Lecture 16

Viruses structure, laboratory d t ti detection, b bacteriophages t i h

Objectives
Understand the nature of viruses and how they differ from prokaryotic & eukaryotic cells Describe the different types of viral genomes Describe the different types of structure of viruses Describe how viruses are grown and quantified in the laboratory Describe the steps involved in virus replication Describe the one-step growth curve seen in virus replication Describe the potential outcomes of viral infection of a cell Understand the principles of virus classification Understand the nature of bacteriophages Differentiate virulent and temperate phages Describe the lytic and lysogenic responses/cycles resulting from bacteriophage infection of bacteria List the uses of bacteriophage
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Introduction
Viruses are genetic elements that replicate p y of a cells chromosomes but not independently independently of cells themselves In order to multiply viruses have to get into a cell in which they can replicate a host cell Viruses take advantage of the host cells metabolic machinery encoded by the host cells chromosome Virus particles vary in size 0.02 to 0.4 m Often measured in nanometres (1000 nm = 1 m) Small pox virus (one of largest viruses) 200 nm; poliovirus (one of the smallest) 28 nm.
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General properties of viruses

Extracellular state Minute particle containing nucleic acid surrounded by protein & occasionally other macromolecules In this state virus particle (virion) is metabolically inert Virion Vi i is i the th structure t t b by which the viral gemome is transferred from the host cell in which it was made to another host cell Intracellular Once inside a host cell viral nucleic acid released Viral replication occurs
New copies of genome produced Viral coat components synthesised

Infection process that occurs when a virus enters a cell and replicates
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Viral genome
Different from prokaryotic and eukaryotic g genomes Viral genome Mostly have either DNA or RNA DNA and RNA can be double stranded or single stranded Some viruses have both DNA and RNA but at different stages of their reproductive cycle All viruses use the host cell translational machinery ie viral mRNA must be generated that can be translated on the host cell ribosome
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Structure of viruses
Core of genetic material (nucleic acid) 2. Protein coat or capsid Protects viral genes from inactivation by adverse environmental factors Core + capsid = nucleocapsid In many viruses important in attachment of viruses to specific receptors on host cells Composed of a large number of subunits capsomeres 3. Many animal virus particles are surrounded by a lipoprotein envelope 1.

Capsomeres
Subunit structure is important Economy of genetic information reduces the number of different proteins genome has to encode if the viral coat is made up of repeating units of a single protein Allows for construction of the virus particles by a process of self assembly into structures held together by non-covalent bonds as occurs in the process of crystallisation so no need for enzyme-catalysed reactions for coat assembly Intracellular release of the viral genome only requires dissociation of non-covalent bonds rather than degradation of a protein coat

Geometry of capsomeres
Icosahedral symmetry

1. Helical symmetry 2 Icosahedral 2. I h d l symmetry 3. Complex structure

Papilloma virus

Papilloma virus

Complex structure pox virus

Helical symmetry

Enveloped viruses
Capsid (containing nuclear material) surrounded by a complex membranous envelope Envelope consists of a lipid bilayer with proteins (usually glycoproteins) imbedded in it
Lipids derived from host cell Proteins encoded by the virus

Envelope is important in determining host cell specificity & some aspects of cell penetration are affected by it

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Additional components
Some virions carry enzymes that are important in the early stages of the infection process
Some bacteriophages carry lysozyme that makes a small hole in the bacterial cell wall to allow the phage nucleic acid to enter the cell Some human viruses contain their own nucleic acid polymerases eg reverse transcriptase in retroviruses Some human viruses (eg influenza viruses) contain neuraminidases break down glycoproteins and glycolipids and aid release of virus from animal cells
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Growth and quantification of viruses

Viruses need to be grown in their host cell animal, i l plant, l t b bacteria t i .. Animal viruses cell culture cells from animals grown artificially in the laboratory
Often in monolayers on glass or plastic overlaid with suitable liquid q media for the cells to grow
Complex medium amino acids, vitamins, salts, pH buffers, glucose, antibiotics, pH indicator ..
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Tissue culture flasks

T-flasks Petri dishes coverslips 6 well, 12, 48 or 96 wells

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Isolation of cells

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Cell culture
Some cells will grow indefinitely permanent cell lines; others will remain alive only for a short period primary cell lines.

