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Life’s Gray Zone: Viruses and Prions 5 It almost seems that not a day goes by without news about some new disease that threatens humanity. Ebola and Zika virus are just two of the more recent entries into the rogues’ gallery of dangerous maladies. And it is not just humans that are vulnerable to such new threats. Elephant endotheliotropic herpesvirus (EEHV) is killing baby Asian elephants. First documented in 1995, there have been over 50 cases in zoos across North America and Europe, only nine of which were successfully treated. Wild Asian elephants are also at risk. Although there have also been serious cases reported in adults, most infected older elephants do not develop life-threatening illness. In affected elephants, extensive damage to the cells lining the capillaries causes blood loss and hemorrhaging, which ultimately leads to shock. The disease can be treated with the rapid application of appropriate drugs, but this has only been successful in around a third of cases. Young elephants apparently contract the disease through physical contact with infected adults. Meanwhile, deer and elk across North America are succumbing to chronic wasting disease (CWD). First recognized in 1967 in Colorado, this invariably fatal disease has spread to 23 US states and two Canadian provinces—most recently to Maryland in 2011. Michigan, with a single case on a captive breeding farm in 2008, reported its first case in a wild deer in 2015. The disease is characterized by weight loss ending in death, and behavioral symptoms such as repetitive walking in a set pattern. It is thought to spread from animal to animal through saliva. There is no treatment. None of the diseases mentioned above are caused by prokaryotic or eukaryotic microbes. EEHV, for instance, is a virus, related to those herpesviruses that plague humans. CWD is a prion disease. Neither viruses nor prions are composed of cells, nor do they display many of those characteristics of living things discussed in Chapter 1. Rather, they occupy a nebulous position, not really alive in the conventional sense, but certainly not the same as nonliving matter either. Here in Chapter 5, we will explore exactly what they are, what characteristics they possess, and how they affect our wellbeing in so many ways. All case studies have a few questions at the end, the answers to which will become apparent as you read the sections following the case. 92 Chapter 5: Life’s Gray Zone: Viruses and Prions Viruses are acellular parasites, requiring a host cell in which to replicate CASE: “DEATH” IN THE RUE MORGUE Edgar Allan Poe died October 7, 1849, at the age of 39. His death has always been attributed to the effects and complications of alcoholism and drug abuse. Michael Benitez at the University of Maryland, on the other hand, believes Poe died of rabies. Benitez came to this conclusion by studying records of Poe’s symptoms at the time of his death. He was first delirious with tremors and hallucinations and then slipped into a coma. He emerged from his coma and was calm and lucid before he lapsed back into delirium. After 4 days, he died. These symptoms, say Benitez, are telltale for rabies but are not typical for alcoholism. Additionally, records show that Poe had abstained from drinking for 6 months prior to his death. We will never know for certain if rabies killed one of the greatest American authors (Figure 5.1), but can we continue to definitively blame Poe’s death on the bottle? “Nevermore.” 1. What type of infection is rabies? How is the agent that causes rabies different from the other prokaryotic or eukaryotic organisms we have already discussed? 2.Why are the symptoms associated with a rabies infection primarily neurological? https://scholar.vt.edu/access/content/group/97b91a99-7258-44a2-8002-9 b7c83a84bd5/WebDev/Website/Gallery/ EnglishGallery/ePGalleryPatrickM/index _files/Edgar_Allan_Poe_2.jpg Figure 5.1 Edgar Allan Poe. Rabies, caused by rabies virus, has been recognized as a serious disease since ancient times. As far back as 4000 years ago, the Mesopotamians had written laws detailing the responsibilities of dog owners, designed to reduce the risk of this dreaded illness. But the doctors who treated Edgar Allan Poe can be forgiven for not understanding what was affecting the author. At the time of Poe’s death, it would still be over 40 years before anyone even suggested that something called a virus existed. What exactly are viruses and how do they differ from other infectious agents? Nobel Prize-winning immunologist Sir Peter Medawar described them as “bad news wrapped in protein,” which is a fairly good definition. First of all, most viruses are small (Figure 5.2a). A typical bacterium might have a length of approximately 5 micrometers (μm; 1,000,000 μm = 1 meter). Viruses range in size from about 20 to several thousand nanometers (nm) in length (1000 nm = 1 μm, so 1,000,000,000 nm = 1 meter). The largest human virus, ironically the smallpox virus, is approximately 300 nm long and is barely visible with the strongest light microscope. The rabies virus is 170 nm long and about 70 nm wide. Not all viruses are quite so tiny, however. In the past 15 years or so, several giant viruses, all of which infect protozoa, have been discovered (Figure 5.2b). Second, unlike prokaryotes and eukaryotes, viruses are acellular—they are not composed of cells. At its simplest, a viral particle consists of only nucleic acid, enclosed in a protein coat, much as Medawar described them. As we will see shortly, many viruses are somewhat more complex than that, but viruses are far simpler than the cell-based microorganisms we have discussed so far. Third, we have previously noted that all living things use DNA to code for the proteins they need to survive. Some viruses also encode genetic information Most viruses have one of a few basic structures (a) 93 (b) Mammalian red blood cell 10,000 nm Typical bacillus length = 3000 nm Poliovirus 30 nm HIV 50 nm Influenza virus 85 nm Rabies virus ~ 70 ¥ 170 nm Smallpox virus ~300 ¥ 200 ¥ 100 nm 1 μm Figure 5.2 Sizes of viruses. (a) Most viruses are significantly smaller than typical bacterial and animal cells shown for comparison. (b) In recent years, several very large viruses have been discovered. Pithovirus is the largest known virus, 1.5 micrometers in length and 0.5 micrometers in width. It is as large as some small bacteria, and it is larger than the smallest eukaryotic cells, found in a type of alga. Discovered in Siberia in 2014, pithovirus infects certain amebas. in DNA, but many rely on RNA as genetic material. Consequently, viruses are often broadly classified as either DNA or RNA viruses. Fourth, all viruses are obligate intracellular