) carried by the virus. This DNA copy is then transcribed into viral mRNA by the regular host cell RNA polymerase (polymerase II). Retroviruses are the only family of viruses that are diploid (i.e., that have two copies of their genome RNA).

These differences explain why some viruses yield infectious nucleic acid and others do not. Viruses that do not require a polymerase in the virion can produce infectious DNA or RNA. By contrast, viruses such as the poxviruses, the negative-stranded RNA viruses, the double-stranded RNA viruses, and the retroviruses, which require a virion polymerase, cannot yield infectious nucleic acid. Several additional features of viral mRNA are described in the “Viral mRNA” box.

Note that two families of viruses utilize a reverse transcriptase (an RNA-dependent DNA polymerase) during their replicative cycle, but the purpose of the enzyme during the cycle is different. As described in Table 29–4, retroviruses, such as HIV, use their genome RNA as the template to synthesize a DNA intermediate early in the replicative cycle. However, hepadnaviruses, such as hepatitis B virus (HBV), use an RNA intermediate as the template to produce their DNA genome late in the replicative cycle.

TABLE 29–4 Comparison of Reverse Transcriptase Activity of HIV (Retroviruses) and HBV (Hepadnaviruses)

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Once the viral mRNA of either DNA or RNA viruses is synthesized, it is translated by host cell ribosomes into viral proteins, some of which are early proteins (i.e., enzymes required for replication of the viral genome) and others of which are late proteins (i.e., structural proteins) of the progeny viruses. (The term early is defined as occurring before the replication of the genome, and late is defined as occurring after genome replication.) The most important of the early proteins for many RNA viruses is the polymerase that will synthesize many copies of viral genetic material for the progeny virus particles. No matter how a virus makes its mRNA, most viruses make a virus-encoded polymerase (a replicase) that replicates the genome (i.e., that makes many copies of the parental genome that will become the genome of the progeny virions). Table 29–5 describes which viruses encode their own replicase and which viruses use host cell polymerases to replicate their genome.

TABLE 29–5 Origin of the Genes That Encode the Polymerases That Synthesize the Viral Genome

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VIRAL MESSENGER RNA

There are four interesting aspects of viral mRNA and its expression in eukaryotic cells. (1) Viral mRNAs have three attributes in common with cellular mRNAs: on the 5′ end there is a methylated GTP “cap,” which is linked by an “inverted” (3´-to-5´) bond instead of the usual 5´-to-3´ bond; on the 3´ end there is a tail of 100–200 adenosine residues [poly(A)]; and the mRNA is generated by splicing from a larger transcript of the genome. In fact, these three modifications were first observed in studies on viral mRNAs and then extended to cellular mRNAs. (2) Some viruses use their genetic material to the fullest extent by making more than one type of mRNA from the same piece of DNA by “shifting the reading frame.” This is done by starting transcription one or two bases downstream from the original initiation site. (3) With some DNA viruses, there is temporal control over the region of the genome that is transcribed into mRNA. During the beginning stages of the growth cycle, before DNA replication begins, only the early region of the genome is transcribed, and therefore, only certain early proteins are made. One of the early proteins is a repressor of the late genes; this prevents transcription until the appropriate time. (4) Three different processes are used to generate the monocistronic mRNAs that will code for a single protein from the polycistronic viral genome:

(1) Individual mRNAs are transcribed by starting at many specific initiation points along the genome, which is the same mechanism used by eukaryotic cells and by herpesviruses, adenoviruses, and the DNA and RNA tumor viruses.

(2) In the reoviruses and influenza viruses, the genome is segmented into multiple pieces, each of which codes for a single mRNA.

(3) In polioviruses, the entire RNA genome is translated into one long polypeptide, which is then cleaved into specific proteins by a protease.

Some viral mRNAs are translated into precursor polypeptides that must be cleaved by proteases to produce the functional structural proteins (Figure 29–4 and Table 29–6), whereas other viral mRNAs are translated directly into structural proteins. A striking example of the former occurs during the replication of picornaviruses (e.g., poliovirus, rhinovirus, and hepatitis A virus), in which the genome RNA, acting as mRNA, is translated into a single polypeptide, which is then cleaved by a virus-coded protease into various proteins. This protease is one of the proteins in the single polypeptide, an interesting example of a protease acting on its own polypeptide.

