Characterizing and Classifying Viruses, Viroids, and Prions

Characterizing and Classifying Viruses, Viroids, and Prions Microengineer a Better Mosquito? Insects in the family Culicidae, commonly called mosquitos, are not just annoying arthropod pests. These blood-sucking insects are major vectors for the spread of infectious diseases, including a leading worldwide killer, malaria, dengue, and birth defects caused by the newly emerging Zika virus. The importance of such vectors and their diseases is indicated by the fact that epidemiologists have given a special name to viruses spread by arthropods, the arboviruses, and their disease are arboviral diseases. Microbiologists have discovered a genus of bacteria, Wolbachia, that is an obligate intracellular parasite in arthropods, including mosquitos. These bacteria take control over their hosts. Recent research has shown that certain strains of Wolbachia can reduce the ability of Aedes aegypti mosquitos to carry and transmit dengue virus. Since the bacteria can spread between mosquitos, some scientists have proposed releasing Wolbachia-infected insects into the wild to spread Wolbachia, which would spread throughout the mosquitoes and prevent the spread of dengue. Image you are on a scientific team at the Environmental Protection Agency (EPA) charged with reviewing an application to release mosquitos LM 40 µm infected with genetically engineered Wolbachia to prevent the spread of Zika in the United States. 1. Would you approve this application? 2. What additional information is required to make an informed decision? Characteristics of Viruses M13_BAUM7206_05_SE_C13.indd 382 LE A R N I N G O UTC O M E 13.1 Define virus and virion, and describe the parts of a virion. Viruses cause most of the diseases that still plague the industrialized world: common colds, influenza, herpes, and AIDS, to name a few. Although we have immunizations against many viral diseases and are adept at treating the symptoms of others, the characteristics of viruses and the means by which they attack their hosts make cures for viral diseases elusive. Throughout this section, we consider the clinical implications of viral characteristics. We begin by looking at the characteristics viruses have in common. A virus is a minuscule, acellular, infectious agent having one or several pieces of nucleic acid—either DNA or RNA. The nucleic acid is the genetic material (genome) of the virus. Being acellular, viruses have no cytoplasmic membrane (though, as we will see, some viruses possess a membrane like envelope). Viruses also lack cytosol and functional organelles. ­ ­ ­ Not all pathogens are cellular. Many infections of humans, animals, and plants (and even of bacteria) are caused by acellular (noncellular) agents, including viruses and other pathogenic particles called viroids and prions. Although these agents are similar to eukaryotic and prokaryotic pathogens in that they cause disease when they invade susceptible cells, they are simple compared to a cell—lacking cell membranes and composed of only a few organic molecules. In addition to lacking a cellular structure, they lack most of the characteristics of life: They cannot carry out any metabolic pathway; they can neither grow nor respond to the environment; and they cannot reproduce independently but instead must utilize the chemical and structural components of the cells they infect. They must recruit the cell’s metabolic pathways in order to increase their numbers. In this chapter, we first examine a range of topics concerning viruses: what their characteristics are, how they are classified, how they replicate, what role they play in some kinds of cancers, how they are maintained in the laboratory, and whether viruses are alive. Then we consider the nature of viroids and prions. Go to the study area to solve the problem. Go to the study area to solve the problem. 11/23/16 3:31 AM       Characteristics of Viruses They are not capable of metabolic activity on their own; instead, once viruses have invaded a cell, they take control of the cell’s metabolism to produce more molecules of viral nucleic acid and viral proteins, which then assemble into new viruses via a process we will examine shortly. A biological virus is similar to a computer virus—it is basically a code that is inactive and harmless until it infects its host; then, it can be devastating. Viruses have an extracellular and an intracellular state. Outside a cell, in the extracellular state, a virus is called a virion (vir’ē-on). Basically, a virion consists of a protein coat, called a capsid, surrounding a nucleic acid core (FIGURE 13.1a). Together, a viral nucleic acid and its capsid are also called a nucleocapsid, which in many cases can crystallize like crystalline chemicals (FIGURE 13.1b). Some virions have a phospholipid membrane called an envelope surrounding the nucleocapsid. The outermost layer of a virion (capsid or envelope) provides the virus both protection and recognition sites that bind to complementary chemicals on the surfaces of their specific host cells. Once a virus is inside, the intracellular state is initiated, and the capsid (and envelope, if there is one) is removed. A virus without its capsid exists solely as nucleic acid but is still referred to as a virus. Now that we have examined ways in which viruses are alike, we will consider the characteristics that are used to distinguish different viral groups. Viruses differ in the type of genetic material they contain, the kinds of cells they attack, their size, the nature of their capsid coat, their shapes, and the presence or absence of an envelope. Capsid (sectioned to show interior) (a) Genetic Material of Viruses (b) 13.2 Discuss viral genomes in terms of dsDNA, ssDNA, ssRNA, dsRNA, and number of segments of nucleic acid. 625 nm ▲ FIGURE 13.1 Virions are complete virus particles and include a nucleic acid, a capsid, and in some cases an envelope. (a) A drawing of a nonenveloped polyhedral virus containing DNA. (b) A transmission electron microscope image of crystallized tobacco mosaic virus. Like many chemicals and unlike cells, some viruses can form crystals. 1000 protein-encoding genes, whereas the genome of virus MS2 has only three. FIGURE 13.2 compares the physical size of the genome of a virus with the genome of its bacterial host, Escherichia coli (esh-ĕ-rik’e-ă kō’lī). ­ Viruses show more variety in the nature of their genomes than do cells. Whereas the genome of every cell is double-stranded DNA, a viral genome is either DNA or RNA. The primary way in which scientists categorize and classify viruses is based on the type of genetic material that makes up the viral genome. Some viral genomes, such as those of herpesvirus and smallpox virus, are double-stranded DNA (dsDNA), like the genomes of cells. Other viral genomes are either single-stranded RNA (ssRNA), single-stranded DNA (ssDNA), or doublestranded RNA (dsRNA). Cells never use these molecules for genomes—in fact, ssDNA and dsRNA are almost nonexistent in cells. Further, the genome of any particular virus may be either linear and composed of several molecules of nucleic acid, as in eukaryotic cells, or circular and singular, as in most prokaryotic cells. For example, the genome of an influenzavirus is composed of eight linear segments of single-stranded RNA, whereas the genome of poliovirus is one molecule of singlestranded RNA. Viral genomes are usually smaller than the genomes of cells. For example, the genome of the smallest bacterium that infects humans (a species of Chlamydia) has almost TEM ­ LE A R N I N G O U TC O M E Nucleic acid (viral genome) Hosts of Viruses LE A R N I N G O UTC O M E S 13.3 Explain the mechanism by which viruses are specific for their host cells. M13_BAUM7206_05_SE_C13.indd 383 and bacteria. ­ ­ ­ Most vir uses i n fect on ly part ic ular hosts’ cells. T h is specificity is due to the precise affinity of viral attachment molecules—proteins or glycoproteins on the viral surface—for complementary proteins or glycoproteins on the surface of the ­ ­ ­ 13.4 Compare and contrast viruses of fungi, plants, animals, 11/23/16 3:31 AM hapter       C 13    Characterizing and Classifying Viruses, Viroids, and Prions Partial genome of E. coli Viral genome (a) Ruptured E. coli TEM 400 nm ­ ▲ FIGURE 13.2 The relative sizes of genomes. A cell of Escherichia coli, just visible at lower left, ruptured, releasing DNA. (b) TEM 0.5 µm ­ host cell. Viruses may be so specific that they infect not only a particular host but also a particular kind of cell in that host. For example, HIV (human immunodeficiency virus, the agent that causes AIDS) specifically attacks helper T lymphocytes (a type of white blood cell) in humans and has no effect on, say, human muscle or bone cells. By contrast, some viruses are generalists; they infect many kinds of cells or many different hosts. An example of a generalist virus is West Nile virus, which can infect humans, most species of birds, several mammalian species, and some reptiles. All types of organisms are susceptible to some sort of viral attack. There are viruses that infect archaeal, bacterial, plant, protozoan, fungal, and animal cells (FIGURE 13.3). There are even tiny viruses that appear to attack specific larger viruses. Most viral research and scientific study has focused on bacterial and animal viruses. A virus that infects bacteria is referred to as a bacteriophage (bak-tēr’ē-ō-fāj) or simply a phage (fāj). Scientists have determined that the number of bacteriophages is more than all bacteria, archaea, and eukaryotes put together. We will return our attention to bacteriophages and animal viruses later in this chapter. Viruses of plants are less well known than bacterial and animal viruses, even though viruses were first identified and isolated from tobacco plants. Plant viruses infect many food crops, including corn, beans, sugarcane, tobacco, and potatoes, resulting in billions of dollars in losses each year. Viruses of M13_BAUM7206_05_SE_C13.indd 384 (c) SEM 100 nm ▲ FIGURE 13.3 Some examples of plant, bacterial, and human hosts of viral infections. (a) Tobacco mosaic virus—the first virus isolated—causes yellow discolorations of tobacco leaves. (b) A bacterial cell (purple) under attack by bacteriophages (pink). (c) A human white blood cell’s cytoplasmic membrane, to which HIV (pink) is attached. 11/23/16 3:31 AM       Characteristics of Viruses ­ ­  ­ ­ ­ ­ Sizes of Viruses “viruses,” which is a Latin word for “poison.” Most viruses are small; for example, 100 million polioviruses could fit side by side on the period at the end of this sentence. Even smaller viruses have diameters as small as 17 nm, while one of the larger viruses, Megavirus, is about 500 nm in diameter— about the diameter of many bacterial cells. FIGURE 13.4 compares the sizes of selected viruses to E. coli and a human red blood cell. In 1892, Russian microbiologist Dmitri Ivanowski (1864–1920) first demonstrated that viruses are acellular with an experiment designed to elucidate the cause of tobacco mosaic disease. He filtered the sap of infected tobacco plants through a porcelain filter fine enough to trap even the smallest of cells. Viruses, however, were not trapped but instead passed through the filter with the liquid, which remained infectious to tobacco plants. This experiment proved the existence of an acellular disease-causing entity smaller than a bacterium. Tobacco mosaic virus (TMV) was isolated and characterized in 1935 by an American chemist, Wendell Stanley (1904–1971). The invention of electron microscopy allowed scientists to finally see TMV and other viruses. ­ plants are introduced into plant cells either through abrasions of the cell wall or by plant parasites, such as nematodes and aphids. After entry, plant viruses follow the replication cycle discussed below for animal viruses. Fungal viruses have been little studied. We do know that fungal viruses are different from animal and bacterial viruses in that fungal viruses appear to exist only within cells; that is, they seemingly have no extracellular state. Presumably, fungal viruses cannot penetrate a thick fungal cell wall. However, because fusion of cells is typically a part of a fungal life cycle, viral infections can easily be propagated by the fusion of an infected fungal cell