Lecture 17 Viruses Bacteriophages life cycle Learning objectives: Characteristics of Virus

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1 Lecture 17 Viruses Bacteriophages life cycle Learning objectives: The aim of this lecture is to introduce the basic facts about the virology and how virus multiplies on the host virus and understand their life cycle. Characteristics of Virus A. Technically non-living since they cannot self-replicate, thus not a kingdom B. consists of just a protein coat for protection and a nucleic acid (RNA or DNA) for information how to make more copies of the same virus C. Cellular parasite- uses cell machinery of a host cell to replicate and so produce more viruses D. Wide variety of : EM pictures: sizes and shapes E. Cause numerous diseases of plants, animals (including humans), bacteria, fungi 1. examples of human viruses: a. herpes virus, b. hepatitis virus, c. rabies, d. ebola, -emerging epidemics (video and pictures of 1995 outbreak in Zaire) e. influenza, -recombination between avian(bird) and pig viruses and human viruses can produce new strains which spread world-wide- e.g. the Hong Kong avian flu of Fall Flu fact sheet- CDC. Dealing with the flu f. HIV (AIDS) (infection process-cartoon HIV info ). 2. Examples of plant viruses: a. potyvirus causing streaking, b. Tobacco Mosaic Virus causing mosaic (mottling) symptoms, c. rose mosaic symptoms, d. spots on fruit F. Virus-like agents 1. Viroids- Segments of RNA which are not packaged into a protein coat 2. Prion- infectious proteins. e.g. Mad Cow Disease, (WHO FAQ, less technical description) Kuru, CJ, Viruses may consist of circles, ovals, long thick or thin rods, flexible or stiff rods and ones with distinctive heads and tail components. The smallest viruses are around 20 nm in diameter and the largest around 250 nm. Viruses are unique from all other life forms in that they can contain only one form of nucleic acid. Some viruses use RNA as their genetic material and other use DNA, but never do they contain both. Further, this nucleic acid polymer may either exist as double stranded (DS) DNA or RNA or as single stranded (SS) DNA or RNA. Each of these characteristics is a constant for a particular virus and is part of it description. All viruses are covered with a protein coat. This protein coat is mainly composed of a few types of proteins of which there are many copies per virus; something like the

2 individual threads in a shirt. These identical protein subunits are called capsomeres and they are made so that they spontaneously come together (ASSEMBLE) in a PREDETERMINED way to produce the virus coat which is called the capsid. A NAKED virus An ENVELOPED virus If a virus has only a protein capsid covering it, it is termed a naked capsid virus or a naked virus. However, some viruses pick up a lipid membrane from the host cell when it is released, that surrounds the capsid. The lipid membrane is called an envelope and such viruses are termed enveloped viruses. All virus contain one more important proteins type; this is attachment protein or docking proteins. The attachment protein is needed by the virus to attach to its target cell before it can enter that cell. Obviously this attachment protein must lie on the outer surface of the virus so that it is available to contact the appropriate receptor sites on the target host cells. These attachment proteins are often called spikes because they can extend away from the cell so as to better be able to contact the host receptor. A BACTERIA PHAGE. Definition - Bacteriophage (phage) are obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery (i.e., viruses that infect bacteria.). There are many similarities between bacteriophages and animal cell viruses. Thus, bacteriophage can be viewed as model systems for animal cell viruses. In addition a knowledge of the life cycle of bacteriophage is necessary to understand one of the mechanisms by which bacterial genes can be transferred from one bacterium to another. Y WODS COMPOSITION AND STRUCTURE OF BACTERIOPHAGE Composition - Although different bacteriophages may contain different materials they all contain nucleic acid and protein. Depending upon the phage, the nucleic acid can be either DNA or RNA but not both and it can exist in various forms. The nucleic acids of phages often contain unusual or modified bases. These modified bases protect phage nucleic acid from nucleases that break down host nucleic acids during phage infection. The size of the nucleic