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FIGURE 29–4 Synthesis of viral precursor polypeptides. A: Poliovirus mRNA is translated into a full-length precursor polypeptide, which is cleaved by the virus-encoded protease into the functional viral proteins. B: Retroviral mRNAs are translated into precursor polypeptides, which are then cleaved by the virus-encoded protease into the functional viral proteins. The cleavage of the Gag-Pol precursor polyprotein by the virion protease occurs in the immature virion after it has budded out from the cell membrane. The cleavage produces the capsid protein (p24), the matrix protein (p17), and enzymes such as the reverse transcriptase and the integrase. The cleavage of the Env polyprotein is carried out by a cellular protease, not by the virion protease. Inhibitors of the virion protease are effective drugs against human immunodeficiency virus.

TABLE 29–6 Virus-Encoded Proteases of Medically Important Viruses

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Another important family of viruses in which precursor polypeptides are synthesized is the retrovirus family. For example, the gag and pol genes of HIV are translated into precursor polypeptides, which are then cleaved by a virus-encoded protease. It is this protease that is inhibited by the drugs classified as protease inhibitors. Flaviviruses, such as hepatitis C virus and yellow fever virus, also synthesize precursor polypeptides that must be cleaved to form functional proteins by a virus-encoded protease. In contrast, other viruses, such as influenza virus and rotavirus, have segmented genomes, and each segment encodes a specific functional polypeptide rather than a precursor polypeptide.

Replication of the viral genome is governed by the principle of complementarity, which requires that a strand with a complementary base sequence be synthesized; this strand then serves as the template for the synthesis of the actual viral genome. The following examples from Table 29–7 should make this clear: (1) poliovirus makes a negative-strand intermediate, which is the template for the positive-strand genome; (2) influenza, measles, and rabies viruses make a positive-strand intermediate, which is the template for the negative-strand genome; (3) rotavirus makes a positive strand that acts both as mRNA and as the template for the negative strand in the double-stranded genome RNA; (4) retroviruses use the negative strand of the DNA intermediate to make positive-strand progeny RNA; (5) hepatitis B virus uses its mRNA as a template to make progeny double-stranded DNA; and (6) the other double-stranded DNA viruses replicate their DNA by the same semiconservative process by which cell DNA is synthesized.

TABLE 29–7 Complementarity in Viral Genome Replication

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As the replication of the viral genome proceeds, the structural capsid proteins to be used in the progeny virus particles are synthesized. In some cases, the newly replicated viral genomes can serve as templates for the late mRNA to make these capsid proteins.

Assembly & Release

The progeny particles are assembled by packaging the viral nucleic acid within the capsid proteins. Little is known about the precise steps in the assembly process. Surprisingly, certain viruses can be assembled in the test tube by using only purified RNA and purified protein. This indicates that the specificity of the interaction resides within the RNA and protein and that the action of enzymes and expenditure of energy are not required.

Virus particles are released from the cell by either of two processes. One is rupture of the cell membrane and release of the mature particles; this usually occurs with nonenveloped viruses. The other, which occurs with enveloped viruses, is release of viruses by budding through the outer cell membrane (Figure 29–5). (An exception is the herpesvirus family, whose members acquire their envelopes from the nuclear membrane rather than from the outer cell membrane.) The budding process begins when virus-specific proteins enter the cell membrane at specific sites. The viral nucleocapsid then interacts with the specific membrane site mediated by the matrix protein. The cell membrane evaginates at that site, and an enveloped particle buds off from the membrane. Budding frequently does not damage the cell, and in certain instances the cell survives while producing large numbers of budding virus particles.

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FIGURE 29–5 Budding. Most enveloped viruses derive their lipoprotein envelope from the cell membrane. The matrix protein mediates the interaction between the viral nucleocapsid and the viral envelope. (Reproduced with permission from Mims CA. The Pathogenesis of Infectious Disease. 3rd ed, Academic Press. Copyright 1987 Elsevier.)

LYSOGENY

The typical replicative cycle described above occurs most of the time when viruses infect cells. However, some viruses can use an alternative pathway, called the lysogenic cycle, in which the viral DNA becomes integrated into the host cell chromosome and no progeny virus particles are produced at that time (Figure 29–6). The viral nucleic acid continues to function in the integrated state in a variety of ways.

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FIGURE 29–6 Comparison of the lytic and lysogenic cycles of bacteriophage (phage) replication. In the lytic cycle, replication of the phage is completed without interruption. In the lysogenic cycle, replication of the phage is interrupted, and the phage DNA integrates into the bacterial DNA. The integrated DNA is called a prophage and can remain in the integrated state for long periods. If the bacteria are exposed to certain activators such as ultraviolet (UV) light, the prophage DNA is excised from the bacterial DNA and the phage enters the lytic cycle, which ends with the production of progeny phage.