parasites; they can reproduce only if they invade a host cell. A viral particle has only a small number of genes. This genetic material codes primarily for the structural proteins of the coat and other required proteins, including some enzymes needed for replication. Everything else is supplied by the host cell. Outside of a host cell, viruses cannot replicate and they show few if any of the properties commonly attributed to living things. However, once inside an appropriate cell, viruses essentially commandeer the host cell’s machinery and use it to replicate themselves. In this manner, an infected cell becomes a viral factory. The virus uses host enzymes and other resources, including amino acids, as well as host structures like ribosomes, to make copies of itself. Newly assembled viral particles are released from the host cell, which frequently kills the cell or seriously compromises normal functioning. Because they are acellular, and because they lack many fundamental characteristics of living things, are viruses even alive? The answer to this question, however, really depends on how we define “life.” Viruses certainly replicate and evolve as other living things do, and the way we treat viral disease is the same whether or not we consider them to be alive. Perhaps the most accurate way to view viruses is to think of them, as implied in this chapter’s title, as straddling the border between the living and nonliving world. Most viruses have one of a few basic structures An individual, complete viral particle is called a virion. As previously stated, all viral particles are composed of genetic material surrounded by a protein coat. Depending on the type of virus, the genetic material may be DNA or RNA. The protein coat is called the capsid, and the individual subunits that make up the capsid are called capsomeres. Each capsomere is composed of one or more proteins. Collectively, the nucleic acid and the capsid are called the nucleocapsid. Most capsids have one of two basic shapes. Many capsids are composed of 20 capsomeres, all in the shape of equilateral triangles. Such viruses have the appearance of geodesic spheres and are known as icosahedral (20-sided) viruses (Figure 5.3a). Helical viruses (Figure 5.3b) have capsomeres that fit together to form a spiral around the enclosed nucleic acid. Apart from these 94 Chapter 5: Life’s Gray Zone: Viruses and Prions (a) (b) (c) (d) Nucleocapsid Envelope Capsomere Nucleic acid Capsid Glycoprotein Nonenveloped icosahedral nucleocapsid Nonenveloped helical nucleocapsid Enveloped icosahedral nucleocapsid Enveloped helical nucleocapsid Figure 5.3 Viral structure. The two main shapes for viral capsids are (a) icosahedral and (b) helical. Both of these shapes may be enveloped, (c) and (d), surrounded by a phospholipid bilayer that is embedded with glycoproteins. two most common shapes, some viruses have a complex structure. Smallpox virus, for example, has a capsid that is composed of multiple layers of protein, neither icosahedral nor helical in shape. Many viruses are surrounded by an envelope that is composed of a phospholipid bilayer in which viral proteins called glycoproteins are embedded (Figures 5.3c and 5.3d). Other viruses are nonenveloped and lack this structure. As we will see, the presence or absence of an envelope and its associated glycoproteins tells us a great deal about how a particular virus enters and leaves a host cell. Many viral nucleocapsids contain a small number of enzymes. These are enzymes lacking in the host cell, which are required for successful viral replication. Specific viruses usually infect only certain hosts and certain cells within those hosts An important characteristic of a virus is its host range—the spectrum of hosts that it is able to infect. Some viruses infect only animals, while others infect only plants, fungi, and so forth. Even within a group such as animal viruses, however, a given virus cannot infect all animal species. For example, cats are susceptible to feline leukemia virus (FLV). While dangerous to cats, this virus is incapable of infecting humans. On the other hand, while humans are susceptible to Epstein–Barr virus, the virus responsible for mononucleosis (mono), cats are not. Even within an appropriate host, only certain types of cells can be infected. For example, the rabies virus that may have killed Edgar Allan Poe can also kill dozens of other mammalian species. However, rabies virus is able to infect only certain cell types within these hosts. The specificity of the virus describes the cell types a particular virus can successfully infect. What determines specificity? Rabies virus is an enveloped virus. The glycoproteins that are embedded in the viral envelope can bind to proteins found in the plasma membrane of nerve cells (neurons). The protein in the plasma membrane of the neuron is similar to a lock, while the glycoprotein is like a key. Only cells that have the proper membrane protein can be infected by this virus (Figure 5.4). This explains why the symptoms accompanying rabies are largely neurological. Hepatitis B virus, on the other hand, lacks Viruses must first attach to and enter an appropriate host cell (a) Viral glycoprotein Capsid Envelope Nucleic acid Host cell membrane protein Host cell plasma membrane (b) the necessary glycoproteins to bind to neurons and therefore cannot infect this cell type. Hepatitis B virus has a different envelope glycoprotein, one that allows it to bind to proteins on the surface of liver cells. While some viruses are very specific, others can infect many cell types; the host molecule to which they attach is widespread and found on a variety of different cells. Nonenveloped viruses attach to target host cells via components of their capsid. In poliovirus, for instance, the point where three capsomeres come together forms a region called a canyon. Cells lining the intestine have proteins in their membranes that can fit snugly into these canyons, permitting infection by the virus. Replication of animal viruses proceeds through a series of defined steps The infection of a susceptible animal cell by a virus can conveniently be divided into several steps, which we will consider in sequence. Many of the differences that occur in the replicative cycle of different viruses depend on whether or not the virus has an envelope, and whether its genetic material is DNA or RNA. Viruses must first attach to and enter an appropriate host cell The first step in the infection process, called attachment, is the contact between a virion and its target host cell. A virion is not motile. It is merely 95 Figure 5.4 Viral host-cell specificity. The ability of a virus to infect specific host cells is due in large part to the ability of the virus to bind to proteins found in the host-cell membrane. (a) The virus has glycoproteins that match the three-dimensional structure and chemical properties of the host-cell membrane protein. Chemical attraction between the virus and the host proteins binds the virus to the host cell. The virus can then enter the cell via one of several mechanisms. (b) The same virus cannot bind a different cell type, because proteins found on this cell are not complementary to the viral glycoproteins. Consequently, this cell type is resistant to infection by this specific virus. 