Chapter 12: Acellular Agents: Viruses, Viroids and Prions Viruses Viruses are acellular infectious agents that are much smaller than bacteria and are usually measured in nanometers (Figure 12.1). They have either DNA or RNA genomes and never have both types of nucleic acids within the virus capsid. Viruses cause many infections of humans, animals, plants, and bacteria and cause most of the diseases that plague the industrialized world (HIV, SARS, herpes, polio). Most viruses infect only certain host cells and can be said to be species specific. Some viruses may be so specific they only infect a certain cell type (ex. HIV infect human T lymphocytes). There are plenty of viruses however that cross species barriers and can infect multiple hosts, these are referred to as generalists (ex. Influenza virus and Rabies virus). Figure 12. 1 Comparative sizes of viruses to E. coli, which is approximately 2 microns long. There is some debate on whether viruses are living organisms or not. Some believe they are the least complex living entity, others that they are complex pathogenic chemicals, which is supported by the numerous non-living characteristics. The arguments for both sides are outlined below.
Living Characteristics Can Reproduce (only in living cells) Can Mutate Non-Living Characteristics Acellular (no cytoplasm or organelles) Do not grow or respond to the environment Have ability to take control of host cell Have DNA or RNA never both Cannot reproduce independently Do not metabolize chemicals for energy Have an intracellular and an extracellular state Recruit the cells metabolic pathways to increase numbers Animal Viruses Virus Structure: Viruses have two states: the extracellular state and the intracellular state. In the extracellular state the virus is called a virion. The virion can either be characterized as Naked or Enveloped. A Naked virion is made up of a nucleic acid genome surrounded by a protein "coat" called the capsid. The genome + the capsid together make up the nucleocapsid. The viral genome may be made of single-stranded DNA (ssdna), double-stranded DNA (dsdna), ssrna or dsrna. The capsid is composed of smaller protein subunits, the capsomeres and can vary in structure: helical (tube/rod shaped), polyhedral, or complex, meaning the shape varies or has multiple parts. Enveloped virions have a phospholipid bilayer that surrounds the capsid. The envelope is often derived from the host cell it is infecting and provides an extra layer of protection (Figure 12.2). The outermost layer of the virus (the envelope if it enveloped, or the capsid if it is naked) protects the viral genome and contains attachment proteins, also known as "spikes". These proteins bind to receptors on the cells of the virus host and are responsible for virus-host specificity. Viruses are classified based on the shape of their capsid, they type of nucleic acid they have, the presence/absence of an envelope and their size. In the intracellular state of the virus exists solely as nucleic acid. Once the virus infects the cell the capsid is degraded and the virus will begin the process to take over the internal cell components.
Figure 12.2. Enveloped virus structure. A naked virus is similar but lacks the envelope. Replication/life cycle of Animal Viruses Viral replication is dependent on host cell organelles and enzymes therefore they must invade the cell in order to replicate. The steps for viral replication are outlined below (Figure 12.3): 1. Recognition and Attachment: capsid or envelope proteins recognize host cell receptors and the virus attaches to the cell 2. Entry: The animal virus can enter by fusion with cell membrane (enveloped only) or endocytosis (naked and enveloped) 3. Synthesis: DNA viruses replicate in the nucleus and viral proteins are translated in the cytoplasm. 4. Assembly: Viral proteins are assembled along with replicated viral DNA in the cytoplasm. 5. Release: The release mechanisms vary depending on the virus the 3 basic release mechanisms are budding, exocytosis, or lysis of the host cell. Because of the way virus particles are made, one cell can make hundreds to thousands of copies of a single virus.
Figure 12.3. Outline of animal virus replication. Influenza Virus Replication Animantion : http://www.npr.org/blogs/krulwich/2011/06/01/114075029/flu-attack-how-a-virus-invades-yourbody Latency and Latent Infections vs. Acute Infections The capability of enveloped viruses to leave the host cell by budding and not killing the host cell allows some viruses to cause latent infections. Latency occurs when the virus is present in the cell but dormant for long periods and then becomes active, going through a cycle of viral replication and producing symptoms in the host. Some latent viruses incorporate their viral DNA into the host cell DNA, causing the entire host cell progeny to become infected, while other latent viruses keep their genome separate from that of the host. Examples of latent infections include herpes (both genital and oral), in which the virus can be dormant for months or years and become active for a short time, producing symptoms, and chickenpox, in which a person contracts the virus (usually as a child) and is symptom free for decades before the virus becomes active and symptoms appear as shingles. People who develop latent infections do not develop immunity and can experience symptoms again and again. Viruses that do not become latent cause acute infections. Acute infections are characterized by sudden onset symptoms that are short in duration with active replication of the virus by the host cell. Infections by these types of viruses usually result in long lasting future immunity for the host. Examples of acute infections include, the cold, influenza and norovirus.