One of the most important functions of lysogeny from a medical point of view is the synthesis of several exotoxins in bacteria, such as diphtheria, botulinum, cholera, and erythrogenic toxins, encoded by the genes of the integrated bacteriophage (prophage). Lysogenic conversion is the term applied to the new properties that a bacterium acquires as a result of expression of the integrated prophage genes (Figure 29–7). Lysogenic conversion is mediated by the transduction of bacterial genes from the donor bacterium to the recipient bacterium by bacteriophages. Transduction is the term used to describe the transfer of genes from one bacterium to another by viruses (see Figures 29–7 and 29–8 and page 20).

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FIGURE 29–7 Lysogenic conversion. In the left-hand panel, transduction of the diphtheria toxin gene by beta bacteriophage results in lysogenic conversion of the nonlysogenized, nonpathogenic Corynebacterium diphtheriae. In the right-hand panel, the recipient lysogenized bacterium can now produce diphtheria toxin and can cause the disease diphtheria. Note that no progeny phages are made within the lysogenized bacterium because the diphtheria toxin gene has replaced some of the beta-phage genes required for replication. The beta phage therefore cannot replicate. The lysogenized bacterium is not killed by the phage and can multiply, produce diphtheria toxin, and cause disease.

The lysogenic or “temperate” cycle is described for lambda bacteriophage, because it is the best-understood model system (Figure 29–8). Several aspects of infections by tumor viruses and herpesviruses are similar to the events in the lysogenic cycle of lambda phage.

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FIGURE 29–8 Lysogeny. The linear lambda (λ) phage DNA is injected into the bacterium, circularizes, and then integrates into the bacterial DNA. When integrated, the phage DNA is called a prophage. When the prophage is induced to enter the replicative cycle, aberrant excision of the phage DNA can occur (i.e., part of the phage DNA and part of the bacterial DNA including the adjacent gal gene are excised). The gal gene can now be transduced to another bacterium. Transduction is also described in Figure 4–4. (Reproduced with permission from Jawetz E et al. Review of Medical Microbiology. 17th ed. Originally published by Appleton & Lange. Copyright 1986 McGraw-Hill.)

Infection by lambda phage in E. coli begins with injection of the linear, double-stranded DNA genome through the phage tail into the cell. The linear DNA becomes a circle as the single-stranded regions on the ends pair their complementary bases. A ligating enzyme makes a covalent bond in each strand to close the circle. Circularization is important because it is the circular form that integrates into host cell DNA.

The choice between the pathway leading to lysogeny and that leading to full replication is made as early protein synthesis begins. Simply put, the choice depends on the balance between two proteins, the repressor produced by the c-I gene and the antagonizer of the repressor produced by the cro gene (Figure 29–9). If the repressor predominates, transcription of other early genes is shut off and lysogeny ensues. Transcription is inhibited by binding of the repressor to the two operator sites that control early protein synthesis. If the cro gene product prevents the synthesis of sufficient repressor, replication and lysis of the cell result. One correlate of the lysogenic state is that the repressor can also prevent the replication of additional lambda phages that infect subsequently. This is called “immunity” and is specifically directed against lambda phage because the repressor binds only to the operator sites in lambda DNA; other phages are not affected.

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FIGURE 29–9 Control of lysogeny. Shortly after infection, transcription of the N and cro genes begins. The N protein is an antiterminator that allows transcription of c-II and c-III and the genes to the right of c-II and to the left of c-III. The c-II protein enhances the production of the c-I repressor protein. c-I has two important functions: (1) It inhibits transcription at PROR and PLOL, thereby preventing phage replication, and (2) it is a positive regulator of its own synthesis by binding to PRM. The crucial decision point in lysogeny is the binding of either c-I repressor or the cro protein to the OR site. If c-I repressor occupies OR, lysogeny ensues; if cro protein occupies OR, viral replication occurs. N, antiterminator gene; c-I, repressor gene; c-II and c-III, genes that influence the production of c-I; PLOL, left promoter and operator; PROR, right promoter and operator; PRM, promoter for repressor maintenance; cro, gene that antagonizes the c-I repressor.

The next important step in the lysogenic cycle is the integration of the viral DNA into the cell DNA. This occurs by the matching of a specific attachment site on the lambda DNA to a homologous site on the E. coli DNA and the integration (breakage and rejoining) of the two DNAs mediated by a phage-encoded recombination enzyme. The integrated viral DNA is called a prophage. Most lysogenic phages integrate at one or a few specific sites, but some, such as the Mu (or mutator) phage, can integrate their DNA at many sites, and other phages, such as the P1 phage, never actually integrate but remain in a “temperate” state extrachromosomally, similar to a plasmid.