96 Chapter 5: Life’s Gray Zone: Viruses and Prions transported passively until it happens to interact with a cell bearing the proper receptor. For enveloped viruses, the glycoproteins in the envelope bind the proper receptor in the host-cell plasma membrane. In nonenveloped viruses, proteins forming part of the capsid often function as attachment sites. The receptor molecule to which a particular virus attaches is typically a membrane protein of some sort. Only cells bearing the correct receptor are vulnerable to a specific virus. For instance, human immunodeficiency virus (HIV) can infect only cells that bear proteins called CD4 on their surface. Following attachment, the virion must enter the host cell in a step called penetration (Figure 5.5). For nonenveloped viruses, entry occurs via endocytosis (Figure 5.5a) (see Chapter 3, pp. 63–64, for a discussion of endocytosis). The binding of the virion to the receptor causes the cell to transport the virus across the membrane and into the cytoplasm inside a membranebound vesicle. Enveloped viruses may also enter a cell via endocytosis, but many employ a different strategy called fusion (Figure 5.5b). During fusion, viral attachment brings the plasma membrane and the viral envelope into close proximity. The two lipid bilayers actually fuse together, releasing the nucleocapsid into the cell cytoplasm. Many of the details of fusion remain unclear, but it appears that when viral glycoproteins bind to their receptors, (a) Envelope Viral glycoprotein Capsid Nucleic acid Host-cell membrane protein Host-cell plasma membrane (b) Figure 5.5 Entry of an animal virus into a host cell. (a) Entry via endocytosis. Following viral attachment to host-cell membrane receptors, the host cell transports the virus into its cytoplasm. Once endocytosis is complete, the viral particle is released into the cytoplasm. Both enveloped and nonenveloped viruses may enter a cell via endocytosis. (b) Entry via fusion. Following viral attachment, the host-cell plasma membrane and the viral envelope are brought into close proximity. Following fusion of the two lipid bilayers, the viral nucleocapsid is released into the host-cell cytoplasm. Only enveloped viruses enter host cells by fusion. The final two stages are the assembly of new virions and their release from the host cell the glycoproteins change their shape due to the interaction. This disrupts the envelope and plasma membrane, allowing them to fuse. Once they have entered the cell, viruses release their protein coat prior to replication Before it replicates, the virus must shed its capsid (Figure 5.6). This process, called uncoating, may occur in endocytotic vesicles, where the pH is low. In other cases, host enzymes called proteases digest the viral protein coat. Once a virus has uncoated, we say that it has entered the eclipse phase. An intact viral particle no longer exists; the eclipse phase ends only when newly replicated virions are assembled at the end of the replicative cycle. Following uncoating, two important tasks must be completed before new viral particles are assembled. First, the nucleic acid must be replicated, and second, new structural proteins must be produced (see Figure 5.6). These two important processes are collectively called the synthesis stage. Different viruses achieve these two goals in various ways that primarily reflect the type of nucleic acid they carry. We will return to this topic shortly, following our completion of the viral replicative cycle. The final two stages are the assembly of new virions and their release from the host cell Once new viral nucleic acid and structural proteins are made, new virions are formed in a process called assembly (see Figure 5.6). In some viruses, the capsomeres first assemble to form the capsids, and the genetic material is then inserted. In others, the capsomeres latch onto the genetic material, ultimately producing the new viral particle. The final step in the viral replicative cycle is the release of newly formed virions from the infected cell. Nonenveloped viruses are generally released by cell lysis. The host cell literally explodes, releasing the newly assembled virions. The released virions may now contact other host cells, to begin a new round of viral replication. Lysis invariably kills the host cell. Host-cell plasma membrane Host-cell cytoplasm Newly replicated viral DNA Uncoating Virion that has successfully penetrated host cell Viral RNA Newly synthesized viral proteins Newly assembled virions Figure 5.6 Uncoating, synthesis, and assembly of a DNA virus. Once a virion successfully penetrates a host cell, it must release its nucleic acid. This may occur either due to the acidity of an endocytic vesicle or the activity of host enzymes. The subsequent synthesis stage consists of two events: The viral DNA is replicated into many new DNA copies and the DNA is transcribed into viral RNA, which in turn is used to produce new viral proteins. The new viral proteins then assemble around the newly synthesized DNA, producing new progeny virions. 97 98 Chapter 5: Life’s Gray Zone: Viruses and Prions (a) Figure 5.7 Viral release by budding. (a) Stages in the release of an enveloped helical virion are shown. Enveloped viruses acquire their envelope as they bud through the plasma membrane of the infected host cell. The envelope is actually a portion of the host-cell plasma membrane. Viral glycoproteins, already produced during the synthesis stage (see Figure 5.6), become embedded in the plasma membrane and are acquired along with the lipid bilayer as newly assembled viral particles leave the cell. (b) Electron micrograph of several vesicular stomatitis virus (VSV) particles budding from the surface of an infected cell. Closely related to rabies virus, VSV affects cattle, horses, and pigs as well as humans. It is transported by biting insects. (b) Viral glycoproteins embedded in host-cell plasma membrane Helical nucleocapsid Cytoplasm New enveloped virion Enveloped viruses, on the other hand, are typically released from infected cells by budding (Figure 5.7), wherein the assembled nucleocapsid pushes through the plasma membrane. In doing so, it becomes coated with plasma membrane material