The Role of Viruses in Cancer Animal genes contain sequences of DNA called protooncogenes (literally pre-cancer genes). These protooncogenes are bound by repressor proteins, which effectively keep the genes off (in the pre-cancer state). Viruses contribute to the development of cancer in a two-step fashion: they insert promoters in front of the protooncogene (remember, in order for a gene to be transcribed, mrna binds to the promoter), then, they disrupt or eliminate the gene for the repressor protein. Without the repressor, the protooncogene can be turned "on", thereby becoming an oncogene. Viruses cause 20 25% of human cancers Some viruses carry copies of oncogenes as part of their genomes Some viruses promote oncogenes already present in host Some viruses interfere with tumor repression Specific viruses are known to cause ~15% of human cancers Burkitt's lymphoma Hodgkin's disease Kaposi's sarcoma Cervical cancer Environmental factors often combine with viruses to contribute to the activation of oncogenes. Examples include: Ultraviolet light Radiation Carcinogens Retroviruses Retroviruses are viruses that have RNA as their genome. This RNA can be used directly as mrna to initiate protein synthesis and make viral capsids and other viral components. The RNA of retroviruses is transcribed into DNA by the viral enzyme, reverse transcriptase. This viral DNA can then be incorporated into the host cell genome. This makes the virus a permanent part of the host cell and any host cell progeny. This splicing may also disrupt repressor genes and contribute to the development of cancer within the host. An example of a prominent retrovirus is Human Immunodeficiency Virus (HIV), the causative agent of AIDS.
Evolution of Animal Viruses Animal viruses evolve via two mechanisms: antigenic drift and antigenic shift. Before we discuss these in detail, let's take a closer look at the influenza virus, which we will use as a model for studying drift and shift: The two major types of influenza are Type A and Type B. Both types have envelopes and 8 segments of RNA as their genome. Both types also have 2 major types of spikes on their envelopes: Hemagglutinin: allows the virus to bind to receptors on pulmonary epithelial cells and facilitates endocytosis by those cells, there are 16 varients of hemagglutinin Neuraminidase: helps the virus penetrate the lung by hydrolyzing mucus in the lungs, and also helps facilitate the release of new virus particles from infected host cells, there are 9 varients of neuraminidase Influenza viral strains are named based on the varients of hemagglutinin and neuraminidase they have. For example, the 2009 flu epidemic was caused by H1N1. The so-called "Avian Flu" is H5N1. Antigenic drift occurs when point mutations occur in the RNA coding for the neuraminidase and hemagglutinin. These point mututions change the virus just enough so that the immune system of a person infected with it would not recognize and remember the virus. The virus would be seen as something new upon re-exposure, and would cause illness while the immune system goes through a primary immune response. Antigenic Shift is a larger change in the genome, usually due to the mixing of genetic information from two strains of virus that are infecting a host simultaneously. In the case of the influenza virus, this may involve the infection of an intermediate host, such as a pig or a bird. Antigenic shift leads to the creation of entirely new strain of virus. Replication/life cycle of Bacteriophages A bacteriophage (or phage for short) is a virus that infects bacteria. Felix d Herelle coined the term bacteriophage, meaning bacteria eater in 1917. There are many kinds of bacteriophage, each infecting a specific type of bacteria. Bacteriophages such as the T-even phage used in this exercise have a complex structure comprised of a polyhedral head called the capsid that contains DNA. These phages first attach to the bacterial cell and then inject their DNA into the bacterium. The tail sheath of the phage contracts and drives the tail core through the bacterial cell wall (sort of like a hypodermic syringe injection). There are two outcomes of this injection, depending on whether the phage is lytic or lysogenic. The cycles are outlined below: Lytic replication
Replication cycle results in lysis and death of host cell about 30 minutes after infection. Lysis releases approximately 100-200 progeny. Lysogenic replication (Figure 12.4) Modified lytic cycle where the viral genome is incorporated into the host cell genome. The bacteria will reproduce for many generations until the virus is induced. Induction is the process in which the viral genome is cut out of the host cell genome and enters into the lytic cycle. Induction can be caused by numerous environmental factors such as DNA damaging chemicals, UV light, and other sub optimal conditions. Bacteria that contain the DNA of the phage usually cannot be re-infected or lysed by the same type of phage. Basic stages of lytic replication cycle 1. Recognition and Attachment: Bacteriophage spikes recognize complementary receptors on bacteria surface. 2. Entry: Injection of viral genome 3. Chromosome degraded: virally coded enzymes degrade the host chromosome. 4. Synthesis: Replication of viral genome and translation of viral proteins 5. Assembly: Note: the gene transfer mechanism of transduction relies on mistakes when the virus is assembling. Short fragments of bacterial chromosome or plasmids may be packaged inside the viral capsid. Once released the bacteriophage will infect another bacteria with the bacterial DNA. 6. Release: is by lysis of the cell resulting in cell death. Figure 12.4. Image showing lysogenic cycle and how induction causes the lytic cycle. Note: the color of the viral genome is red and the host chromosome is in blue. Notice how during assembly some of the phage particles have blue bacterial DNA in the capsid. This is how the gene transfer mechanism of transduction works.