with an uninfected one. Not all viruses are deleterious. The box Beneficial Microbes: Good Viruses? Who Knew? illustrates some useful aspects of viruses in the environment. In the late 1800s, scientists hypothesized that the cause of many diseases, including polio and smallpox, was an agent smaller than a bacterium. They named these tiny agents E. coli (bacterium) (1000 nm x 3000 nm) Red blood cell (10,000 nm in diameter) 2500 nm 750 nm Bacterial ribosomes (25 nm) Poliovirus (30 nm) 300 nm Bacteriophage MS2 (24 nm) Smallpox virus (200 nm x 300 nm) Bacteriophage T4 (50 nm x 225 nm) Tobacco mosaic virus (15 nm x 300 nm) ▲ FIGURE 13.4 Sizes of selected virions. Selected viruses are compared in size to a bacterium, Escherichia coli, and to a human red blood cell. How can viruses be so small and yet still be pathogenic? Figure 13.4 Viruses utilize a host cell’s enzymes, organelles, and membranes to complete their replication cycle. M13_BAUM7206_05_SE_C13.indd 385 11/23/16 3:31 AM hapter       C 13    Characterizing and Classifying Viruses, Viroids, and Prions Capsid Morphology LE A R N I N G O U TC O M E 13.5 Discuss the structure and function of the viral capsid. As we have seen, viruses in their extracellular state as virions have capsids—protein coats that provide both protection for viral nucleic acid and a means by which many viruses attach to their hosts’ cells. The capsid of a virus is composed of proteinaceous subunits called capsomeres (or capsomers). Some capsomeres are composed of only a single type of protein, whereas others are composed of several different kinds of proteins. Recall that viral nucleic acid surrounded by its capsid is termed a nucleocapsid. (a) TEM (b) TEM 35 nm Viral Shapes The shapes of virions are also used to classify viruses. There are three basic types of viral shapes: helical, polyhedral, and complex (FIGURE 13.5). The capsid of a helical virus is composed of capsomeres that bond together in a circling fashion to form a tube around the nucleic acid. The capsid of a polyhedral virus is roughly spherical, with a shape similar to a geodesic dome. The most common type of polyhedral capsid is an icosahedron, which has 20 sides. Complex viruses have capsids of many different shapes that do not readily fit into either of the other two categories. An example of a complex virus is smallpox virus, which has several covering layers (including lipid) and no easily identifiable capsid. The complex shapes of many bacteriophages include icosahedral heads, which contain the genome, attached to helical tails with tail fibers. The complex capsids of such bacteriophages somewhat resemble NASA’s lunar lander (FIGURE 13.6). 50 nm Enveloped capsid Fiber layer Cell‘s vesicle The Viral Envelope LE A R N I N G O U TC O M E 13.6 Discuss the origin, structure, and function of the viral (c) TEM (d) TEM 250 nm envelope. ­ All viruses lack cell membranes (after all, they are not cells), but some, particularly animal viruses, have an envelope similar in composition to a cell membrane surrounding their capsids. Other viral proteins called matrix proteins fill the region between capsid and envelope. A virion with a membrane is an enveloped virion (FIGURE 13.7); a virion without an envelope is called a nonenveloped or naked virion. An enveloped virus acquires its envelope from its host cell during viral replication or release (discussed shortly). Indeed, the envelope of a virus is a portion of the membrane system of a host cell. Like a cytoplasmic membrane, a viral envelope is composed of a phospholipid bilayer and proteins. Some of the proteins are virally coded glycoproteins, which appear as spikes protruding outward from the envelope’s surface (see Figure 13.7). Host DNA carries the genetic code required for the assembly of the phospholipids and some of the proteins in the envelope, while the viral genome specifies the other membrane proteins. M13_BAUM7206_05_SE_C13.indd 386 60 nm ▲ FIGURE 13.5 The shapes of virions. (a) A helical virus, tobacco mosaic virus. The tubular shape of the capsid results from the tight arrangement of several rows of helical capsomeres. (b) Polyhedral virions of a virus that causes common colds. (c) Complex shape of Megavirus, which is shown inside a cell’s vesicle. These viruses have a layer of protein and sugar fibers outside the capsid. (d) The complex shape of rabies virus, which results from the shapes of the capsid and bullet-shaped envelope. 11/23/16 3:31 AM       Classification of Viruses ◀ FIGURE 13.6 The complex Head shape of bacteriophage T4. It includes an icosahedral head and an ornate tail that enables viral attachment and penetration. Tail fibers Tail Base plate TEM 45 nm (b) An envelope’s proteins and glycoproteins often play a role in the virion’s recognition of host cells. A viral envelope does not perform other physiological roles of a cytoplasmic membrane, such as endocytosis or active transport. An envelope provides some protection to the virus from the immune system; after all, enveloped viruses are carrying membrane from host cells and thus are more chemically similar to the host. However, membranes are more susceptible to detergents, alcohol, and drying out, so enveloped viruses are more fragile than naked ones. Naked viruses are more stable outside a host than are enveloped viruses, but the naked capsid exposes more viral proteins to the environment and are thus more susceptible to recognition and attack by the immune system once inside a host. TABLE 13.1 on p. 388 summarizes the novel properties of viruses and how those properties differ from the corresponding characteristics of cells. Next we turn our attention to the criteria by which virologists classify viruses. TELL ME WHY Why are naked icosahedral viruses able to crystallize? Classification of Viruses LE A R N I N G O UTC O M E 13.7 List the characteristics by which viruses are classified. Virologists established the International Committee on Taxonomy of Viruses (ICTV) in 1966 to provide a single taxonomic scheme for viral classification and identification. They classify viruses by ­ (a) BENEFICIAL MICROBES Good Viruses? Who Knew? Some important ways viruses affect our world: ­ • Scientists have found a previously unknown virus that attacks a tiny marine alga that forms algal “blooms” consisting of hundreds of thousands to millions of algal cells per milliliter of water, which are visible from space (see photo). Algal blooms like these can deplete the water of oxygen at night, potentially harming fish and other marine life. The newly discovered virus stops blooms by killing the alga, a result that is good for animal life. • When the algal cells die by this means, they release an airborne sulfate compound that acts to seed clouds. The resulting increase in cloudiness noticeably shades the ocean, measurably lowering water temperature. Thus, a marine virus both protects wildlife and helps to reduce global warming! • Researchers have also discovered a bacteriophage of oceanic cyanobacteria that transfers genes for photosynthetic machinery into its hosts’ cells so that the cells’ photosynthetic rate increases. There are up to 10 million of these viruses in a single milliliter of seawater, so the scientists estimate that much of the oxygen we breathe may be attributable to the action of this virus on blue-green bacteria. ­ Viruses, though normally pathogenic to their host An algal bloom off the coast of Seattle, cells, do have positive influences, including what appear to be extensive roles in the environment. Washington. M13_BAUM7206_05_SE_C13.indd 387 11/23/16 3:31 AM 13    Characterizing and Classifying Viruses, Viroids, and Prions hapter       C ◀ FIGURE 13.7 Enveloped virion. Artist’s rendition and electron micrograph of severe acute respiratory syndrome (SARS) virus, an enveloped virus with a helical capsid. Glycoproteins Helical capsid Matrix protein Envelope TEM Enveloped virus with helical capsid TABLE 13.1 50 nm The Novel Properties of Viruses Viruses Cells Inert macromolecules outside a cell but become active inside a cell Metabolize on their own Do not divide or grow Divide and grow Acellular Cellular Obligate intracellular parasites Most are free-living Contain either DNA or RNA Contain both DNA and RNA Genome can be dsDNA, ssDNA, dsRNA, + ssRNA, or - ssRNA Genome is dsDNA Usually ultramicroscopic in size, ranging from 10 nm to over 500 nm 200 nm to 12 cm in diameter Have a proteinaceous capsid around genome; some have an envelope around the capsid Surrounded by a phospholipid membrane and often a cell wall Replicate in an assembly-line manner using the enzymes and organelles of a host cell Self-replicating by asexual and/or sexual means ­ the type of nucleic acid, presence of an envelope, shape, and size. So far, virologists have established families for all viral genera, but only seven viral orders are described. Taxonomists have not described kingdoms, divisions, and classes for viruses because the relationships among viruses are not well understood. Some researchers consider viruses to be a fourth domain of life (in addition to Bacteria, Archaea, and Eukarya). Family names are typically derived either from special characteristics of viruses within the family or from the name of an important member of the family. For example, family Picornaviridae contai ns ver y small 1 RNA vir uses, a nd Hepadnaviridae contains a DNA virus that causes hepatitis B. Family Herpesviridae is named for herpes simplex, a virus that can cause genital herpes. TABLE 13.2 lists the major families of human viruses, grouped according to the type of nucleic acid each contains. Specific epithets for viruses are their common English designations written in italics. Generally, viruses are known solely 1Pico means one-trillionth, 10 -12. M13_BAUM7206_05_SE_C13.indd 388 by their English names. The nomenclature for two important viral pathogens, HIV and rabies virus, is as follows: HIV Rabies Virus Order Not yet assigned Mononegavirales Family Retroviridae Rhabdoviridae Genus Lentivirus (len’ti-vī-rŭs) Lyssavirus (lis’a-vī-rŭs) Specific Epithet human immunodeficiency virus rabies virus TELL ME WHY What characteristics of the genomes of parvoviruses and of reoviruses make them very different from cells? Viral Replication As previously noted, viruses cannot reproduce themselves because they lack the genes for all the enzymes necessary for replication; in addition, they do not possess functional 11/23/16 3:31 AM       Viral Replication TABLE 13.2 Family Families of Human Viruses Strand Type Representative Genera (Diseases) Poxviridae Double Orthopoxvirus (smallpox) Herpesviridae Double Simplexvirus (herpes type 1: fever blisters, respiratory infections; herpes type 2: genital infections); Varicellovirus (chicken pox); Lymphocryptovirus, Epstein-Barr virus (infectious mononucleosis, Burkitt’s lymphoma); Cytomegalovirus (birth defects); Roseolovirus (roseola) Papillomaviridae Double Papillomavirus (benign tumors, warts, cervical and penile cancers) Polyomaviridae Double Polyomavirus (progressive multifocal leukoencephalopathy) Adenoviridae Double Mastadenovirus (conjunctivitis, respiratory infections) Hepadnaviridae Partial single and partial double Orthohepadnavirus (hepatitis b) Parvoviridae Single Erythrovirus (erythema infectiosum) Picornaviridae Single, + a Enterovirus (polio); Hepatovirus (hepatitis A) Caliciviridae Single, + Norovirus (gastroenteritis) Astroviridae Single, + Astrovirus (gastroenteritis) Hepeviridae Single, + Hepevirus (hepatitis E) Togaviridae Single, + Alphavirus (encephalitis); Rubivirus (rubella) Flaviviridae Single, + Flavivirus (yellow fever, Japanese encephalitis); Hepacivirus (hepatitis C) Coronaviridae Single, + Coronavirus (common cold, severe acute respiratory syndrome) DNA Viruses RNA Viruses Retroviridae Single, + , segmented Deltaretrovirus (leukemia); Lentivirus (AIDS) Orthomyxoviridae Single, - b segmented Influenzavirus (flu) Paramyxoviridae Single, - Paramyxovirus (common cold, respiratory infections); Pneumovirus (pneumonia, common cold); Morbillivirus (measles); Rubulavirus (mumps) Rhabdoviridae Single, - Lyssavirus (rabies) Bunyaviridae Single, - , segmented Bunyavirus (California encephalitis virus); Hantavirus (pneumonia) Filoviridae Single, - Filovirus (Ebola hemorrhagic fever); Marburgvirus (hemorrhagic fever) Arenaviridae