that now forms the viral envelope. The virus has already coded for envelope glycoproteins, which are already present in the plasma membrane, and the new virus will acquire them along with its envelope. Unlike lysis, budding does not necessarily result in cell death. The synthesis step described above is substantially different for DNA and RNA viruses. In the next sections we consider some of the details in the synthesis stage for these two viral groups. DNA viruses must replicate their DNA and use it to direct synthesis of viral proteins Once a DNA virus releases its DNA into the cell, some of the viral genes begin to code for protein. This process is similar to what occurs in both eukaryotic and prokaryotic cells and involves two crucial steps. First, the viral genes, composed of DNA, are used to produce RNA that carries the genes’ genetic instructions. Second, the RNA associates with host ribosomes, and the genetic message is converted into the specified viral protein. The details of this two-step process will be explored in Chapter 7. Some of the proteins that are made first are those needed to replicate the viral DNA. The viral DNA is then replicated, and hundreds or even thousands of new DNA molecules are produced. Finally, another round of protein synthesis takes place, primarily using host enzymes and resources. This time the proteins being produced are the structural proteins that will make up the completed virions. Examples of DNA viruses that replicate in this manner include Retroviruses use their RNA to produce DNA Viral enzymes needed to replicate DNA Newly replicated viral DNA Viral RNA Newly assembled virions Viral DNA Host-cell cytoplasm Newly synthesized capsid proteins Figure 5.8 Synthesis in a DNA virus. To produce new progeny viral particles, the infecting virion must both replicate its DNA and synthesize necessary proteins. Initially, enzymes that the virus needs to replicate its DNA are produced. DNA replication then follows. Additional structural proteins including capsid proteins are then produced, permitting the assembly of new virions. the herpesviruses and the papillomaviruses that cause warts. The synthesis stage of a DNA virus is illustrated in Figure 5.8. RNA viruses rely on RNA rather than DNA as their genetic material Unlike any prokaryote or eukaryote, RNA viruses encode their genetic information in RNA. For some RNA viruses, DNA plays no role whatsoever in their synthesis stage. Although many of the details vary for different RNA viruses, a general overview of their synthesis stage is provided in Figure 5.9a. For these viruses, the RNA is replicated many times to provide the genetic material for newly synthesized virions. The RNA also guides the production of viral proteins, including capsid proteins, which will assemble around the newly replicated RNA genetic material. RNA viruses are unique in their ability to make copies of their RNA in this way. To do so, they require an enzyme called RNA-dependent RNA polymerase, which is unique to RNA viruses. A few familiar RNA viruses include poliovirus, rhinoviruses (which cause colds), measles virus, and influenza virus. The rabies virus introduced in our case is likewise an RNA virus. Retroviruses use their RNA to produce DNA Although they are RNA viruses, retroviruses use a very different replication strategy in their synthesis stage. Retroviruses also carry RNA in their nucleocapsid, but instead of replicating it into new RNA molecules, they convert their RNA to DNA in a process called reverse transcription (Figure 5.9b). Reverse transcription requires an enzyme called reverse transcriptase. The newly synthesized DNA is then transcribed back to many copies of the viral RNA. This RNA serves as the genetic material for new virions and is translated into structural proteins. By far, the most important human retrovirus is HIV, the causative agent of AIDS. Because reverse transcriptase is not used by our cells, it makes an attractive target for antiviral drugs. Ideally, interfering with this enzyme should have no adverse consequences for the host cell. Many of the important currently available anti-HIV medications act by targeting this enzyme. The topic of antiviral drugs will be more fully explored in Chapter 13. 99 100 Chapter 5: Life’s Gray Zone: Viruses and Prions Production of new viral proteins (a) Assembly Production of new RNA molecules by RNAdependent RNA polymerase Newly assembled virion Viral RNA Production of new viral proteins (b) Infecting retrovirus virion Production of DNA using reverse transcriptase Assembly Production of new RNA DNA RNA New retrovirus virion Figure 5.9 Replication of RNA viruses. (a) RNA viruses use RNA, both to copy into new RNA molecules and to guide the production of new viral proteins. These newly produced proteins assemble around the newly synthesized RNA molecules to form new virions. (b) In retroviruses, RNA is converted into DNA, which is then used to make many copies of RNA. The newly synthesized RNA is used both for protein production and for incorporation into new progeny virions. Not all viral infections cause symptoms or kill host cells Not all viral infections affect the host in the same way. If the virus undergoes repeated rounds of replication, the infection is termed acute; infected cells essentially devote themselves to viral production and ultimately die as a consequence. Some viruses, such as influenza and rabies viruses, cause acute infections only. The viruses keep reproducing and infecting new cells until either the host immune system eliminates the intruder or the host dies (Figure 5.10). Chronic infections are characterized by a slow release of viral particles that may or may not kill the host cell; if such infections do result in individual cell death, host-cell replication is able to keep up with cell death, and there may be few or no signs and symptoms of disease in an infected individual. A good Relative amount of virus in host tissues Time highlighted in pink corresponds to when signs and symptoms of disease are apparent. Viral reactivation of previously latent infection Acute infection Chronic infection following initial acute infection Latent infection following initial acute infection Time (days, months, or years depending on virus) Figure 5.10 Types of viral infections. Symptoms of acute infections are associated with that period of time when levels of virus are high. Chronic infections typically start with an acute infection; viral levels then diminish, and viral replication continues at a lower rate. The amount of virus produced during the course of a chronic infection may or may not be sufficient to cause symptoms. Latent infections also typically begin as acute infections. Following the acute infection phase, the virus