Because the integrated viral DNA is replicated along with the cell DNA, each daughter cell inherits a copy. However, the prophage is not permanently integrated. It can be induced to resume its replicative cycle by the action of ultraviolet (UV) light and certain chemicals that damage DNA. UV light induces the synthesis of a protease, which cleaves the repressor. Early genes then function, including the genes coding for the enzymes that excise the prophage from the cell DNA. The virus then completes its replicative cycle, leading to the production of progeny virus and lysis of the cell.

Relationship of Lysogeny in Bacteria to Latency in Human Cells

Members of the herpesvirus family, such as herpes simplex virus (HSV), varicella-zoster virus, cytomegalovirus (CMV), and Epstein–Barr virus, exhibit latency—the phenomenon in which no or very little virus is produced after the initial infection but, at some later time, reactivation and full virus replication occur. The parallel to lysogeny with bacteriophage is clear.

What is known about how the herpesviruses initiate and maintain the latent state? Shortly after HSV infects neurons, a set of “latency-associated transcripts” (LATS) are synthesized. These are noncoding, regulatory RNAs that suppress viral replication. The precise mechanism by which they do so is unclear. Reactivation of viral replication at a later time occurs when the genes encoding LATS are excised.

CMV employs different mechanisms. The CMV genome encodes microRNAs that inhibit the translation of mRNAs required for viral replication. Also, the CMV genome encodes both a protein and an RNA that inhibit apoptosis in infected cells. This allows the infected cell to survive.

PEARLS

Viral Growth Curve

• One virion infects a cell and hundreds of progeny virions are produced within hours. This is a remarkable amplification and explains the rapid spread of virus from cell to cell.

• The eclipse period is the time when no virus particles are detected within the infected cell. It occurs soon after the cell is infected.

• Cytopathic effect (CPE) is the term used to describe the damage, both morphologic and functional, inflicted on the cell by the virus. In the clinical laboratory, the presence of a virus in the patient’s specimen is often detected by seeing a CPE in cell culture.

Viral Growth Cycle

• Attachment: The interaction of proteins on the surface of the virus with specific receptor proteins on the surface of the cell is one of the main determinants of both the species specificity and the organ specificity of the virus.

• Infectious nucleic acid is viral genome DNA or RNA, purified free of all proteins, that can undergo the entire replicative cycle within a cell and produce infectious progeny viruses. Infectious nucleic acid, because it has no associated protein, can enter and replicate within cells that the intact virion cannot.

• Polarity of viral genome RNA: Genome RNA that has the same base sequence as the mRNA is, by definition, positive-polarity RNA. Most positive-polarity genomes are translated into viral proteins without the need for a polymerase in the virion. The exception is the retroviruses, which use reverse transcriptase in the virion to transcribe the genome RNA into DNA. Genome RNA that has a base sequence complementary to mRNA has, by definition, negative polarity. A virus with a negative-polarity RNA genome must have an RNA polymerase in the virion to synthesize its mRNA.

• Viral gene expression: All viruses require virus-specific messenger RNA to synthesize virus-specific proteins.

• RNA viruses: Some RNA viruses, such as poliovirus, have a positive-polarity RNA genome that serves as the mRNA (i.e., the genome is the mRNA). Other viruses, such as influenza virus, have a negative-polarity RNA genome and have an RNA polymerase in the virion that synthesizes the viral mRNA. Rotavirus has a double-stranded RNA genome and has an RNA polymerase in the virion that synthesizes the viral mRNA. Retroviruses, such as HIV, have a positive-polarity RNA genome and have a DNA polymerase in the virion that synthesizes a DNA copy of the RNA genome. This DNA is the template used by the host cell RNA polymerase to synthesize the viral mRNA.

• DNA viruses: Most DNA viruses, such as herpesviruses, adenoviruses, and papillomaviruses, have a double-stranded DNA genome and use the host cell RNA polymerase to synthesize the viral mRNA. Poxviruses have a double-stranded DNA genome but have an RNA polymerase in the virion that synthesizes the viral mRNA. Poxviruses have an RNA polymerase in the virion because they replicate in the cytoplasm and do not have access to the host cell RNA polymerase in the nucleus.

• Viral replication: All DNA viruses replicate in the nucleus, except poxviruses, which replicate in the cytoplasm. All RNA viruses replicate in the cytoplasm, except retroviruses, influenza virus, and hepatitis D virus, which require an intranuclear step in their replication. Many viruses encode a replicase, which is a DNA or RNA polymerase that synthesizes the many copies of the progeny viral genomes.