3 acid varies depending upon the phage. The simplest phages only have enough nucleic acid to code for 3-5 average size gene products while the more complex phages may code for over 100 gene products. The number of different kinds of protein and the amount of each kind of protein in the phage particle will vary depending upon the phage. The simplest phage have many copies of only one or two different proteins while more complex phages may have many different kinds. The proteins function in infection and to protect the nucleic acid from nucleases in the environment. Structure - Bacteriophage come in many different sizes and shapes. The basic structural features of bacteriophages are illustrated in Figure 1, which depicts the phage called T4. 1. Size - T4 is among the largest phages; it is approximately 200 nm long and nm wide. Other phages are smaller. Most phages range in size from nm in length. 2. Head or Capsid - All phages contain a head structure which can vary in size and shape. Some are icosahedral (20 sides) others are filamentous. The head or capsid is composed of many copies of one or more different proteins. Inside the head is found the nucleic acid. The head acts as the protective covering for the nucleic acid. 3. Tail - Many but not all phages have tails attached to the phage head. The tail is a hollow tube through which the nucleic acid passes during infection. The size of the tail can vary and some phages do not even have a tail structure. In the more complex phages like T4 the tail is surrounded by a contractile sheath which contracts during infection of the bacterium. At the end of the tail the more complex phages like T4 have a base plate and one or more tail fibers attached to it. The base plate and tail fibers are involved in the binding of the phage to the bacterial cell. Not all phages have base plates and tail fibers. In these instances other structures are involved in binding of the phage particle to the bacterium. III. INFECTION OF HOST CELLS A. Adsorption - The first step in the infection process is the adsorption of the phage to the bacterial cell. This step is mediated by the tail fibers or by some analogous structure on those phages that lack tail fibers and it is reversible. The tail fibers attach to specific receptors on the bacterial cell and the host specificity of the phage (i.e. the bacteria that it is able to infect) is usually determined by the type of tail fibers that a phage has. The nature of the bacterial receptor varies for different bacteria. Examples include proteins on the outer surface of the bacterium, LPS, pili, and lipoprotein. These receptors are on the bacteria for other purposes and phage have evolved to use these receptors for infection. B. Irreversible attachment - The attachment of the phage to the bacterium via the tail fibers is a weak one and is reversible. Irreversible binding of phage to a bacterium is mediated by one or more of the components of the base plate. Phages lacking base plates have other ways of becoming tightly bound to the bacterial cell. T4C. Sheath Contraction - The irreversible binding of the phage to the bacterium results in the contraction of the sheath (for those phages which have a sheath) and the hollow tail fiber is pushed through the bacterial envelope (Figure 2). Phages that don't have contractile sheaths use other mechanisms to get the phage particle

4 through the bacterial envelope. Some phages have enzymes that digest various components of the bacterial envelope. D. Nucleic Acid Injection - When the phage has gotten through the bacterial envelope the nucleic acid from the head passes through the hollow tail and enters the bacterial cell. Usually, the only phage component that actually enters the cell is the nucleic acid. The remainder of the phage remains on the outside of the bacterium. There are some exceptions to this rule. This is different from animal cell viruses in which most of the virus particle usually gets into the cell. This difference is probably due to the inability of bacteria to engulf materials. PHAGE MULTIPLICATION CYCLE A. Lytic or Virulent Phages 1. Definition - Lytic or virulent phages are phages which can only multiply on bacteria and kill the cell by lysis at the end of the life cycle. 2. Life cycle - The life cycle of a lytic phage is illustrated in Figure 3. a. Eclipse period - During the eclipse phase, no infectious phage particles can be found either inside or outside the bacterial cell. The phage nucleic acid takes over the host biosynthetic machinery and phage specified m-rna's and proteins are made. There is an orderly expression of phage directed macromolecular synthesis, just as one sees in animal virus infections. Early m-rna's code for early proteins which are needed for phage DNA synthesis and for shutting off host DNA, RNA and protein biosynthesis. In some cases the early proteins actually degrade the host chromosome. After phage DNA is made late m-rna's and late proteins are made. The late proteins are the structural proteins that comprise the phage as well as the proteins needed for lysis of the bacterial cell. b. Intracellular Accumulation Phase - In this phase the nucleic acid and structural proteins that have been made are assembled and infectious phage particles accumulate within the cell. c. Lysis and Release Phase - After a while the bacteria begin to lyse due to the accumulation of the phage lysis protein and intracellular phage are released into the medium. The number of particles released per infected bacteria may be as high as Assay for Lytic Phage a. Plaque assay - Lytic phage are enumerated by a plaque assay. A plaque is a clear area which results from the lysis of bacteria (Figure 4). Each plaque arises from a single infectious phage. The infectious particle that gives rise to a plaque is called a pfu (plaque forming unit). B. Lysogenic or Temperate Phage 1. Definition - Lysogenic or temperate phages are those that can either multiply via the lytic cycle or enter a quiescent state in the cell. In this quiescent state most of the phage genes are not transcribed; the phage genome exists in a repressed state. The phage DNA in this repressed state is called a prophage because it is not a phage but it has the potential to produce phage. In most cases the phage DNA actually integrates into the host chromosome and is replicated along with the host chromosome and passed on to the daughter cells. The cell harboring a prophage is not adversely affected by the presence of the prophage and the lysogenic state may persist indefinitely. The cell harboring a prophage is termed a lysogen.