enters a period of dormancy (the latent period), during which it does not replicate. This latent period lasts indefinitely; reactivation does not necessarily occur. If reactivation does occur, there will be a new acute episode. Viruses damage host cells in several ways example is a hepatitis B virus (HBV) infection. A person infected with HBV initially suffers through an acute phase with severe illness. In many individuals, the virus is destroyed by the immune system and the patient returns to the uninfected state. In others, the immune system can eventually suppress but not entirely eliminate the virus. Consequently, low levels of virus continue to be produced over a prolonged period of time, sometimes lasting many years. Some DNA viruses and retroviruses cause latent infections. Latent infections begin as acute infections, but then the virus enters a quiescent period during which there is no additional production of viral progeny (see Figure 5.10). During this latent phase, the viral DNA codes for few if any viral proteins. Furthermore, it does not replicate independently of the host DNA. If the host cell divides, however, the viral DNA is duplicated along with the host DNA prior to cell division. Consequently, if a cell infected with a latent virus divides, the two newly produced daughter cells will both be infected with the virus. Viruses may persist in this latent state for many years or in some cases for the entire life of the host. However, a sudden change in the health of the host or certain environmental factors can cause viral reactivation. A reactivated virus is a previously latent virus that has resumed replication. During this time, symptoms may become apparent in an infected individual. Herpesviruses undergo replication cycles of this type. For instance, human herpesvirus type 1 (HHV-1) is the causative agent of cold sores. When a person is first infected, an acute infection causes cell death, resulting in the oral lesion. An effective immune response eventually destroys replicating viruses. However, the virus will remain latent and may be reactivated at any time. The reappearance of the cold sore reflects such reactivation. If you are infected with HHV-1, you will remain infected for life, even if you never experience any symptoms after the initial infection. If you get a cold sore a second time in the same spot, it is a consequence of the same initial infection event. This is quite unlike an infection with influenza virus; once you recover from a bout of the flu, the virus is gone. If you suffer through another case of the flu in the future, you can blame it on a second infection. EEHV, the virus discussed in the introduction, is a herpesvirus specific to elephants. The baby elephants that die due to EEHV succumb during the early, acute phase. If they survive that period, the elephants remain infected for life, with the virus entering a latent phase. Young elephants apparently become infected when they interact with adults who carry the latent virus. In these adults, the virus may reactivate in a few cells—not enough to cause symptoms in the adult, but enough to infect the young elephants. We are only just beginning to understand the factors that might cause a latent virus to reactivate. Anything that suppresses the host immune system, such as cancer chemotherapy or HIV infection, makes reactivation more likely. In the case of HHV-1, factors such as exposure to UV light seem to play a role. Viruses damage host cells in several ways Disease-causing viruses create problems for cells by interfering with their normal functions. Any such interference is known as the virus’ cytopathic effects. One important cytopathic effect is the ability of many viruses to interfere with the host-cell plasma membrane. Recall from Chapter 3 that the plasma membrane maintains cell integrity and is important in regulating transport into or out of the cell. In virally infected cells, the budding of 101 102 Chapter 5: Life’s Gray Zone: Viruses and Prions enveloped viruses can significantly compromise the ability of the plasma membrane to carry out these functions. Viruses can also damage cells by interfering with the ability of the cell to carry out its own protein synthesis or by simply using resources that the cell requires for its own needs. Some viruses prevent the host cell from copying its DNA. Others can initiate a process that ultimately leads to cancer. Let us return to Edgar Allan Poe and the rabies virus that may have killed him. Oddly, in spite of the extreme symptoms exhibited by rabies victims, examination of infected neurons reveals little damage. There are, however, large, prominent clumps of viral proteins visible in the cytoplasm. These Negri bodies, as they are called, are often the only visible sign of infection. Presumably, Negri bodies interfere with normal neuron function, resulting in the neurological symptoms typical of rabies. Like animals, plants are susceptible to many viral infections Unlike animal cells, plant cells are surrounded by a rigid cell wall and plant viruses are unable to reach the plasma membrane when the cell wall is intact. Consequently, plant viruses infect cells through breaks or wounds in the cell wall. In many cases this damage is caused by insects, bacteria, or fungi. In other respects, plant viruses are similar to animal viruses. Like animal viruses, they may carry DNA or RNA. Their mode of replication is essentially the same, as is their overall morphology. In one important respect, however, viral infections in plants are quite different. Plant cells are connected to one another via small junctions, which essentially form connections between the cytoplasm of adjacent cells. In effect, all cells linked by these junctions have a common cytoplasm. Progeny of plant viruses can spread to new host cells through these junctions and through the phloem, which functions as the plant’s circulatory system. Because of this, plant infections are often systemic. In other words, whereas animal viruses usually affect only specific organs or tissues, plant viruses can affect cells throughout the entire organism. Many important plant diseases are caused by viruses. Plant viruses take a large economic toll in crops such as corn and wheat, but they cause even greater problems in perennial plants like potatoes. Plants suffering from viral infections are often stunted (Figure 5.11a). Irregular coloration often appears on leaves and stems, and green pigments may be lost. Unlike animals, plants rarely recover from a viral infection because, as mentioned previously, the entire plant is often infected. They also lack the ability to mount a highly specific immune response. In a few happy cases, viral infection in plants is actually desirable. The