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Figure 13.8

Cell morphology

3T3 [low]

ECs

VSM

Monocytes y

3T3 [high]

Intestinal

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Embryonated egg inoculation

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Quantification of viruses
Quantify by measuring their effect on host cells ll Virus infectious unit smallest unit that causes a detectable effect Bacteriophage plaque assay (lysis of bacterial cells on an agar plate) Animal viruses cell monolayers overlaid with agar plaque assay
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Bacteriophage plaque assay lytic bacterophage

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Animal virus plaque assay

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Virus replication
1. Attachment adsorption of the virion to a susceptible host cell 2. Penetration (injection) of the virion or its i nucleic l i acid id i into the h cell ll 3. Synthesis of nucleic acid & protein Early phase virus redirects cell metabolism to synthesise new viral nucleic acid and proteins Late phase structural proteins that are subunits of the viral coat are synthesised 4. Assembly of structural subunits (and membrane components in enveloped viruses) and packaging of nucleic acid into new virus particles 5. Release of mature virions from the cell Virus replication is a one-step growth curve

One step growth curve of virus replication

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Virus-host cell interactions

Four main scenarios 1. Multiplication of the virus and destruction of the host cell 2. Elimination of the virus from the cell and infection aborted without any recognisable effect on the cells 3. Survival of the infected cell in an unchanged state but it continues to carry the virus in a latent state 4. Survival of the infected cell in a dramatically altered or transformed state eg transformation of a normal cell to one having the properties of a cancerous cell

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Viral hosts and taxonomy

Viruses can be classified on the basis of the y infect hosts they
Animal viruses Plant viruses Bacterial viruses (bacteriophages)
Provide a useful model system for studying molecular biology and genetics of virus reproduction

There is a formal system of viral taxonomy that organises viruses within hierarchical taxon levels
Order, family, genus, species
Increased phylogenetic studies contributing to this

Baltimore scheme (see next lecture) based on how the viral nucleic acid replicates
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Bacteriophages
Bacteriophages are viruses that infect bacteria Important in study of virology as the mechanisms of viral replication were first worked out using bacteriophage (phage) All species of bacteria probably susceptible to phage Phages are highly host specific
Eg Staph aureus phage will not attack a Staph epidermidis bacterium Many phages strain specific eg different phages attack bovine strains of Staph aureus from those that attack human strains
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Phage
Most phage are tadpole-shaped
Heads - contain n nucleic cleic acid Tails attach the virus to the host cell

There are some simple icosahedral and helically symmetrical phages Phages vary in size Majority of phages ds DNA some small icosahedral & helical phages have ss DNA or RNA
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Virus Structure
Bacteriophage T4.

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Phages
Phages classified on the basis of the response they produce in the host cell p 1. Virulent phage As a result of infection of a bacterium with phage, phage particles replicate in the cell and the cell is lysed as the particles are released lytic response 2. Temperate phage After infection the viral genome does not take over the host cell cellular activity; the cell survives; viral DNA is incorporated into host cell genome (prophage) Cells carrying viral genes in this way lysogenic Lysogenic infections can proceed to lytic infections
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Replication of virulent bacteriophage

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Lysogeny
Infection of bacterial cells with temperate bacteriophage Integration of the prophage into the bacterial chromosome ensures that each daughter cell will receive a set of viral genes each cell division In normally growing cells the host cells generally keep the prophage in a dormant state but in a few cells multiplication of the virus and lysis of the cells will occur Exposure of lysogenic cultures to certain chemical & physical agents (eg H2O2, mitomycin C, UV light) y & production p of leads to mass lysis lots of phage ie a lytic response/cycle process called induction

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Lysogeny
When a lysogenic cell is exposed to the same type of phage as it carries the activity of the invading viral genes are repressed by the same mechanisms that are keeping the prophage dormant Lysogeny is generally a very stable state but cells can lose their prophage cured cells are once more susceptible to infection with that phage Lysogeny is a common phenomenon most bacteria carry one or more prophages eg Staph aureus - some strains 4 or 5 prophages Sometimes prophage can pick up some bacterial chromosomal genes when they move out of the chromosome potential to spread bacterial genes from one bacterium to another (transduction) Some phages carry virulence factors eg the -phage of Corynebacterium diphtheriae encodes for diphtheria toxin ie strains without the prophage are non-toxigenic.

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Lytic and lysogenic cycles

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Figure 13.12

Use of phages
Studies on viral replication Molecular biology transduction transfer of genes from one bacterium to another; transfer of virulence factors Typing of bacteria strains of the same species of bacteria can be differentiated by sensitivity to sets of phages
Staphylococcus aureus Salmonella spp

Ph Phage therapy th t to treat t t infections i f ti eg MRSA, MRSA Pseudomonas aeruginosa-infected burns Treatment of ready-to-eat food products to remove pathogens eg Listeria monocytogenes, E coli O157
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