5 2. Events Leading to Lysogeny - The Prototype Phage: Lambda a. Circularization of the phage chromosome - Lambda DNA is a double stranded linear molecule with small single stranded regions at the 5' ends. These single stranded ends are complementary (cohesive ends) so that they can base pair and produce a circular molecule. In the cell the free ends of the circle can be ligated to form a covalently closed circle as illustrated in Figure 5. b. Site-specific recombination - A recombination event, catalyzed by a phage coded enzyme, occurs between a particular site on the circularized phage DNA and a particular site on the host chromosome. The result is the integration of the phage DNA into the host chromosome as illustrated in Figure 6. c. Repression of the phage genome - A phage coded protein, called a repressor, is made which binds to a particular site on the phage DNA, called the operator, and shuts off transcription of most phage genes EXCEPT the repressor gene. The result is a stable repressed phage genome which is integrated into the host chromosome. Each temperate phage will only repress its own DNA and not that from other phage, so that repression is very specific (immunity to superinfection with the same phage). 3. Events Leading to Termination of Lysogeny Anytime a lysogenic bacterium is exposed to adverse conditions, the lysogenic state can be terminated. This process is called induction. Conditions which favor the termination of the lysogenic state include: desiccation, exposure to UV or ionizing radiation, exposure to mutagenic chemicals, etc. Adverse conditions lead to the production of proteases (rec A protein) which destroy the repressor protein. This in turn leads to the expression of the phage genes, reversal of the integration process and lytic multiplication. 4. Lytic vs Lysogenic Cycle The decision for lambda to enter the lytic or lysogenic cycle when it first enters a cell is determined by the concentration of the repressor and another phage protein called cro in the cell. The cro protein turns off the synthesis of the repressor and thus prevents the establishment of lysogeny. Environmental conditions that favor the production of cro will lead to the lytic cycle while those that favor the production of the repressor will favor lysogeny. 5. Significance of Lysogeny a. Model for animal virus transformation - Lysogeny is a model system for virus transformation of animal cells b. Lysogenic conversion - When a cell becomes lysogenized, occasionally extra genes carried by the phage get expressed in the cell. These genes can change the properties of the bacterial cell. This process is called lysogenic or phage conversion. This can be of significance clinically. e.g. Lysogenic phages have been shown to carry genes that can modify the Salmonella O antigen, which is one of the major antigens to which the immune response is directed. Toxin production by Corynebacterium diphtheriae is mediated by a gene carried by a phage. Only those strain that have been converted by lysogeny are pathogenic. There are two primary types of bacteriophages: lytic bacteriophages and temperate bacteriophages.