attractive variegated color of some tulips is due to a viral infection that is transmitted through the bulbs (Figure 5.11b). (a) (b) Bacteriophages are viruses that infect bacteria Even bacteria do not get a free ride when it comes to pathogens. They are subject to infection with bacteria-attacking viruses called bacteriophages or phages for short. The most intensively studied phages are those that infect E. coli. A specific phage known as T4 provides an illustrative example. A T4 virion has a complex structure, with an icosahedral head containing DNA and a hollow, helical tail (Figure 5.12). The tail region is associated with other characteristic structures called the tail fibers, tail pins, and base plate. Figure 5.11 Plant viruses. (a) Tobacco mosaic virus. The leaf on the right is from a tobacco plant infected with tobacco mosaic virus. Note its stunted and discolored appearance compared with the leaf from an uninfected plant on the left. (b) The attractive variegated color in some tulips is the result of a viral infection. Bacteriophages are viruses that infect bacteria (a) Figure 5.12 Structure of T4, a representative bacteriophage. (a) Note the complex structure, including the icosohedral head encompassing the nucleic acid, and the helical tail, which makes initial contact with the host cell. (b) An electron micrograph of a bacteriophage. (b) Nucleic acid Icosahedral head Capsid 103 Collar Sheath or tail Tail pins Tail fibers Base plate The initial step in the infection cycle of bacteria involves the contact and adherence to the bacterial surface by the phage (Figure 5.13a). This process, called adsorption, cannot occur on just any bacterial cell. To successfully adhere, molecules on the phage tail and tail fibers must match specific molecules on the bacterial surface that serve as receptors. A bacterium lacking these molecules is resistant to infection. (a) Adsorption (f) Release (b) Penetration (c) Replication of nucleic acid and production of phage proteins (e) Maturation (d) Assembly Figure 5.13 Replicative cycle of bacteriophage T4. This bacteriophage is able to infect E. coli cells. (a) Adsorption: The phage makes contact and adheres to the bacterial surface. (b) Penetration: The helical sheath contracts, forcing the viral DNA into the cell cytoplasm. (c) Replication involves both replication of the viral DNA and inhibition of hostcell activity. (d) Assembly of new phage particles. (e) Maturation of phages into newly produced infective viral particles. (f ) Lysis of the host cell and release of newly produced, mature phages. 104 Chapter 5: Life’s Gray Zone: Viruses and Prions Once the phage has attached, it injects its DNA into the bacterium in a step called penetration (Figure 5.13b). This involves the contraction of the helical sheath, which forces the hollow tube into the cell cytoplasm, much like a microscopic syringe. In the process, the viral DNA is released into the cell’s interior. The viral capsid does not enter the cell. It remains as an empty shell, attached to the cell exterior. Once DNA has entered the host cell, the metabolic activity of the cell is blocked and the cell begins producing proteins coded for by the viral DNA (Figure 5.13c). These include proteins that block normal host-cell activity, enzymes required for viral replication, and structural proteins needed to construct new capsids. The viral DNA is also repeatedly copied. All energy required for these processes is provided by the host cell. Phage DNA enters host cell As new viral DNA and structural proteins are produced, they spontaneously assemble into new virions (Figure 5.13d). Up to several hundred new phages may ultimately be produced (Figure 5.13e). Eventually, the host cell bursts, releasing the progeny phages (Figure 5.13f). These newly released phages may then contact and infect other susceptible cells. The T4 replicative cycle we have just described is termed a lytic cycle, because it results in bacterial cell lysis and death. Not all phages, however, undergo a lytic cycle. Infection of E. coli with phage lambda, for instance, does not necessarily cause lysis. After releasing its DNA into a susceptible bacterium, lambda does not immediately reproduce. Rather, it enters an inactive period called the prophage state (Figure 5.14). In the prophage state, the viral DNA is actually incorporated into the bacterial DNA. If the bacterium itself divides, the viral DNA will also replicate along with it, but otherwise, the infected cell remains largely unaffected. This state of viral inactivity inside an infected cell is called lysogeny. Under certain conditions, however, the virus may begin to replicate, returning to the lytic cycle. Ultraviolet light or exposure to certain chemicals is known to promote a return to the lytic cycle in phage lambda. Phage DNA integrates into host-cell DNA, entering prophage stage When host cell replicates its DNA, phage DNA is also replicated Phages can influence the number of bacteria and even the diseases that they cause Although they do not infect animals or plants, bacteriophages affect humans in several important direct and indirect ways. First, the number of phages in the environment is almost inconceivable. A single milliliter of sea water may contain close to 50 million of them, and virologists have found similar numbers in soil. According to some estimates, in the oceans alone, up to 40% of all bacteria may be destroyed each day by phages. Consequently, they may influence the entire world’s food supply by limiting the numbers of bacteria available for other organisms to eat. The vast numbers of killed bacteria no doubt impact nutrient cycling in the ocean and other environments. Of perhaps more immediate concern to humans is the effect lysogenic phages have on the virulence of some bacteria. For example, cholera is caused by Vibrio cholerae, a Gram-negative species (Figure 5.15). This often deadly disease is caused by a toxin produced by the bacteria that results in massive diarrhea. However, the bacterium cannot make the toxin by itself. Rather, lysogenic phage genes inside the cell instruct the host to create the toxin. Bacteria that are uninfected with these phages do not cause disease. Yet other phages may actually provide us with an invaluable service— namely, protection from potentially pathogenic bacteria. It has recently Host cell divides; phage DNA is present in both daughter cells Figure 5.14 Lysogenic cycle of bacteriophage lambda. Once phage DNA is released into the cytoplasm of the host cell, it incorporates into the host DNA. During the lysogenic cycle, no new progeny phages are produced. If the bacterial cell replicates, the phage DNA, called the prophage, is replicated along with the host DNA. Under appropriate conditions, prophages