Single, - , segmented Lassavirus (hemorrhagic fever) Reoviridae Double, segmented Orbivirus (encephalitis); Rotavirus (diarrhea); Coltivirus (Colorado tick fever) aPositive-sense (+ RNA) is equivalent to mRNA; that is, it instructs ribosomes in protein translations. (- RNA) is complementary to mRNA; it cannot be directly translated. bNegative-sense ­ • Attachment of the virion to the host cell • Entry of the virion or its genome into the host cell • Synthesis of new nucleic acids and viral proteins by the host cell’s enzymes and ribosomes • Assembly of new virions within the host cell • Release of the new virions from the host cell ▶ ­ANIMATION: Viral Replication: Overview In the following sections, we examine the events that occ ur in the replicat ion of bacteriophages and animal viruses. We begin with lytic replication in bacteriophages; M13_BAUM7206_05_SE_C13.indd 389 then we t urn to lysogenic replication (a modification of replication); and finally, we consider the replication of animal viruses. ­ ribosomes for protein synthesis. Instead, viruses depend on their hosts’ enzymes and organelles to produce new virions. Once a host cell falls under control of a viral genome, it is forced to replicate viral genetic material and translate viral proteins, including viral capsomeres and viral enzymes. The replication cycle of a virus usually results in the death and lysis of the host cell. Because the cell undergoes lysis near the end of the cycle, this type of replication is called a lytic replication cycle. In general, a lytic replication cycle consists of the following five stages: Lytic Replication of Bacteriophages LE A R N I N G O UTC O M E 13.8 Sketch and describe the five stages of the lytic replication cycle as it typically occurs in bacteriophages. Studies of phages revealed the basics of viral biology. Indeed, bacteriophages make excellent tools for the general study of viruses because bacteria are easier and less expensive to culture than animal or human cells. Beneficial Microbes: Prescription Bacteriophages? on p. 392 is an interesting side note on the use of bacteriophages as an alternative to antibiotics. Here, we examine the replication of a much-studied dsDNA phage of E. coli called type 4 (T4). T4 virions are complex, having the polyhedral heads and helical tails seen in many bacteriophages. We begin with attachment, the first stage of replication (FIGURE 13.8). 11/23/16 3:31 AM hapter       C 13    Characterizing and Classifying Viruses, Viroids, and Prions Attachment Bacteriophage genome Entry Tail sheath Outer membrane Peptidoglycan Cytoplasmic membrane Bacterial chromosome 2 Entry 1 Attachment Phage DNA Lytic replication cycle of bacteriophage 5 Release 3 Synthesis Phage proteins 4 Assembly Assembly Sheath DNA Capsid Mature head ▲ FIGURE 13.8 The lytic replication cycle in bacteriophages. The phage shown in this illustration is T4, and the bacterium shown is E. coli. The circular bacterial chromosome is represented diagrammatically; in reality, it would be much longer. M13_BAUM7206_05_SE_C13.indd 390 Tail fibers Mature virion V T ideo Tail utor Base 11/23/16 3:31 AM       Viral Replication Entry Release ­ 1 2 Once phage T4 has attached to the bacterium’s cell wall, it must still overcome the formidable barrier posed by the cell wall and cytoplasmic membrane if it is to enter the cell. T4 overcomes this obstacle in an elegant way. Upon contact with E. coli, T4 releases lysozyme (li’sō-zīm), a protein enzyme carried within the capsid that weakens the peptidoglycan of the cell wall. The phage’s tail sheath then contracts, forcing an internal hollow tube within the tail through the cell wall and membrane, much as a hypodermic needle penetrates the skin. The phage injects its genome through the tube and into the bacterium. The empty capsid, having performed its task, is left on the outside of the cell looking like an abandoned spacecraft. After entry, viral enzymes (which either are carried within the capsid and delivered with the viral genome or are coded by viral genes and made by the bacterium) degrade the bacterial DNA into its constituent nucleotides. 4  ­ Scientists do not understand completely how phages are assembled inside a host cell, but it appears that as capsomeres accumulate within the cell, they spontaneously attach to one another to form new capsid heads. Likewise, tails M13_BAUM7206_05_SE_C13.indd 391 Replication: Virulent Bacteriophages 3 of virion 10 1 Entry Synthesis and assembly 0 Attachment s by lys Burst size 100 is 1000 5 10 15 20 Time (minutes) Release After losing its chromosome, the bacterium stops synthesizing its own molecules and begins synthesizing only viral parts under control of the viral genome. For dsDNA viruses like T4, protein synthesis is straightforward and similar to cellular transcription and translation, except that mRNA is transcribed from viral DNA instead of cellular DNA. Translation by the host cell’s ribosomes results in viral proteins, including capsomeres, components of the tail, viral DNA polymerase (which replicates viral DNA), and lysozyme (which weakens the bacterial cell wall from within, enabling the virions to leave the cell once they have been assembled). Assembly 5 Newly assembled virions are released from the cell as lysozyme completes its work on the cell wall and the bacterium disintegrates. Areas of disintegrating bacterial cells in a lawn of bacteria in a Petri plate look as if the lawn were being eaten, and it was the appearance of these plaques that prompted early scientists to give the name bacteriophage, “bacterial eater,” to these viruses. For phage T4, the process of lytic replication takes about 25 minutes and can produce as many as 100 to 200 new virions for each bacterial cell lysed. For any phage undergoing lytic replication, the period of time required to complete the entire process, from attachment to release, is called the burst time, and the number of new virions released from each lysed bacterial cell is called the burst size (FIGURE 13.9). ▶ ANIMATION: Viral Number of infective virions in medium (log scale) Synthesis  ­ Because phages, like all virions, are nonmotile, contact with bacteria occurs by purely random collision, brought about as molecular bombardment and currents move virions through the environment. The tail fibers are responsible for the attachment of T4 to its host bacterium. Attachment proteins on the tail fibers precisely fit to complementary receptor proteins on the surface of the cell wall of E. coli. The specificity of the attachment proteins for the receptors ensures that the virus will attach only to E. coli. Various bacteriophages may attach to receptor proteins on their host cells’ walls, flagella, or pili. Phage attachment can be so specific that a phage might infect only one particular strain of a bacterial species. Thus, scientists can use phage attachment for bacterial identification in the lab. (Chapter 4 discusses so-called phage typing.) assemble and attach to heads, and tail fibers attach to tails, forming mature virions. Such capsid assembly is a spontaneous process, requiring little or no enzymatic activity. For many years, it was assumed that capsids form around a genome; however, research has shown that for some viruses, enzymes pump the genome into the assembled capsid under high pressure— five times that used in a paintball gun. This process resembles stuffing a strand of cooked spaghetti into a matchbox through a single small hole. Sometimes a capsid assembles around leftover pieces of host DNA instead of viral DNA. A virion formed in this manner is still able to attach to a new host by means of its tail fibers, but instead of inserting phage DNA, it transfers DNA from the first host into a new host. This process is known as transduction (described in more detail in Chapter 7). Attachment 25 30 35 Burst time ▲ FIGURE 13.9 Pattern of virion abundance in lytic cycle. Shown is virion abundance over time for a single lytic replication cycle. New virions are not observed in the culture medium until synthesis, assembly, and release (lysis) are complete, at which time (the burst time) the new virions are released all at once. Burst size is the number of new virions released per lysed host cell. 11/23/16 3:31 AM 13    Characterizing and Classifying Viruses, Viroids, and Prions hapter       C BENEFICIAL MICROBES Prescription Bacteriophages? LE A R N I N G O U TC O M E 13.9 Compare and contrast the lysogenic replication cycle of viruses with the lytic cycle. Not all viruses follow the lytic pattern of phage T4 we just examined. Some bacteriophages have a modified replication cycle in which infected host cells grow and reproduce normally for many generations before they lyse. Such a replication cycle is called a lysogenic replication cycle or lysogeny (lī-soj’ĕ-nē), and the phages are called temperate phages or lysogenic phages. Here, we examine lysogenic replication as it occurs in a much-studied temperate phage, lambda phage, which is another parasite of E. coli. A lambda phage has a linear molecule of dsDNA in a complex capsid consisting of an icosahedral head attached to a tail that lacks tail fibers (FIGURE 13.10). TEM 100 nm ▲ FIGURE 13.10 Bacteriophage lambda. Note the absence of tail fibers. Phage T4 attaches by means of molecules on its tail fibers. How does lambda phage, which lacks fibers, attach? Figure 13.10 Lambda has attachment molecules at the end of its tail rather than on its tail fibers. M13_BAUM7206_05_SE_C13.indd 392 ­ FIGURE 13.11 illustrates lysogeny with lambda phage. First, the virion randomly contacts an E. coli cell and attaches via its tail 1 . The viral DNA enters the cell, just as occurs with phage T4, but the host cell’s DNA is not destroyed, and the phage’s genome does not immediately assume control of the cell. Instead, the virus remains inactive. Such an inactive bacteriophage is called a prophage (pro’fāj) 2 . A prophage remains inactive by coding for a protein that suppresses prophage genes. A side effect of this repressor protein is that it renders the bacterium resistant to additional infection by other viruses of the same type. Another difference between a lysogenic cycle and a lytic cycle is that the prophage inserts into the DNA of the bacterium, becoming a physical part of the bacterial chromosome 3 . For DNA viruses like lambda phage, this is a simple process of fusing two pieces of DNA: One piece of DNA, the virus, is fused to another piece of DNA, the chromosome of the cell. Every time the cell replicates its infected chromosome, the prophage is also replicated 4 . All daughter cells of a lysogenic cell are thus infected with the nearly inactive virus. A prophage and its descendants may remain a part of bacterial chromosomes for generations or even forever. Lysogenic phages can change the phenotype of a bacterium, for example, from a harmless form into a pathogen—a process called lysogenic conversion. Bacteriophage genes are responsible for toxins and other disease-evoking proteins found in the bacterial agents of diphtheria, cholera, rheumatic fever, and certain severe cases of diarrhea caused by E. coli. At some later time, a prophage might be excised from the chromosome by recombination or some other genetic event; it then reenters the lytic phase. The process whereby a prophage is excised from the host chromosome is called induction 5 . Inductive agents are typically the same physical and chemical agents that damage DNA molecules, including ultraviolet light, X rays, and carcinogenic chemicals. After induction, the lytic steps of synthesis 6 , assembly 7 , and release 8 resume from the point at which they stopped. The cell becomes filled with virions and breaks open. ­ Lysogenic Replication of Bacteriophages growing problem of antibiotic-resistant bacteria, scientists in the United States and Western Europe have renewed interest in investigating phage therapy. A phage reproduces by inserting genetic material into a bacterium, causing the bacterium to build copies of the virus that burst out of the cell to infect other bacteria. A single phage can become 10 trillion phages within 2 hours, killing 99.9% of its host bacteria. Since each type of phage attacks a specific strain of bacteria, phage treatment is effective only if the phages are carefully matched to the disease-causing bacterium. It also means that phage treatment, unlike the use of antibiotics, can be effective without killing the body’s helpful bacteria. ­ ­ 50 nm ­ In 1917, Canadian biologist Felix d’Herelle published a paper announcing the discovery of bacteriophages, Escherichia coli viruses that prey on bacteria. In fact, half the infected with bacteria on Earth succumb to phages every two bacteriophages. days! D’Herelle said that phages could be natural weapons against bacterial pathogens. Phage therapy was used in the early 1900s to combat dysentery, typhus, and cholera but was largely abandoned in the 1940s in the United States, eclipsed by the development of antibiotics, such as penicillin. Phage therapy continued in the Soviet Union and Eastern Europe, where research is still centered. Today, motivated by the SEM 11/23/16 3:31 AM       Viral Replication 1 Attachment 3 Prophage in chromosome 2 Entry Lambda phage Lytic cycle Lysogeny 6 Synthesis 8 Release 4 Replication of chromosome and virus; cell division 7 Assembly ­ ▲ FIGURE 13.11 The lysogenic replication cycle in bacteriophages. 