6 1. Bacteriophages that replicate through the lytic life cycle are called lytic bacteriophages, and are so named because they lyse the host bacterium as a normal part of their life cycle. 2. Bacteriophages capable of a lysogenic life cycle are termed temperate phages. When a temperate phage infects a bacterium, it can either replicate by means of the lytic life cycle and cause lysis of the host bacterium, or, it can incorporate its DNA into the bacterium's DNA and become a noninfectious prophage. Prophage is the term for a phage DNA in its dormant state, typically integrated into the host bacteria's chromosome, but also can include the rare cases where the phage exists as a stable plasmid within the host cell. Temperate phages are those phages able to undergo both a lytic cycle and a lysogenic cycle, such as the lambda phage. They lyse a small fraction of bacterial cells; in the remaining majority of the bacteria, the phage DNA becomes integrated into the bacterial chromosome and replicates along with it. In this lysogenic state, the information contained in the viral nucleic acid is not expressed. The lysogenic bacterium multiples normally until some environmental induction, such as ionizing radiation or ultraviolet light threatens the bacterial cell and induces the prophage to initiate the lytic cycle. Lytic Cycle of a Virulent Phage: Life cycle involves following steps- Attachment or adsorption of Phage to bacterial cell. Injection or penetration of viral genetic material (DNA) into host cell. Eclipse period during which synthesis of new phage DNA and protein coats takes place. Assembly of Phage DNA into protein coats. Lysis of host cell and release of infective progeny phages. Such a phage is called virulent or lytic phage since it has infectiousness and it causes death of host cell by lysis. The adsorption of the phage to its host is due to random chemical collision between the two. Reactive groups called Adsorption protein or Pilot protein are present at the end of the tail of phage and can join with a receptor site of the bacterium. Long tail fibres are the first to attach to the cell. Once it has taken its position, injection can take place involving a movement of phage through hollow core of tail into host. The protein coat remains outside the cell. Once inside the host, phage DNA becomes a vegetative phage. Hence, phage genes takes over the metabolic machinery of the cell and direct it to produce replicas of the infecting virus. The cell continues to take raw materials from the environment. Phage genes allow only viral component to built. Further, either the normal ability of the host to control cell is lost or the host DNA is completely destroyed. Hence, phage DNA is both replicated and transcribed. First the enzymes needed for synthesis of phage DNA are translated, then capsid proteins are translated.

7 Phage particles are assembled around cores of the complete phage nucleic acid. Lastly, the lytic enzymes coded by phage, break open the bacterium and release new phage particles which diffuse in surrounding in search of new host to infect and replicate. STEPS of Lytic phage cycle Adsorption during the Lytic Life Cycle of a Lytic Bacteriophage: The bacteriophage injects its genome into the bacterium's cytoplasm Penetration during the Lytic Life Cycle of a Lytic Bacteriophage: The bacteriophage genome replicates and bacteriophage components begin to be produced by way of the host bacterium's metabolic machinery. Early Replication during the Lytic Life Cycle of a Lytic Bacteriophage: The production of bacteriophage components and enzymes progresses. Late Replication during the Lytic Life Cycle of a Lytic Bacteriophage:The bacteriophage components assemble. Maturation during the Lytic Life Cycle of a Lytic Bacteriophage: Phage-coded lysozyme breaks down the bacterial peptidoglycan causing osmotic lysis and release of the intact bacteriophages Release during the Lytic Life Cycle of a Lytic Bacteriophage Reinfection - 50 to 200 phages may be produced per infected bacterium. Lysogenic Cycle of a Virulent Phage: Certain bacteriophage such as P1 and lambda ( λ) phages, have entirely different pattern of life cycle than the virulent phages. This pattern is called Lysogeny and is characterised by delayed lysis after phage infection. A virus with this capacity is called temperate virus. The infected host cell is said to be lysogenic because dormant virus may at any time become active and begin directing the synthesis of new virus particle. In lysogeny, the process of adsorption and nucleic acid injection are similar to lytic cycle. The next step is unique to lysogeny. The nucleic acid is neither extensively replicated nor extensively transcribed. The virus generally expresses one or a few genes which code for a repressor proteins that turn off the expression of other genes of the virus. Virus is not replicated, but phage DNA remains in the bacterium and replicate in a way that when lysogenic bacteria divides, each daughter cell receives at least one phage genome in addition to the bacterial genome. There are 2 ways to the persistence of this phage DNA: The phage chromosome may exist as a fragment of DNA outside the host s chromosome (in cytoplasm) as a plasmid (P1 bacteriophage). Or it may attach itself to the host s chromosome as an episome ( λ phage). In λ phage, DNA becomes circular as both cohesive ends joined and integrated into circular DNA of the bacterium. This integrated and dormant viral genome is called Provirus or Prophage.

8 The infection of E.coli with λ phage renders that cell immune to further attack by phage of the same type. Steps in Lysogenic phage cycle Differences Between Lytic and Lysogenic Cycles Lytic Cycle Viral DNA destroys Cell DNA takes over cell functions and destroys the cell The Virus replicates and produces progeny phages symptoms of viral infection Virtulant viral infection takes place Lysogenic Cycle Viral DNA merges with Cell DNA does not destroy the cell The Virus does not produce progeny no symptoms of viral infection Temperate viral replication takes place