in the lysogenic cycle may return to the lytic cycle. Prions are infectious proteins 105 been proposed that phages in our intestine help keep intestinal bacteria in check, and thus unable to easily cause disease. These phages typically adhere in almost astronomical numbers to the mucus lining the intestine. Researchers have speculated that these phages stand ready to attack potentially disease-causing bacteria that attempt to penetrate the mucus to reach the underlying intestinal lining. The phages benefit from this relationship because of their access to a steady stream of bacteria in which to reproduce. In other words, this may be an odd and ancient partnership between animals and phages that is beneficial to both. Finally, because they destroy specific bacteria, some have suggested that bacteriophages could be utilized therapeutically to treat bacterial infections. This idea was first proposed in the early twentieth century. Spotty results, however, combined with the development of powerful antibiotics dampened interest in phage therapy. However, in recent years, with the development of antibiotic resistance by many bacteria, some researchers are taking a fresh look at this approach. We will return to this topic in Chapter 13. Prions are infectious proteins CASE: LAST LAUGH FOR THE “LAUGHING DEATH” Up through the 1950s, 2–3% of the Fore people of New Guinea mysteriously died of the “laughing death” each year. The disease, also called kuru, started with increased clumsiness, headaches, and a tendency to giggle inappropriately. Within a few months, victims could no longer walk. Total incapacitation, followed by death, occurred within a year. Curiously, the disease affected women and young children almost exclusively (Figure 5.16). Older boys and men were spared. The reason for this was unknown until 1957, when Carleton Gajdusek, of the US National Institutes of Health, determined how the disease was transmitted. The Fore people practiced ritual cannibalism as part of their burial ceremony. When a tribal member died, it was customary for female family members to prepare the body and eat parts of the deceased’s brain. Small children also consumed brain tissue, but older boys and adult men did not participate. Gajdusek postulated that an infectious agent was transmitted through the brain tissue, explaining why only those who had consumed such tissue were affected. Kuru has now been eradicated, because cannibalism has not been practiced since the 1960s. Figure 5.15 Cholera. This 1912 painting by the French painter Jean-Loup Charmet depicts the horror associated with cholera epidemics. 1. What exactly causes kuru? 2. How is this infectious agent unlike any other that we have thus far discussed? 3. What other diseases are caused by similar agents? Figure 5.16 A young Fore child infected with kuru. This photo was taken in the early 1950s, before measures to eradicate kuru were initiated. Kuru may be gone, but other diseases, such as Creutzfeldt–Jakob disease (CJD) and bovine spongiform encephalopathy (BSE), also known as mad cow disease, are still with us. Likewise, chronic wasting disease (CWD), discussed in the introduction, continues to plague deer and elk in North America. These diseases all cause neurological symptoms and they are all uniformly fatal. They are all caused by highly unusual infectious agents called prions. Prions are infectious proteins. Their very existence was not proposed until 1982, and it is only since the 1990s that most of the scientific community has accepted the notion that a protein could cause infection. The concept of prions was hotly contested because their existence violates a basic tenet of biology—that only nucleic acids can replicate themselves and serve as genetic material. 106 Chapter 5: Life’s Gray Zone: Viruses and Prions But prions do not replicate in the traditional sense. Prions seem to be abnormally folded forms of a protein that already exists in the brain (Figure 5.17). The best-studied prion disease is scrapie, which affects sheep (see Figure 1.6b). It is named for the behavior of infected animals, which includes scraping themselves bare against fence posts or other solid objects. On the surface of the sheep’s brain is a protein called PrPc. Occasionally this protein folds up incorrectly. It is then called PrPSc. This aberrant protein is able to interact with normal PrPc proteins, forcing them to refold in the abnormal three-dimensional shape (Figure 5.18). In other words, PrPSc acts like a mold or template, which forces PrPc to take on the disease-causing shape. Eventually, the PrPSc proteins build up to such an extent that they begin to form abnormal plaque on the surface of brain cells. Whether or not it is these plaque deposits that are the direct cause of the disease remains to be seen. Other prion diseases, including kuru, CJD, and CWD are also believed to be caused by improper folding of PrPc and the ability of the aberrant protein to cause normal proteins to adopt the abnormal shape. PrPc Prion disease can be caused by mutation of the gene coding for PrPc, but it can also be transmitted between individuals. Creutzfeldt–Jakob disease, for instance, can be transmitted via transplants or surgical instruments. Ninety percent of all prion disease in humans has been identified as CJD. Typical age of onset is approximately 65, and the disease is characterized by progressive dementia. Like all known prion diseases, it is always fatal. Bovine spongiform encephalopathy (BSE) is a prion disease of cattle. The disease is named for the brain’s spongy appearance in affected cows (Figure 5.19). It is also popularly called mad cow disease because of the erratic behavior of affected cattle. It is believed that the disease was spread by the previously common practice of adding bone meal to cattle feed. Bone meal often has nerve tissue attached to it, and such tissue may contain infectious prion particles. The disease came to the attention of the general public when it suddenly appeared in British cattle in 1980. There were over 180,000 cases and millions of cattle were slaughtered in an effort to contain the epidemic. The use of animal products in cattle feed was banned. Many were concerned that humans who consumed meat from infected cows might develop a similar disease. There was also speculation that CJD was a human form of mad cow disease. This is probably not the case, but disturbingly, in 1996, a new form of CJD called variant CJD (vCJD) was first reported in Great Britain. Unlike CJD, vCJD has an average age of onset of 27. Furthermore, the disease kills its victims in just over a year, as opposed to about 3 years for CJD. About 230 people have died of vCJD, mostly in Great Britain. Could vCJD be the human equivalent of mad