5 Induction Further replications and cell divisions ­ The phage shown in this illustration is phage lambda, and the bacterium shown is E. coli. The circular bacterial chromosome is represented diagrammatically; in reality, it would be much longer. How is a lysogenic cycle different from a lytic cycle? Figure 13.11 In a lysogenic cycle, the virus is inserted into the bacterial chromosome, and it is replicated and passed on to all daughter cells until it is induced to leave the chromosome; a lytic cycle is a replication cycle that results in cell death. Bacteriophages T4 and lambda demonstrate two replication strategies that are typical for many DNA viruses. RNA viruses and enveloped viruses present variations on the lytic and lysogenic cycles we have examined. We will next examine some variations seen in replication of animal viruses. ▶ ANIMATION: Viral Replication: Temperate Bacteriophages Replication of Animal Viruses LE A R N I N G O U TC O M E S 13.10 Explain the differences between bacteriophage replica- tion and animal viral replication. 13.11 Compare and contrast the replication and synthesis of DNA, -RNA, and +RNA viruses. 13.12 Compare and contrast the release of viral particles by lysis and budding. 13.13 Compare and contrast latency in animal viruses with phage lysogeny. M13_BAUM7206_05_SE_C13.indd 393 Animal viruses have the same five basic steps in their replication pathways as bacteriophages—that is, attachment, entry, synthesis, assembly, and release. However, there are significant differences in the replication of animal viruses that result in part from the presence of envelopes around some of the viruses and in part from the eukaryotic nature of animal cells and the lack of an animal cell wall. In this section, we examine the replication processes that are shared by DNA and RNA animal viruses, compare these processes with those of bacteriophages, and discuss how the synthesis of DNA and RNA viruses differ. Attachment of Animal Viruses As with bacteriophages, attachment of an animal virion is dependent on the chemical attraction and exact fit between attachment molecules on the virion and complementary receptors on the animal cell’s surface. Unlike the bacteriophages we have examined, animal viruses lack both tails and tail fibers. Instead, animal viruses often have glycoprotein spikes on their capsids or envelopes. 11/23/16 3:31 AM hapter       C 13    Characterizing and Classifying Viruses, Viroids, and Prions Entry and Uncoating of Animal Viruses 1 Viral genome inside capsid 2 3 Empty capsid Receptors on cytoplasmic membrane Viral genome (a) Direct penetration Viral glycoproteins remain in cytoplasmic membrane Viral glycoproteins 1 2 ­ ­ ­ ­ ­ Animal viruses enter a host cell shortly after attachment. Even though entry of animal viruses is not as well understood as entry of bacteriophages, there are at least three different mechanisms: direct penetration, membrane fusion, and endocytosis. Some naked viruses enter their hosts’ cells by direct penetration—a process in which the viral capsid attaches and sinks into the cytoplasmic membrane, creating a pore through which the genome alone enters the cell, leaving the empty capsid on the surface (FIGURE 13.12a). Poliovirus infects host cells via direct penetration. With other animal viruses, by contrast, the entire capsid and its contents enter a host cell by either membrane fusion or endocytosis. With viruses using membrane fusion, such as measles virus, the viral envelope and the host’s cytoplasmic membrane fuse, releasing the capsid into the cell’s cytoplasm while leaving the envelope glycoproteins as part of the cell membrane (FIGURE 13.12b). Most enveloped viruses and some naked viruses enter host cells by triggering endocytosis. Attachment of the virus to receptor molecules on the cell’s surface stimulates the cell to endocytize the entire virus (FIGURE 13.12c). For example, adenoviruses (naked) and herpesviruses (enveloped) enter human host cells via endocytosis. For a virus that penetrates a host cell with its capsid intact, the capsid must be removed to release the genome before replication of the virus can continue. The process, called uncoating, is poorly understood. Uncoating occurs by different means in different viruses; some viruses are uncoated within vesicles by cellular enzymes, whereas others are uncoated by enzymes within the cell’s cytosol. Envelope 3 4 Receptors on cytoplasmic membrane of host 5 Viral genome ­ Uncoating capsid (b) Membrane fusion Cytoplasmic membrane of host engulfs virus (endocytosis) Synthesis of DNA Viruses of Animals 2 1 ­ Synthesis of animal viruses also differs from synthesis of bacteriophages. Each type of animal virus requires a different strategy for synthesis that depends on the kind of nucleic acid involved—whether it is DNA or RNA and whether it is double stranded or single stranded. Some DNA animal viruses enter the cell’s nucleus, whereas other DNA viruses and most RNA animal viruses are replicated in the cytoplasm. Further, in synthesis of enveloped viruses, some viral proteins are inserted into cellular membranes. These membranes become the viral envelopes while the proteins become the envelope’s spikes. As we discuss the synthesis and assembly of each type of animal virus, consider the following two questions: 3 4 ­ 6 Uncoating capsid (c) Endocytosis ▲ FIGURE 13.12 Three mechanisms of entry of animal viruses. (a) Direct penetration, a process whereby naked virions inject their genomes into their animal cell hosts. (b) Membrane fusion, in which fusion of the viral envelope and cell membrane dumps the capsid into the cell. (c) Endocytosis, in which attachment of a naked or an enveloped virus (as shown here) stimulates the host cell to engulf the entire virus. After penetration, many animal viruses must be uncoated, but bacteriophages need not be. Why is this so? ­ • How is mRNA—needed for the translation of viral proteins—synthesized? • What molecule serves as a template for nucleic acid replication? 5 Viral genome M13_BAUM7206_05_SE_C13.indd 394 Figure 13.12 Generally, bacteriophages inject their DNA during penetration, so the capsid does not enter the cell. dsDNA Viruses Synthesis of new double-stranded DNA (dsDNA) virions is similar to the normal replication of cellular DNA (which is double stranded) and the normal translation of 11/23/16 3:31 AM       Viral Replication ­ polymerase transcribes complementary negative-sense, single-stranded RNA ( -ssRNA) from the +ssRNA genome; -ssRNA then serves as the template for the transcription of multiple +ssRNA genomes (FIGURE 13.13a). Such transcription of RNA from RNA is unique to viruses; no cell transcribes RNA from RNA. ­ Retroviruses Unlike other +ssRNA viruses, the +ssRNA viruses called retroviruses do not use their genome as mRNA. Instead, reverse transcriptase carried within the retrovirus’s capsid transcribes a dsDNA intermediary from the +ssRNA genome. This DNA intermediary then serves as the template for the synthesis of additional +ssRNA molecules, which act both as mRNA for protein synthesis and as genomes for new virions (see Chapter 18, Figures 18.15 and 18.17). Human immunodeficiency virus (HIV) is a prominent retrovirus. Negative-Sense ssRNA Viruses Other single-stranded RNA virions are negative sense (-ssRNA). Their negative-sense single-stranded RNA genomes must overcome a unique problem: In order to synthesize a protein, a cell’s ribosome can use only mRNA, which is positive-sense (i.e., +ssRNA). Ribosomes don’t recognize -ssRNA. The virus overcomes this problem by carrying within its capsid an enzyme, RNA­dependent RNA transcriptase, which is released into the host cell’s cytoplasm during uncoating and then transcribes +ssRNA molecules from the virus’s -ssRNA genome. Translation of proteins can then occur as usual. The newly transcribed +ssRNA also serves as a template for transcription of additional copies of -ssRNA (FIGURE 13.13b). Diseases caused by -ssRNA viruses include rabies and flu. ssDNA Viruses A human virus with a genome composed of single-stranded DNA (ssDNA) is a parvovirus. Cellular RNA polymerase synthesizes mRNA complementary to the viral ssDNA, and cellular ribosomes can then translate viral proteins. Cells do not have ssDNA, so synthesizing ssDNA viruses requires a unique form of DNA replication. The ssDNA of parvovirus folds back on itself to form a hairpin loop that acts as double-stranded DNA, which can be replicated by cellular DNA polymerase. However, when DNA polymerase replicates this “false” dsDNA, the newly replicated strand of DNA is released as ssDNA. This single-stranded DNA is packaged into a viral capsid. ­ ­ ­ ­ Positive-Sense ssRNA Viruses Single-stranded viral RNA that can act directly as mRNA is called positive-sense single-stranded RNA (∙RNA). Ribosomes can translate polypeptides using the codons of such RNA. An example of a +ssRNA virus is poliovirus. In many +ssRNA viruses, a virally coded RNA ­ ­ ­ ­ As previously noted, cells don’t have RNA genes, so it follows that synthesis of RNA viruses must differ significantly from cellular processes and from the replication of DNA viruses as well. There are four types of RNA viruses: positive-sense, single-stranded RNA (designated +ssRNA); retroviruses (a kind of +ssRNA virus); negative-sense, single-stranded R NA (-ssRNA); a nd double - st ra nde d R NA (d sR NA). Scientists designate viral RNA that can act as mRNA as positive-sense RNA; that is, ribosomes can translate polypeptides directly from +ssRNA. Ribosomes cannot directly translate negative-sense RNA. A complementary copy of negative-sense RNA—a positive-sense molecule—must first be transcribed before a ribosome can translate the message carried by -ssRNA. The synthesis processes for the four types of RNA viruses are varied and rather complex. We start with the synthesis of +ssRNA viruses. M13_BAUM7206_05_SE_C13.indd 395 dsRNA Viruses Viruses that have double-stranded RNA (dsRNA) use yet another method of synthesis. The positive strand of the molecule serves as mRNA for the translation of proteins, one of which is an RNA polymerase that transcribes dsRNA. Each strand of the dsRNA acts as a template for transcription of its opposite, which is reminiscent of DNA replication in cells (FIGURE 13.13c) . Double-stranded RNA rotaviruses cause most cases of diarrhea in infants. TABLE 13.3 on p. 396 summarizes the various strategies by which animal viruses are synthesized. ­ Synthesis of RNA Viruses of Animals ­ ­ cellular proteins. The genome of most dsDNA viruses enters the nucleus of the host cell, where cellular enzymes replicate the viral genome in the same manner as they replicate the host DNA—using each strand of viral DNA as a template for its complement. Messenger RNA is transcribed from viral DNA in the nucleus and exported into the cytoplasm, where host ribosomes make capsomere proteins. The capsomeres then enter the nucleus, where new virions spontaneously assemble. This method of replication is seen with herpes and papilloma (wart) viruses. Replication of hepatitis B virus is an exception to this scenario: The genome of hepatitis B viruses is replicated using an RNA intermediary instead of replicating DNA from a DNA template. In other words, the genome of hepatitis B virus is transcribed into RNA, which is then used as a template to make multiple copies of viral DNA genome. The latter process, which is the reverse of normal transcription, is mediated by a viral enzyme, reverse transcriptase. Assembly and Release of Animal Viruses As with bacteriophages, once the components of animal viruses are synthesized they assemble into virions that are then released from the host cell. Most DNA viruses assemble in and are released from the nucleus into the cytosol, whereas most RNA viruses develop solely in the cytoplasm. The number of viruses produced and released depends on both the type of virus and the size and initial health of the host cell. Generally, replication of animal viruses takes more time than replication of bacteriophages. Herpesviruses, for example, require almost 24 hours to replicate, as compared to 25 minutes for bacteriophage T4. 