It is important to study phage and learn to manipulate them for the following reasons. 1. Animal viruses, including human pathogens, are grown in tissue culture cells in the same fashion as phage on bacteria. Tissue cultures are animal cells grown in bottles or on plates. The animal virus can form plaques by causing cells to degenerate or die. It is convenient to learn viral techniques using bacteriophage, because human pathogenic viral manipulation in lab could be disastrous if you used bad aseptic technique. 2. Phages are used in recombinant DNA experiments and are also useful in studying the genetics of bacteria. They are useful tools for transfer of genes and pieces of DNA from cell to cell. 3. Phages are used to identify certain strains of bacteria, because one type of phage will only infect one or a few specific species or strains of bacteria. 4. Lysogeny has served as a model for viruses that insert their DNA into animal cells. The life cycle of lysogenic phage resembles that of retroviruses (e.g. HIV). Lysogenic conversion occurs when the genes carried by the phage alter the phenotype of the bacterial cell in some way. The toxins produced by C. diphtheriae and C. botulinum are the result of lysogenic conversion. Culturing Viruses Culturing of viruses is problematic because they are obligate intracellular parasites: they must be grown in their host cells. In the case of bacteriophage, a plaque assay can be done (Lab 12.1). The phage particles are mixed with bacterial cells then mixed into warm, molten agar which is then poured into Petri plates and allowed to solidify. Mixing the bacteria into the agar this way should create a lawn. Wherever the phage have infected and killed bacterial cells, holes called plaques occur in the lawn. Plaques contain high numbers of bacteriophage which can be cultured again in this manner. Human and animal viruses can be cultured in cell or tissue culture. First, the host cells must be grown in tissue culture media. Sometimes, cell culture lines are available commercially, but some types of cells are not and they must be harvested fresh from the host organisms. I worked with hepatocytes, and they had to be cultured fresh each time. This meant harvesting liver cells from mice and rats each time I needed to perform an experiment. Cell culture is also prone to contamination because the media and much of the equipment is heat-sensitive and cannot be autoclaved. After establishing a culture of host cells, the virus is introduced to the culture and allowed to infect it. Animal viruses can also be cultured in embryonated chicken eggs (Figure 12.5). Chicken eggs are free from microbes and contain nourishing yolk to provide energy for viral replication.
Figure 12.5. Sites of injection for culturing viruses. Other Parasitic Particles: Viroids & Prions Viroids Viroids are extremely small, circular pieces of RNA that are infectious and pathogenic in plants, although little is known about how they infect and cause disease in plants. They are similar to RNA viruses, but lack capsids. It is not known if viroids infect animals or humans. Prions The term "prion" is short for proteinaceousinfectious agent (if you rearrange the letters a bit). Prions are misfolded PrP proteins that can induce other proteins to change their shape to become prion proteins. PrP proteins are made by all mammals, prion PrP proteins convert normal PrP into prion PrP. Prions in a sense sort of act like an enzyme slowly converting good proteins into bad proteins.
Prions affect the brain matter causing spongiform encephalopathies. These result in always fatal neurological degeneration, fibril deposits and loss of brain matter. On examination, the brain appears to have large vacuoles in it, giving it the characteristic spongy appearance. There is still a lot that is unknown regarding prion diseases and their transmission. As of now, there are no diagnostic tests for them beyond autopsy, and there are no treatments. It is known that prions can be transmitted by ingesting contaminated neural tissue or by receiving transplanted tissue from a donor who had a prion disease, but it is not known if prions can be transmitted in any other way. Prion disease include mad cow (cattle), scrapie (sheep), varient Creutzfeld-Jakob syndrome (humans) and chronic wasting disease (deer and elk). In order to destroy prions, they must be autoclaved in 1N NaOH or incinerated. The mad cow outbreak in Great Britain in the mid-1980s was a result of two disturbing practices: 1. using the carcasses of dead cattle to boost protein levels in cattle feed and 2. not rendering the carcasses at high enough temperatures to destroy prions prior to adding them to the feed. The lower temperature was adopted as a way to save on energy costs. Because we do not fully understand the transmission of prions, people from England and Americans who visited/work in England during this time period are not allowed to donate blood in the U.S.