• Viral genome: The genome of all DNA viruses is double-stranded except for that of parvoviruses, which is single-stranded. The genome of all RNA viruses is single-stranded except for that of reoviruses (e.g., rotavirus), which is double-stranded.

• Viral proteins: Early proteins are typically enzymes used in the synthesis of viral nucleic acids, whereas late proteins are typically structural proteins of the progeny viruses. Some viruses, such as poliovirus and retroviruses, translate their mRNA into precursor polyproteins, which must be cleaved by proteases to produce functional proteins.

• Assembly and release: All enveloped viruses acquire their envelope by budding through the external cell membrane as they exit the cell, except herpesviruses, which acquire their envelope by budding through the nuclear membrane. The matrix protein mediates the interaction of the nucleocapsid with the envelope.

• Lysogeny is the process by which viral DNA becomes integrated into host cell DNA, replication stops, and no progeny virus is made. Later, if DNA is damaged by, for example, UV light, viral DNA is excised from the host cell DNA, and progeny viruses are made. The integrated viral DNA is called a prophage. Bacterial cells carrying a prophage can acquire new traits, such as the ability to produce exotoxins such as diphtheria toxin. Transduction is the process by which viruses carry genes from one cell to another. Lysogenic conversion is the term used to indicate that the cell has acquired a new trait as a result of the integrated prophage.

SELF-ASSESSMENT QUESTIONS

1. Many viruses are highly specific regarding the type of cells they infect. Of the following, which one is the most important determinant of this specificity?

(A) The matrix protein

(B) The polymerase in the virion

(C) The protease protein

(D) The surface glycoprotein

(E) The viral mRNA

2. Your summer research project is to study the viruses that cause upper respiratory tract infections. You have isolated a virus from a patient’s throat and find that its genome is RNA. Furthermore you find that the genome is the complement of viral mRNA within the infected cell. Of the following, which is the most appropriate conclusion you could draw?

(A) The genome RNA is infectious.

(B) The genome RNA is segmented.

(C) The virion contains a polymerase.

(D) The virion has a lipoprotein envelope.

(E) A single-stranded DNA is synthesized during replication.

3. The purified genome of certain viruses can enter a cell and elicit the production of progeny viruses (i.e., the genome is infectious). Regarding these viruses, which one of the following statements is most accurate?

(A) Their genome RNA has positive polarity.

(B) Their genome RNA is double-stranded.

(C) They have a polymerase in the virion.

(D) They have a segmented genome.

(E) They require tegument proteins in order to be infectious.

4. Regarding viral replication, which one of the following is most accurate?

(A) The cytopathic effect typically occurs during the eclipse period.

(B) The early proteins are typically enzymes, whereas the late proteins are typically capsid proteins.

(C) The assembly of a nonenveloped virus typically occurs as the virion buds from the cell membrane.

(D) Influenza viruses synthesize their mRNA using host cell-encoded RNA-dependent RNA polymerase.

(E) Retroviruses (e.g., HIV) synthesize their mRNA using an enzyme in the virion called reverse transcriptase.

5. Regarding the viral growth cycle, which one of the following is most accurate?

(A) During the lysogenic phase, the typical result is the production of hundreds of progeny virions.

(B) Hepatitis B virus has an RNA polymerase in the virion that is required to synthesize messenger RNA from the positive strand of the viral DNA.

(C) Herpesviruses have an RNA-dependent DNA polymerase in the virion.

(D) Lysogenic conversion is the process by which bacteria acquire new genes due to transduction by a lysogenic bacteriophage.

(E) Smallpox virus translates its genome into a single polypeptide, which is then cleaved into structural and nonstructural proteins.

6. Which one of the following choices names two viruses that both translate their messenger RNA into precursor polypeptides that must be cleaved by virion-encoded proteases?

(A) Herpes simplex virus and human papillomavirus

(B) Human immunodeficiency virus and poliovirus

(C) Influenza virus and measles virus

(D) Rabies virus and hepatitis B virus

(E) Rotavirus and parvovirus

ANSWERS

1. (D)

2. (C)

3. (A)

4. (B)

5. (D)

6. (B)

PRACTICE QUESTIONS: USMLE & COURSE EXAMINATIONS

Questions on the topics discussed in this chapter can be found in the Basic Virology section of PART XIII: USMLE (National Board) Practice Questions starting on page 700. Also see PART XIV: USMLE (National Board) Practice Examination starting on page 731.

1Positive polarity is defined as an RNA with the same base sequence as the mRNA. RNA with negative polarity has a base sequence that is complementary to the mRNA. For example, if the mRNA sequence is an A-C-U-G, an RNA with negative polarity would be U-G-A-C and an RNA with positive polarity would be A-C-U-G.