11/23/16 3:31 AM P       Chapter 13    Characterizing and Classifying Viruses, Viroids, and rions Receptors on cytoplasmic membrane of host Receptors on cytoplasmic membrane of host –ssRNA virus +ssRNA virus Receptors on cytoplasmic membrane of host dsRNA virus +ssRNA –ssRNA Transcription by viral RNA polymerase dsRNA Transcription by RNA-dependent RNA transcriptase Complementary –ssRNA to act as template Further transcription Translation of viral proteins; genome acts as mRNA Unwinding –ssRNA Complementary +ssRNA to act as template and as mRNA Further transcription Transcription by viral RNA polymerase to make complementary RNA strands Translation of viral proteins Copies of –ssRNA +ssRNA acts as template and as mRNA Translation of viral proteins Copies of +ssRNA Assembly Assembly (a) Positive-sense ssRNA virus Assembly (b) Negative-sense ssRNA virus (c) Double-stranded RNA virus TABLE 13.3 A R A R A R A A R R A R P ▲ FIGURE 13.13 Synthesis of proteins and genomes in animal RNA viruses. (a) ositive-sense ss N virus, in which +ssrNa acts as m N and -ssrNa is synthesized for a viral genome template. (b) Negative-sense ss N virus: transcription forms +ssrNa to serve both as m N and as template for viral -ssrNa genome. (c) ds N virus genome unwinds so that the positive-sense strand serves as m N and each strand serves as a template for its complement. Synthesis Strategies of Animal Viruses What Molecule Is the Template for Genome Replication? ach strand of DN serves as template for its complement (except for hepatitis B, which synthesizes N to act as the template for new DN ) ssDN By N polymerase (in nucleus of cell) Complementary strand of DN is synthesized to act as template + ssrNa Genome acts as m N - rNa complementary to the genome is synthesized to act as template + ssrNa (Retroviridae) DN is synthesized from N by reverse transcriptase; m N is transcribed from DN by N polymerase - ssrNa By N -dependent N transcriptase + rNa (m N ) complementary to the genome ds NA Positive strand of genome acts as m NA Each strand of genome acts as template for its complement M13_BAUM7206_05_SE_C13.indd 396 A R A A DN A R A R A A R A A R A R A A R R A R R R A A A R A Typically by cellular N polymerase (in nucleus) or by viral N polymerase in cytoplasm E dsDNA A How Is mRNA Synthesized? R Genome 06/03/2018 09:44 Enveloped virion Number of infective virions in medium       The Role of Viruses in Cancer 5 Budding of enveloped virus 3 4 2 1 Viral proteins Viral capsid Cytoplasmic membrane of host Synthesis and assembly Release of virions Attachment Time ▲ FIGURE 13.15 Pattern of virion abundance in persistent infections. A generalized curve of virion abundance for persistent infections by budding enveloped viruses. Because the curve does not represent any actual infection, units for the graph’s axes are omitted. ­ ▲ FIGURE 13.14 The process of budding in enveloped viruses. What term describes a nonenveloped virus? Entry ­ Because viral replication uses cellular structures and pathways involved in the growth and maintenance of healthy cells, any strategy for the treatment of viral diseases that involves disrupting viral replication may disrupt normal cellular processes as well. This is one reason it is difficult to treat viral diseases. (The modes of action of some available antiviral drugs are discussed in Chapter 10; Chapters 15 and 16 discuss the body’s naturally produced antiviral chemicals—interferons and antibodies.) Latency of Animal Viruses Some animal viruses, including AIDS, chicken pox, hepatitis B, and herpes viruses, may remain dormant in cells in a M13_BAUM7206_05_SE_C13.indd 397 TELL ME WHY Why are lysogenic and latent viral infections generally longer lasting than lytic infections? The Role of Viruses in Cancer LE A R N I N G O UTC O M E S 13.14 Define the terms neoplasia, tumor, benign, malignant, cancer, and metastasis. ­ Animal Viruses ­ Figure 13.14 Naked. Enveloped animal viruses are released via a process called budding (FIGURE 13.14). As virions are assembled, they are extruded through one of the cell’s membranes—the nuclear, endoplasmic reticulum, or cytoplasmic membrane. Each enveloped virion acquires a portion of a cellular membrane, into which viral proteins were inserted during the synthesis stage. This membrane becomes the viral envelope. Because the animal cell is not quickly lysed, as occurs in bacteriophage replication, budding allows an infected cell to remain alive for some time. Infections with enveloped viruses in which host cells shed viruses slowly and relatively steadily are called persistent infections; a curve showing virus abundance over time during a persistent infection lacks the burst of new virions seen in lytic replication cycles (FIGURE 13.15; compare to Figure 13.9). Naked animal viruses may be released in one of two ways: Either they may be extruded from the cell by exocytosis, in a manner similar to budding but without the acquisition of an envelope, or they may cause lysis and death of the cell, reminiscent of bacteriophage release. ▶ ANIMATION: Viral Replication: process known as latency; the viruses involved in latency are called latent viruses or proviruses. Latency may be prolonged for years with no viral activity, signs, or symptoms. Though latency is similar to lysogeny as seen with bacteriophages, there are differences. Typically, when latent animal viruses are incorporated into host DNA, the condition is permanent; induction does not occur. Thus, an incorporated, latent provirus becomes a permanent, physical part of the host’s chromosome, and all descendants of the infected cell will carry the provirus. Given that RNA cannot be incorporated directly into a chromosome molecule, how does the ssRNA of HIV become a provirus incorporated into the DNA of its host cell? HIV can become a permanent part of a host’s chromosome because it, like all retroviruses, carries reverse transcriptase, which transcribes the genetic information of the +RNA molecule to a DNA molecule—which can then become incorporated into a host cell’s genome. TABLE 13.4 on p. 398 compares the features of the replication of bacteriophages and animal viruses. Next we turn our attention to the part viruses can play in cancer, beginning with a brief consideration of the terminology needed to understand the basic nature of cancer. 13.15 Explain in simple terms how a cell may become cancer- ous, with special reference to the role of viruses. 11/23/16 3:31 AM 13    Characterizing and Classifying Viruses, Viroids, and Prions hapter       C TABLE 13.4 A Comparison of Bacteriophage and Animal Virus Replication Bacteriophage Animal Virus Attachment Proteins on tails attach to proteins on cell wall Spikes, capsids, or envelope proteins attach to proteins or glycoproteins on cell membrane Penetration Genome is injected into cell or diffuses into cell Capsid enters cell by direct penetration, fusion, or endocytosis Uncoating None Removal of capsid by cell enzymes Site of Synthesis In cytoplasm RNA viruses: in cytoplasm; most DNA viruses: in nucleus Site of Assembly In cytoplasm RNA viruses: in cytoplasm; most DNA viruses: in nucleus Mechanism of Release Lysis Naked virions: exocytosis or lysis; enveloped virions: budding Nature of Chronic Infection Lysogeny, always incorporated into host chromosome, may leave host chromosome Latency, with or without incorporation into host DNA; incorporation is permanent ­ ­ Some tumors are benign tumors; that is, they remain in one place and are not generally harmful, although occasionally such noninvasive tumors are painful and rob adjacent normal cells of space and nutrients. Other tumors are malignant tumors, invading neighboring tissues and even traveling throughout the body to invade other organs and tissues to produce new tumors—a process called metastasis (mĕ-tas’tă-sis). Malignant tumors are also called cancers. Cancers rob normal cells of space and nutrients and cause pain; in some kinds of cancer, malignant cells derange the function of the affected tissues, until eventually the body can no longer withstand the loss of normal function and dies. ­ ­ Under normal conditions, the division of cells in a mature multicellular animal is under strict genetic control; that is, the animal’s genes dictate that some types of cells no longer divide at all and that those that can divide are prevented from unlimited division. In this genetic control, either genes for cell division are “turned off,” or genes that inhibit division are “turned on,” or some combination of both these genetic events occurs. However, if something upsets the genetic control, cells begin to divide uncontrollably. This phenomenon of uncontrolled cell division in a multicellular animal is called neoplasia2 (nē-ō-pla’zē-ă). Cells undergoing neoplasia are said to be neoplastic, and a mass of neoplastic cells is a tumor. EMERGING DISEASE CASE STUDY Chikungunya ­ 2From ­ ­ Aedes albopictus (Asian tiger mosquito), which carries the virus, has moved into temperate climates, including Europe and the United States, as the climate has warmed. With the mosquito comes the possibility of viral proliferation—the insects have spread the tropical disease as far north in Europe as France. And our Italian patient? His crippling pain lasted for months, but he survived. Now that he knows about mosquito-borne chikungunya, he insists that his family and friends use mosquito repellent liberally. Officials in the rest of Europe and in the United States join in his concern: With the coming of Ae. albopictus, is incurable chikungunya far behind? ­ ­ ­ ­ ­ ­ ­ An old man arrived at the doctor’s office in Ravenna, Italy, with a combination of signs and symptoms the physician had never heard of: a widespread, severe rash; difficulty in breathing; high fever; nausea; and extreme joint pain. Chikungunya (chik-en-gun’ya) had arrived in Europe. Though scientists had known of chikungunya virus, which is related to equine encephalitis viruses, for over 50 years, most considered the tropical disease benign—a limited, mild irritation, not a catastrophe. Therefore, few researchers studied chikungunya virus or its disease. Now, they know better. Over the past decade, chikungunya virus has spread throughout the nations of the Indian Ocean and across Africa. In 2006, officials on the French-owned island of La Réunion in the Indian Ocean reported 47,000 cases of chikungunya in a single week! That same year chikungunya reemerged in India for the first time in four decades with more than 1.5 million reported cases, and in 2010 it emerged in China. Why? 1. Besides using repellents, how can people protect themselves from mosquito-borne pathogens? 2. Why is Aedes commonly known as the tiger mosquito? 