cow disease, and did British victims become infected by eating contaminated meat? Certainly the sudden appearance vCJD in 1996 is consistent with such a link. Looking back and looking forward We have now completed our examination of our microbial roster. Viruses and prions are far simpler than the cellular life forms reviewed in Chapter 4. Viruses, the ultimate parasites, cannot replicate unless they infect appropriate host cells. Once inside, viruses commandeer the cell, diverting host resources to their own replication, often with dire consequences for the host PrPSc Figure 5.17 Normal and abnormal forms of the PrP protein. The PrPc protein is normally found on brain cells. The normal PrPc sometimes takes on the form of the aberrant PrPSc. The normal protein in its correct threedimensional structure has four spiral-like regions called alpha helices. In the abnormal form, two of these alpha helices, shown in red, lose their helical form and take on a different, elongated structure, indicated by the colored arrows. Looking back and looking forward Normal PrPc protein DNA RNA Normal cell 107 Figure 5.18 Conversion of the normal PrP protein to the abnormal form. The aberrant PrPSc can force normal PrPc proteins to take on the aberrant shape. (1) PrPSc (the red square) may be acquired by consuming contaminated animal products or may be due to a mutation in the gene coding for the protein. (2) The aberrant protein is able to interact with the normal protein (blue circles) and force it to take on the aberrant shape. (3)–(6) As this process continues, more and more of the normal PrPc is converted to PrPSc. High levels of the aberrant protein form abnormal plaque deposits on the surface of brain cells, which may be involved in the nervous disorders seen in individuals suffering from prion disease. Infective PrPSc protein 1. 2. 3. 4. 5. 6. Infected cell cell. Prions, even less complex than viruses, are essentially rogue proteins that force other host proteins into an aberrant shape. As these abnormally folded proteins build up, symptoms of disease appear. So now, the stage is set. We know who the microorganisms are, and we are starting to understand what they do. But before we continue to investigate them, we will consider what they did. In Chapter 6, we examine microbes with a historian’s eye and investigate some of the astounding ways in which they have influenced human affairs throughout history. We will see that empires have fallen and societies altered, all because of microorganisms. We will also review the colorful history of microbiology itself, looking at some of the important discoveries that have advanced the science of microbiology to where it is today. Figure 5.19 Stained cross section of the brain from a cow that died of BSE. The white areas scattered across the brain tissue are places where neurons have died due to the buildup of the prion protein. Brain tissue preparations from individuals that have died of other prion diseases, including CJD and CWD appear similar. 108 Chapter 5: Life’s Gray Zone: Viruses and Prions Garland Science Learning System •• http://garlandscience.rocketmix.com/students/ •• Investigate viral factories and their assembly lines •• Test your knowledge of this chapter by taking the quiz •• Familiarize yourself with the terminology used in this chapter by using the vocabulary review •• Get help with the answers to the Concept questions Concept questions These questions are designed to help you start thinking like a microbiologist. The answers are not always simply found in the text. Instead, you will need to take the concepts about which you have learned and apply them to new situations. Some of the questions may not even have just a single correct answer. Help is provided as part of the GSLS resources, which can be accessed through http://garlandscience.rocketmix.com/students/. 1. What is fundamentally different about the way viruses reproduce compared with cellular forms of life? 2. Referring back to Chapter 1, in which the properties of living things were discussed, make an argument that (a) viruses are not living things or (b) viruses are living things. 3.An anti-HIV drug called Enfuvirtide binds to and interferes with the glycoproteins found in the viral envelope. Which stage of the viral replicative cycle is Enfuvirtide interrupting? 4.RNA-dependent RNA polymerase is a unique viral enzyme produced by many RNA viruses. It is not produced by any prokaryotic or eukaryotic cells. Would this enzyme make a reasonable target for drugs active against the RNA viruses that produce it? Why or why not? 5. If you know that a particular virus is enveloped, what does this information alone suggest about its replicative cycle? 6. What is meant by the “eclipse phase” in the viral replicative cycle? Referring back to the steps described in the replicative cycle here in Chapter 5, after which step does it begin? After which step does it end? 7. Suppose that a particular virus has a wide host range but narrow tissue specificity. What precisely does this mean? What about a narrow host range and broad tissue specificity? 8.Chickenpox is caused by the chickenpox virus. If a child is unvaccinated and exposed to the virus, she might develop clinical signs of this disease. Many years later, this same person may develop a case of shingles, caused by the same viral infection that caused chickenpox as a child. Referring back to the types of viral infections described in this chapter, state the type of infection the individual is experiencing when she had chickenpox. What type of infection does she have in the time between chickenpox and shingles? What kind of infection does she have while she is experiencing shingles? 9.Aphids use a remarkable trick to protect themselves from certain eukaryotic parasites. The aphids have symbiotic bacteria that live in their gut. These bacteria often are infected with phage. The phages produce a protein that is toxic to the parasites. What type of phage infection do the bacteria within the aphid’s digestive system have? 10.Before the idea of prions was proposed, diseases caused by what we now know to be prions were believed to be caused by as-yet unidentified viruses. Some of the evidence used to show that such diseases were not caused by viruses included the use of DNAses (enzymes that digest DNA) and RNAses (enzymes that digest RNA). Experiments were performed in which brain tissue from infected mice was treated with either DNAse or RNAse. Other, previously healthy mice were then exposed to the DNAse- or RNAse-treated tissue. In either case, these newly exposed mice developed the same disease as the original mice from which the brain tissue had been taken. How do these results help demonstrate that a virus was not involved?