3. Why aren’t antibiotics such as penicillin, erythromycin, and ciprofloxacin effective in preventing and treating chikungunya? Greek neo, meaning “new,” and plassein, meaning “to mold.” M13_BAUM7206_05_SE_C13.indd 398 11/23/16 3:31 AM       Culturing Viruses in the Laboratory Several theories have been proposed to explain the role viruses play in the development of cancers. These theories revolve around the presence of protooncogenes (prō-tō-ong’ kō-jēnz)—genes that promote cell growth and division. As long as protooncogenes are appropriately expressed under controlled conditions, no cancer results. However, activity of oncogenes (their name when they are active) or inactivation of oncogene repressors can cause cancer to develop. In most cases, several genetic changes must occur before cancer develops. Put another way, “multiple hits” to the genome must occur for cancer to result (FIGURE 13.16). A variety of environmental factors contribute to the inhibition of oncogene repressors and the activation of oncogenes. Ultraviolet light, radiation, certain chemicals called carcinogens (kar-si’nō-jenz), and viruses have all been implicated in the development of cancer. Viruses may contribute to the development of 20% to 25% of human cancers, and they do so in several ways. Some viruses carry copies of oncogenes as part of their genomes, other viruses insert near to and promote oncogenes already present in the host, and still other viruses interfere with normal tumor repression when they insert (as latent proviruses) into repressor genes. That viruses lead to some animal cancers is well established. In the first decade of the 1900s, virologist F. Peyton Rous (1879–1970) proved that viruses induce cancer in chickens. Though several DNA and RNA viruses are associated with human cancers, proving that viruses cause human cancers has been difficult. Among the human cancers proven to be virally induced are Burkitt’s lymphoma, Hodgkin’s disease, Kaposi’s sarcoma, and cervical cancer. DNA viruses in the families Adenoviridae, Herpesviridae, Hepadnaviridae, Papillomaviridae, and Polyomaviridae and two RNA viruses in the family Retroviridae lead to these and other human cancers. (Chapters 18–24 discuss diseases caused by DNA and RNA viruses.) TELL ME WHY Why are DNA viruses more likely to cause neoplasias than are RNA viruses? Culturing Viruses in the Laboratory LE A R N I N G O UTC O M E S 13.16 Describe some ethical and practical difficulties to over- come in culturing viruses. 13.17 Describe three types of media used for culturing Normal state: DNA Protooncogene Gene for repressor mRNA Scientists must culture viruses in order to conduct research and develop vaccines and treatments. But because viruses cannot metabolize or replicate by themselves, they cannot be grown in standard microbiological broths or on agar plates. Instead, viruses must be cultured inside suitable host cells, a requirement that complicates the detection, identification, and characterization of viruses. Virologists have developed three types of media for culturing viruses: media consisting of mature organisms (bacteria, plants, or animals), embryonated (fertilized) eggs, and cell cultures. We begin by considering the culture of viruses in organisms. ­ Represses viruses. Repressor Result: No cancer First “hit”: Virus inserts promoter DNA Oncogene Represses Gene for repressor Culturing Viruses in Mature Organisms mRNA LE A R N I N G O UTC O M E S Repressor Result: Still no cancer 13.18 Explain the use of a plaque assay in culturing viruses in bacteria. 13.19 List three problems with growing viruses in animals. Second “hit”: Virus inserts into repressor gene DNA Oncogene mRNA Protein No repressor protein because gene is segmented Causes cell division Result: Cancer ▲ FIGURE 13.16 The oncogene theory of the induction of cancer in humans. The theory suggests that more than one “hit” to the DNA (i.e., any change or mutation), whether caused by a virus (as shown here) or various physical or chemical agents, is required to induce cancer. M13_BAUM7206_05_SE_C13.indd 399 In the following sections, we consider the use of bacterial cells as a virus culture medium before considering the issues involved in growing viruses in living animals. Culturing Viruses in Bacteria Most of our knowledge of viral replication has been derived from research on bacteriophages, which are relatively easy to culture because some bacteria are easily grown and maintained. Phages can be grown in bacteria maintained either in liquid cultures or on agar plates. In the latter case, bacteria and phages are mixed with warm (liquid) nutrient agar and poured 11/23/16 3:31 AM hapter       C 13    Characterizing and Classifying Viruses, Viroids, and Prions Injection into chorioallantoic membrane Injection into chorioallantois Bacterial lawn Viral plaques Injection into embryo Injection into amnion ▲ FIGURE 13.17 Viral plaques in a lawn of bacterial growth on the surface of an agar plate. What is the cause of viral plaques? Injection into yolk sac ▲ FIGURE 13.18 Inoculation sites for the culture of viruses in embryonated chicken eggs. Why are eggs often used to grow animal Figure 13.17 Each plaque is an area in a bacterial lawn where bacteria have succumbed to phage infections. viruses? Plant and animal viruses can be grown in laboratory plants and animals. Recall that the first discovery and isolation of a virus was the discovery of tobacco mosaic virus in tobacco plants. Rats, mice, guinea pigs, rabbits, pigs, and primates have been used to culture and study animal viruses. However, maintaining laboratory animals can be difficult and expensive, and this practice raises ethical issues for some people. Growing viruses that infect only humans raises additional ethical complications. Scientists have developed alternative ways of culturing animal and human viruses using fertilized chicken eggs or cell cultures. Culturing Viruses in Embryonated Chicken Eggs Chicken eggs are a useful culture medium for viruses because they are inexpensive, are among the largest of cells, are free of contaminating microbes, and contain a nourishing yolk (which makes them self-sufficient). Most suitable for culturing viruses M13_BAUM7206_05_SE_C13.indd 400 ­ ­ are chicken eggs that have been fertilized and thus contain a developing embryo. Embryonic tissues (called membranes, which should not be confused with cellular membranes) provide ideal inoculation sites for growing viruses (FIGURE 13.18). Researchers inject samples of virus into embryonated eggs at the sites that are best suited for the particular virus’s replication. Vaccines against some viruses can also be prepared in eggs. You may have been asked whether you are allergic to eggs before you received such a vaccine; the reason is that egg protein may remain as a contaminant in the vaccine. ­ Culturing Viruses in Plants and Animals Figure 13.18 Eggs are large, sterile, self-sufficient cells that contain a number of different sites suitable for viral replication. in a thin layer across the surface of an agar plate. During incubation, bacteria infected by phages lyse and release new phages that infect nearby bacteria, while uninfected bacteria grow and reproduce normally. After incubation, the appearance of the plate includes a uniform bacterial lawn interrupted by clear zones called plaques, which are areas where phages have lysed the bacteria (FIGURE 13.17). Such plates enable virologists to estimate phage numbers via a technique called plaque assay, in which they assume that each plaque corresponds to a single phage spreading and multiplying through the original bacterium–virus mixture. Culturing Viruses in Cell (Tissue) Culture LE A R N I N G O UTC O M E 13.20 Compare and contrast diploid cell culture and continu- ous cell culture. Viruses can also be grown in cell culture, which consists of cells isolated from an organism and grown on the surface of a medium or in broth (FIGURE 13.19). Such cultures became practical when antibiotics provided a way to limit the growth of contaminating bacteria. Cell culture can be less expensive than maintaining research animals, plants, or eggs, and it avoids some of the moral problems associated with experiments performed on animals and humans. Cell cultures are sometimes called tissue cultures, but the term cell culture is more accurate because only a single type of cell is used in the culture. (By definition, a tissue is composed of at least two kinds of cells.) 11/23/16 3:31 AM       Other Parasitic Particles: Viroids and Prions ▲ FIGURE 13.19 An example of cell culture. The bag contains a colored nutrient medium for growing cells in which viruses can be cultured. TELL ME WHY Why are viruses seemingly alive and yet not alive? Other Parasitic Particles: Viroids and Prions ­ ­ Cell cultures are of two types. The first type, diploid cell culture, is created from embryonic animal, plant, or human cells that have been isolated and provided appropriate growth conditions. The cells in diploid cell culture generally last no more than about 100 generations (cell divisions) before they die. The second type of culture, continuous cell culture, is longer lasting because it is derived from tumor cells. Recall that a characteristic of neoplastic cells is that they divide relentlessly, providing a never-ending supply of new cells. One of the more famous continuous cell cultures is of HeLa cells, derived from Henrietta Lacks, who died of cervical cancer in 1951. Though she is dead, Mrs. Lacks’s cells live on in laboratories throughout the world. It is interesting that HeLa cells have lost some of their original characteristics. For example, they are no longer diploid, because they have lost many chromosomes. Thus, HeLa cells provide only a semistandard3 human tissue culture medium for studies on cell metabolism, aging, and (of course) viral infection. Now that we have studied the characteristics and replication processes of viruses, let’s ask a question: Are viruses alive? To be able to wrestle with the answer, we must first recall five characteristics of life: growth, self-reproduction, responsiveness, and the ability to metabolize, all within structures called cells. According to these criteria, viruses lack the qualities of living things, prompting some scientists to consider them nothing more than complex pathogenic chemicals. For other scientists, however, at least four observations—viruses use sophisticated methods to invade cells, have the means of taking control of their host cells, possess genomes containing instructions for replicating themselves, and evolve over time—indicate that viruses are the ultimate parasites because they use cells to make more viruses. According to this viewpoint, viruses are the least complex living entities. At the least, viruses are biological entities on the threshold of life—outside cells they do not appear to be alive, but within cells they direct the synthesis and assembly required to make copies of themselves. Viruses are not the only submicroscopic entities capable of causing disorders within cells. In this section, we will consider the characteristics of two molecular particles that infect cells: viroids and prions. Characteristics of Viroids LE A R N I N G O UTC O M E S 13.22 Define and describe viroids. 13.23 Compare and contrast viroids and viruses. TELL ME WHY ­ HIV replicates only in certain types of human cells, and one early problem in AIDS research was culturing those cells. Why are scientists now able to culture HIV? ­ Are Viruses Alive? Viroids are extremely small (as few as 239 nucleotides long), circular pieces of ssRNA that are infectious and pathogenic in plants (FIGURE 13.20). Viroids are similar to RNA viruses except that they lack capsids and viroid RNA does not code for proteins. Viroids appear to cause disease by adhering to complementary sequences of plant mRNA, forming double-stranded RNA, which a plant enzyme degrades as if it were a dsRNA virus. In the case of a viroid disease, the enzyme is destroying the plant’s own mRNA, which results in the disease state. Several plant diseases, including some of coconut palm, chrysanthemum, potato, cucumber, and avocado, are caused by viroids, including the stunting shown in FIGURE 13.21. Viroidlike agents—i n fect ious, pat hogen ic RNA particles that lack capsids but do not infect plants—affect LE A R N I N G O U TC O M E 13.21 Discuss aspects of viral replication that are lifelike and nonlifelike. 3HeLa cells are “semistandard” because various strains have lost different chromosomes and acquired different mutations over the years. The result is that HeLa cells in one laboratory may be slightly different from HeLa cells in another laboratory. M13_BAUM7206_05_SE_C13.indd 401 11/23/16 3:31 AM hapter       C 13    Characterizing and Classifying Viruses, Viroids, and Prions Genome of bacteriophage T7, a virus Characteristics of Prions Viroids LE A R N I N G O UTC O M E S 13.24 Define and describe prions, including their replication process, and contrast them with viruses. 13.25 Describe methods to control and destroy prions. 13.26 List four diseases caused by prions. TEM 500 nm ▲ FIGURE 13.20 The RNA strand of potato viroid. Though ­ ­ circular, viroids can appear linear, as here, because bonding of complementary bases on opposite sides of the circle can flatten the circle. Also shown for comparison is the longer DNA genome of bacteriophage T7. Compare both to the size of a bacterial genome in Figure 13.2. How are viroids similar to and different from viruses? Figure 13.20 Viroids are similar to certain viruses in that they are infectious and contain a single strand of RNA; they are different from viruses in that they lack a proteinaceous capsid. ­ ­ ­ In 1982, Stanley Prusiner (1942–) described a proteinaceous infectious agent that was different from any other known infectious agent in that it lacked instructional nucleic acid. Prusiner named such agents of disease prions (prē’onz), for proteinaceous infective particles. Before his discovery, researchers thought prion diseases were caused by “slow viruses,” which were so named because 60 years might lapse between an infection and the onset of signs and symptoms. Through experiments, Prusiner and his colleagues showed that prions are not viruses because they lack nucleic acid. ▶ ANIMATION: Prions: Overview Some scientists resist the concept of prions because particles that lack any nucleic acid violate the “universal” rule of protein synthesis—that proteins are translated from a molecule of mRNA. Given that prions lack RNA or DNA genes, how can they replicate themselves? All mammals’ brain cells make a cytoplasmic membrane protein called PrP. PrP is anchored in lipid rafts and plays a role in the normal activity of the brain, though the exact function of PrP is unknown. The amino acid sequence in PrP is such that the protein can fold into two stable tertiary structures: The normal, functional structure of cellular PrP (c­PrP) has several prominent α-helices, whereas a disease-causing form—prion PrP (p­PrP)— is characterized by β-pleated sheets (FIGURE 13.22). Scientists have determined that prion PrP acts like a bad influence in a crowd of teenagers, encouraging molecules of normal, cellular PrP to misbehave by refolding into prion PrP molecules. This process is called templating—prion PrP acts as a template to refold molecules of normal, cellular PrP (FIGURE 13.23). As clumps of prion PrP propagate throughout the brain, neurons stop working properly and eventually die, leaving holes and a spongy appearance (FIGURE 13.24). Because of this characteristic, clinicians call prion diseases of the brain spongiform encephalopathies (spŭn’ji-fōrm en-sef’ă-lop’ă-thēz). ▶ ANIMATION: Prions: Characteristics ­ some fungi. (They are not called viroids because they do not infect plants.) No animal diseases are known to be caused by viroidlike molecules, though the possibility exists that infectious RNA may be responsible for some diseases in humans. M13_BAUM7206_05_SE_C13.indd 402 ­ ▲ FIGURE 13.21 One effect of viroids on plants. The potatoes at right are stunted as the result of infection with potato viroids. Why don’t prions develop in all mammals, given that all mammals have PrP? Under normal circumstances, it appears that other nearby proteins and polysaccharides in lipid rafts in the cytoplasmic membrane force PrP into the correct (cellular) shape. Further, human PrP misfolds only if it contains methionine as the 129th amino acid. About 40% of humans have this amino acid at this place in their cellular PrP and are thus susceptible to prion disease. Prion disease can be inherited, sporadic, or infectious. In the inherited form, a mutation in the cellular PrP gene results in the initial formation of prion PrP. Sporadic prion disease develops following a rare random change of c-PrP in the membrane into p-PrP; note that this is a change in protein shape—the PrP gene remains normal in these patients. Infectious prion disease follows ingestion of infected tissue, transplants of infected tissue, or contact between infected tissue and mucous membranes or skin abrasions. 11/23/16 3:31 AM       Other Parasitic Particles: Viroids and Prions ­ ◀ FIGURE 13.22 The two stable, threedimensional forms of prion protein (PrP). (a) Cellular ­ prion protein (normal form) found in functional cells has a preponderance of alpha-helices. (b) Prion PrP (abnormal form), which has the same amino acid sequence, is folded to produce more beta-pleated sheets. (a) Cellular PrP (c-PrP) (b) Prion PrP (p-PrP) p-PrP c-PrP 1 Cell makes c-PrP, which is inserted into the cell’s cytoplasmic membrane. 2 Incorrectly folded p-PrP may infect the brain or may be produced by an altered c-PrP gene (not shown). 3 Prion protein (p-PrP) reacts with c-PrP on the cell surface. 4 p-PrP converts c-PrP to p-PrP. 5 p-PrP molecules continue to convert normal c-PrP into p-PrP. ▲ FIGURE 13.23 Templating action of prions. (a) Normal brain tissue LM 500 mm (b) Spongiform encephalopathy brain LM 500 mm ▲ FIGURE 13.24 Brain tissue. (a) Normal human brain tissue appears nearly solid. (b) Large vacuoles and a spongy appearance are typical in prion-induced diseases. Shown here is the brain from a patient with variant Creutzfeldt-Jacob disease. M13_BAUM7206_05_SE_C13.indd 403 11/23/16 3:31 AM 13    Characterizing and Classifying Viruses, Viroids, and Prions hapter ­ ­ Generally, prions of one host affect only related hosts, though some prions are able to “jump” species. Prions are associated with several diseases, including bovine spongiform encephalitis (BSE), so-called mad cow disease, which can spread from cattle to people); scrapie in sheep; kuru, a human disease that has been eliminated; chronic wasting disease (CWD) in deer and elk; and variant Creutzfeldt­Jakob disease4 (vCJD) in humans. Some scientists suggest that noninfectious prions may also be involved in Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and even some types of cancer. another; however, an epidemic of BSE in Great Britain in the late 1980s resulted in the spread of prions from cattle into humans who ate infected beef from cattle that had fed on nervous tissue from infected sheep. To forestall the spread of prions from one species to another, and resulting infections between and among species, most countries ban the use of animal-derived protein in animal feed. Unfortunately, this step was too late for more than 175 Europeans who developed fatal vCJD. (See Clinical Case Study: Invasion from Within or Without?) ▶ ANIMATION: Prions: Diseases TELL ME WHY Why did scientists initially resist the idea of an infectious protein? ­ Cooki ng doesn’t deact ivate prions, nor do normal sterilization procedures; it takes much more severe heat treatment to destroy prions—heating to 482°C for four hours or autoclaving at 132°C in concentrated sodium hydroxide for one hour. Treatment with a 10% solution of some phenolic compounds for an hour can also be effective. The European Union recently approved the use of enzymes developed using biotechnology to remove prions from medical equipment. There is no standard treatment for any prion disease, though some drugs hold promise. These include the antimalarial drug quinacrine, the antipsychotic drug chlorpromazine, the antihistamine astemizole, and antibodies against p-PrP. These have each mitigated prion diseases in animals. Scientists must perform human trials to see whether these drugs are safe and effective in humans. PrP proteins differ between species, and at one time it was thought unlikely that prions could move from one species to Invasion from Within or Without? A 32-year-old father of two small children lived in the midwestern United States. An avid hunter since childhood, the man visited Colorado annually with family and friends for elk hunting. His job required frequent travel to Europe, where he enjoyed exotic foods. In 1988, his wife recalls, he began having problems. Frequently, he forgot to pick up things from the store or even forgot that his wife had called him. Later that year, he was unable to complete paperwork at his business and had difficulty ­ CLINICAL CASE STUDY In this chapter, we have seen that humans, animals, plants, fungi, bacteria, and archaea are susceptible to infection by acellular pathogens: viruses, viroids, and prions. TABLE 13.5 summarizes the differences and similarities among these pathogenic agents and bacterial pathogens. performing even basic math. In England on business, he had forgotten his home phone number in the United States and couldn’t remember how to spell his name for directory assistance. By September, his wife insisted he seek medical care. All the standard blood tests came back normal. A psychologist diagnosed depression, but a brain scan revealed spongiform changes. His family was told he had about six weeks to live because there is no treatment for this disease. ­       C 1. What is the likely diagnosis? 2. The man’s wife wondered, “Can we catch this disease from my husband?” How would you respond? 3. Where and how was the man probably infected? 4“Variant” because it is derived from BSE prions in cattle, as opposed to the regular form of CJD, which is a genetic disease. M13_BAUM7206_05_SE_C13.indd 404 11/23/16 3:31 AM       Chapter Summary TABLE 13.5 Comparison of Viruses, Viroids, and Prions to Bacterial Cells Bacteria Viruses Viroids Prions Width 200–2000 nm 10–400 nm 2 nm 5 nm Length 200–550,000 nm 20–800 nm 40–130 nm 5 nm Nucleic Acid Both DNA and RNA Either DNA or RNA, never both RNA only None Protein Present Present Absent Present (PrP) Cellular Ye s No No No Cytoplasmic Membrane Present Absent (though some viruses do have a membranous envelope) Absent Absent Functional Ribosomes Present Absent Absent Absent Growth Present Absent Absent Absent Self-Replicating Ye s No No No; transforms PrP protein already present in cell Responsiveness Present Some bacteriophages respond to a host cell by injecting their genomes Absent Absent Metabolism Present Absent Absent Absent 1. The Hantavirus genome consists of three ­ ­ segments of negative-sense, single-stranded RNA. How are Hantavirus proteins synthesized in host cells? 2. Hantavirus is an enveloped virus. How do enveloped viruses acquire their envelopes? What role does the envelope play in viral replication? ­ At the emergency room, David’s white blood cell counts are normal, but he is hypotensive (low blood pressure) and bradycardic (slow heart beat). His respiration rate is low, breathing is strained, and oxygen levels in his blood are low. A cardiac workup reveals no abnormalities, but a chest X-ray exam indicates pneumonia in the left lung. Because David’s condition is so severe, he is admitted to the intensive care unit (ICU). He is given respiratory supportive care to help with his breathing. The normal white blood cell counts suggest that a bacterial infection is not causing David’s pneumonia; the doctor orders blood tests to see whether a virus is the causative agent. The results of the blood work reveal the presence of Hantavirusspecific antibodies. There is no cure for Hantavirus infection; David remains in the ICU on supportive care. After ten days, his condition begins to improve, and he is taken off respiratory support two days later. David makes a complete recovery and is able to return to his job within the next month. ­ Working Too Hard? CHAPTER SUMMARY Visit the MasteringMicrobiology Study Area to challenge your understanding with practice tests, animation quizzes, and clinical case studies! ­ 1. Viruses, viroids, and prions are acellular disease-causing agents that lack cell structure and cannot metabolize, grow, selfreproduce, or respond to their environment. Characteristics of Viruses (pp. 382–387) 1. A virus is a tiny infectious agent with nucleic acid surrounded by proteinaceous capsomeres that form a coat called a capsid. M13_BAUM7206_05_SE_C13.indd 405 A virus exists in an extracellular state and an intracellular state. A virion is a complete viral particle, including a nucleic acid and a capsid, outside a cell. 2. The genomes of viruses include either DNA or RNA. Viral genomes may be dsDNA, ssDNA, dsRNA, or ssRNA. They may exist as linear or circular and singular or multiple molecules of nucleic acid, depending on the type of virus. 11/23/16 3:31 AM 13    Characterizing and Classifying Viruses, Viroids, and Prions 2. The International Committee on Taxonomy of Viruses (ICTV) has recognized viral family and genus names. With the exception of three orders, higher taxa are not established. Viral Replication (pp. 388–397) ­ 1. Viruses depend on random contact with a specific host cell type for replication. Typically, a virus in a cell proceeds with a lytic replication cycle consisting of five stages: attachment, entry, synthesis, assembly, and release. ▶ ANIMATION: Viral Replication: Overview 2. Once the virion has attached to the host cell, the nucleic acid enters the cell. With phages, only the nucleic acid enters the host cell. With animal viruses, the entire virion often enters the cell, where the capsid is then removed in a process called uncoating. ▶ ANIMATION: Viral Replication: Animal Viruses 3. Within the host cell, the viral nucleic acid directs synthesis of more viruses using metabolic enzymes and ribosomes of the host cell. V T utor 5. Virions are released from the host cell either by lysis of the host cell (seen with phages and animal viruses) or by the extrusion of enveloped virions through the host’s cytoplasmic membrane (called budding), a process seen only with certain animal viruses. If budding continues over time, the infection is persistent. An envelope is derived from a cell membrane. ▶ ­ANIMATION: Viral Replication: Virulent Bacteriophages ideo 4. Assembly of synthesized virions occurs in the host cell, typically as capsomeres surround replicated or transcribed nucleic acids to form new virions. ­ ­ 6. Temperate phages (lysogenic phages) enter a bacterial cell and remain inactive in a process called lysogeny or lysogenic replication cycle. Such inactive phages are called prophages and are inserted into the chromosome of the cell and passed to its daughter cells. Lysogenic conversion results when phages carry genes that alter the phenotype of a bacterium. At some point in the generations that follow, a prophage may be excised from the chromosome in a process known as induction. At that point, the prophage again becomes a lytic virus. ▶ ANIMATION: Viral Replication:Temperate Bacteriophages 7. With the exception of hepatitis B virus, dsDNA viruses use their DNA like cellular DNA in transcription and replication. 8. Some ssRNA viruses have positive-sense single-stranded RNA ( + ssRNA), which can be directly translated by ribosomes to M13_BAUM7206_05_SE_C13.indd 406 11. In dsRNA viruses, the positive strand of RNA functions as mRNA, and each strand functions as a template for an RNA complement. 12. In latency, a process similar to lysogeny, an animal virus remains inactive in a cell, possibly for years, as part of a chromosome or in the cytosol. A latent virus is also known as a provirus. A provirus that has become incorporated into a host’s chromosome remains there.  ­ 1. Viruses are classified based on type of nucleic acid, presence of an envelope, shape, and size. 10. Negative-sense single-stranded RNA ( -ssRNA) viruses carry an RNA-dependent RNA transcriptase for transcribing mRNA from the - ssRNA genome, and the mRNA is translated to protein. Transcription of RNA from RNA does not occur in cells. The Role of Viruses in Cancer (pp. 397–399) 1. Neoplasia is uncontrolled cellular reproduction in a multicellular animal. A mass of neoplastic cells, called a tumor, may be relatively harmless (benign tumor) or invasive (malignant tumor). Malignant tumors are also called cancer. Metastasis describes the spreading of malignant tumors. Environmental factors or oncogenic viruses may cause neoplasia. ­ Classification of Viruses (pp. 387–388) ­ 5. Virions can have a membranous envelope or be naked—that is, have no envelope. 9. Retroviruses, such as HIV, are + ssRNA viruses that carry reverse transcriptase, which transcribes DNA from RNA. This reverse process (DNA transcribed from RNA) is reflected in the name retrovirus. ­ 4. All types of organisms can be infected by viruses. A bacteriophage (or phage) is a virus that infects a bacterial cell. synthesize protein. From the positive-sense single-stranded RNA ( ∙ssRNA), complementary negative-sense single-stranded RNA ( -ssRNA) is transcribed to serve as a template for more + ssRNA. ­ 3. Viruses are specific for their hosts’ cells because viral attachment molecules are complementary in shape to specific receptor molecules on the host’s cells. Culturing Viruses in the Laboratory (pp. 399–401) 1. In the laboratory, viruses must be cultured inside mature organisms, in embryonated chicken eggs, or in cell cultures because viruses cannot metabolize or replicate alone. 2. When a mixture of bacteria and phages is grown on an agar plate, bacteria infected with phages lyse, producing clear areas called plaques on the bacterial lawn. A technique called plaque assay is used to estimate phage numbers. 3. Viruses can be grown in two types of cell cultures. Whereas diploid cell cultures last about 100 generations, continuous cell cultures, derived from cancer cells, last longer. Are Viruses Alive? (p. 401) 1. Outside cells, viruses do not appear to be alive, but within cells, they exhibit lifelike qualities, such as the ability to replicate. Other Parasitic Particles: Viroids and Prions (pp. 401–405) 1. Viroids are small circular pieces of RNA with no capsid that infect and cause disease in plants. Similar pathogenic RNA molecules have been found in fungi. 2. Prions are infectious protein particles that lack nucleic acids and replicate by inducing similar, normal proteins to misfold into new prions—a process called templating. Diseases caused by prions are spongiform encephalopathies, which involve fatal neurological degeneration. ▶ ANIMATIONS: Prions: Overview, Characteristics, and Diseases ­ hapter       C 11/23/16 3:31 AM       Questions for Review QUESTIONS FOR REVIEW Answers to the Questions for Review (except Short Answer questions) begin on p. A-I. Multiple Choice Matching 1. Which of the following is not an acellular agent? a. viroid c. rickettsia b. virus d. prion 2. Which of the following statements is true? a. Viruses move toward their host cells. b. Viruses are capable of metabolism. c. Viruses lack a cytoplasmic membrane. d. Viruses grow in response to their environmental conditions. Match each numbered term with its description. A. dormant virus in a eukaryotic cell 1. _____ uncoating 2. _____ prophage B. a virus that infects a bacterium 3. _____ retrovirus 4. _____ bacteriophage 5. _____ capsid C. transcribes DNA from RNA D. protein coat of virus E. a membrane on the outside of a virus 3. A virus that is specific for a bacterial host is called a __________________. a. phage c. virion b. prion d. viroid 6. _____ envelope 4. A naked virus __________________. a. has no membranous envelope b. has injected its DNA or RNA into a host cell c. is devoid of capsomeres d. is one that is unattached to a host cell 9. _____ benign tumor 7. _____ virion F. complete viral particle 8. _____ provirus G. inactive virus within bacterial cell H. removal of capsomeres from a virion 10. _____ cancer I. invasive neoplastic cells J. harmless neoplastic cells ­ 5. Which of the following statements is false? c. Viral DNA may be linear. a. Viruses may have circular d. Typically, viruses have DNA. DNA or RNA but not b. dsRNA is found in bacteria both. more often than in viruses. 6. When a eukaryotic cell is infected with an enveloped virus and sheds viruses slowly over time, this infection __________________. a. is called a lytic infection b. is a prophage cycle c. is called a persistent infection d. is caused by a quiescent virus VISUALIZE IT 1. Label each step in the bacteriophage replication cycle below. 7. Another name for a complete virus is __________________. a. virion c. prion b. viroid d. capsid 8. Which of the following viruses can be latent? a. HIV c. herpesviruses b. chicken pox virus d. all of the above b. ____________ a. ____________ 9. Which of the following is not a criterion for specific family classification of viruses? a. the type of nucleic acid c. capsid type present b. envelope structure d. lipid composition 10. A clear zone of phage infection in a bacterial lawn is __________________. a. a prophage c. naked b. a plaque d. a zone of inhibition M13_BAUM7206_05_SE_C13.indd 407 e. ____________ d. ____________ c. ____________ 11/23/16 3:31 AM hapter       C 13    Characterizing and Classifying Viruses, Viroids, and Prions 2. Identify the viral capsid shapes. Short Answer 1. Compare and contrast a bacterium and a virus by writing either “Present” or “Absent” for each of the following structures. Structure Bacterium Virus Cell membrane Functional ribosome Cytoplasm Nucleic acid Nuclear membrane 2. Describe the five phases of a generalized lytic replication cycle. a. ___________________________ b. ___________________________ 3. Why is it difficult to treat viral infections? 4. Describe four different ways that viral nucleic acid can enter a host cell. 5. Contrast lysis and budding as means of release of virions from a host cell. 6. What is the difference between a virion and a virus particle? 7. How is a provirus like a prophage? How is it different? 8. Describe lysogeny. c. ___________________________ d. ___________________________ 9. How are viruses specific for their host’s cells? 10. Compare and contrast diploid cell culture and continuous cell culture. CRITICAL THINKING ­ 1. Larger viruses usually have a double-stranded genome, whereas small viruses typically have a single-stranded genome. What reasonable explanation can you offer for this observation? 2. What are the advantages and disadvantages to bacteriophages of the lytic and lysogenic reproductive strategies? 3. How are computer viruses similar to biological viruses? Are computer viruses alive? Why or why not? 4. Compare and contrast lysogeny by a prophage and latency by a provirus. 5. An agricultural microbiologist wants to stop the spread of a viral infection of a crop. Is stopping viral attachment a viable option? Why or why not? 6. Some viral genomes, composed of single-stranded RNA, act as mRNA. What advantage might these viruses have over other kinds of viruses? 7. In some viruses, the capsomeres act enzymatically as well as structurally. What advantage might this provide the virus? 8. Why has it been difficult to develop a complete taxonomy for viruses? 9. If a colony of 1.5 billion E. coli cells were infected with a single phage T4 and each lytic replication cycle of the phage produced M13_BAUM7206_05_SE_C13.indd 408 200 new phages, how many replication cycles would it take for T4 phages to overwhelm the entire bacterial colony? (Assume for the sake of simplicity that every phage completes its replication cycle in a different cell and that the bacteria themselves do not reproduce.) 10. What differences would you expect in the replication cycles of RNA phages from those of DNA phages? (Hints: Think about the processes of transcription, translation, and replication of nucleic acids. Also, note that RNA is not normally inserted into a DNA molecule.) 11. Although many + ssRNA viruses use their genome directly as messenger RNA, +ssRNA retroviruses do not. Instead, their + RNA is transcribed into DNA by reverse transcriptase. What advantage do retroviruses gain by using reverse transcriptase? 12. If an enveloped virus were somehow released from a cell without budding, it would not have an envelope. What effect would this have on the virulence of the virus? Why? 13. A latent virus that is incorporated into a host cell’s chromosome is never induced; that is, it never emerges from the host cell’s chromosome to become a free virus. Given that it cannot emerge from the host cell’s chromosome, can such a latent virus be considered “safe”? Why or why not? 11/23/16 3:31 AM       Questions for Review CONCEPT MAPPING Using the following terms, fill in the following concept map that describes the replication of animal viruses. Release ssDNA Synthesis Uncoating Viral nucleic acid Viral proteins dsRNA Endocytosis Exocytosis Fusion Host cell New virions +ssRNA +ssRNA retrovirus −ssRNA Attachment Budding dsDNA Replication of animal viruses involves 1 Entry to 3 2 by Assembly of of by 15 8 5 Direct penetration 17 16 varies depending on 6 18 or Viral genome Lysis 7 9 M13_BAUM7206_05_SE_C13.indd 409 4 10 11 12 13 14 3/6/18 4:59 PM