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Lecture 16

Viruses structure, laboratory

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e ec on, ac er op ages

Objectives Understand the nature of viruses and how they differ from

prokaryotic & eukaryotic cells

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

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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 replicateindependently of a cells chromosomes but notindependently of cells themselves

In order to multiply viruses have to get into a cell inwhich they can replicate a host cell

Viruses take advantage of the host cells metabolicmachinery encoded by the host cells chromosome

Virus particles vary in size 0.02 to 0.4 m

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Often measured in nanometres (1000 nm = 1 m)

Small pox virus (one of largest viruses) 200 nm;poliovirus (one of the smallest) 28 nm.

General properties of virusesExtracellular state

Minute article containin

Intracellular

Once inside a host cellnucleic acid surroundedby protein & occasionallyother macromolecules

In this state virus particle(virion) is metabolicallyinert

viral nucleic acid released

Viral replication occurs New copies of genome

produced

Viral coat componentssynthesised

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r on s t e structure ywhich the viral gemome istransferred from the hostcell in which it was madeto another host cell

Infection process thatoccurs when a virusenters a cell andreplicates

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Viral genome

Different from prokaryotic and eukaryoticenomes

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

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All viruses use the host cell translational

machinery ie viral mRNA must be generated thatcan be translated on the host cell ribosome

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Structure of viruses1. Core of genetic material

(nucleic acid)

2. Protein coat or capsid

Protects viral genes frominactivation by adverse

environmental factors

Core + capsid =

nucleocapsid

In many viruses important in

attachment of viruses tospecific receptors on host

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cells

Composed of a large

number of subunits capsomeres

3. Many animal virus particles are

surrounded by a lipoprotein

envelope

Capsomeres Subunit structure is important

Economy of geneticinformation reduces thenumber of different proteinsgenome has to encode if theviral coat is made up ofrepeating units of a singleprotein

Allows for construction of thevirus particles by a process ofself assembly into structuresheld together by non-covalentbonds as occurs in the

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process of crystallisation sono need for enzyme-catalysedreactions for coat assembly

Intracellular release of the viralgenome only requiresdissociation of non-covalentbonds rather than degradationof a protein coat

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Geometry of capsomeres

1. Helical symmetryIcosahedral symmetry

. cosa e rasymmetry

3. Complex structure

Papilloma virus

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Papilloma virus

Complex structure pox virus Helical symmetry

Enveloped viruses Capsid (containing nuclear

material) surrounded by acomp ex mem ranousenvelope

Envelope consists of a lipidbilayer with proteins (usuallyglycoproteins) imbedded in it

Lipids derived from host cell

Proteins encoded by the virus

Envelope is important in

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determining host cell specificity& some aspects of cellpenetration are affected by it

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Additional components

Some virions carry enzymes that are important

Some bacteriophages carry lysozyme that makes asmall hole in the bacterial cell wall to allow the phagenucleic acid to enter the cell

Some human viruses contain their own nucleic acidpolymerases eg reverse transcriptase in retroviruses

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Some human viruses (eg influenza viruses) containneuraminidases break down glycoproteins andglycolipids and aid release of virus from animal cells

Growth and quantification ofviruses

Viruses need to be grown in their host cell an ma , p an , ac er a ..

Animal viruses cell culture cells fromanimals grown artificially in the laboratory

Often in monolayers on glass or plasticoverlaid with suitable li uid media for the cells

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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.

16Figure 13.8

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Cell morphology

3T3 [low]

VSM

ECs

Monoc tes

173T3 [high] Intestinal

Embryonated egg inoculation

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Quantification of viruses

Quantify by measuring their effect on hostce s

Virus infectious unit smallest unit thatcauses a detectable effect

Bacteriophage plaque assay (lysis of

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Animal viruses cell monolayers overlaidwith agar plaque assay

Bacteriophage plaque assay lyticbacterophage

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Animal virus plaque assay

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Virus replication1. Attachment adsorption of the

virion to a susceptible host cell2. Penetration (injection) of the virion

or its nucleic acid into the cell3. Synthesis of nucleic acid & protein Early phase virus redirects cell

metabolism to synthesise new viralnucleic acid and proteins

Late phase structural proteins thatare subunits of the viral coat aresynthesised

4. Assembly of structural subunits (andmembrane components inenvelo ed viruses and acka in

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of nucleic acid into new virusparticles5. Release of mature virions from the cell

Virus replication is a one-step growthcurve

One step growth curve of virus replication

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Virus-host cell interactions

Four main scenarios

.cell

2. Elimination of the virus from the cell and infectionaborted 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

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transformed state eg transformation of a normal cell toone having the properties of a cancerous cell

Viral hosts and taxonomy Viruses can be classified on the basis of the

hosts the infect Animal viruses Plant viruses Bacterial viruses (bacteriophages)

Provide a useful model system for studying molecular biologyand genetics of virus reproduction

There is a formal system of viral taxonomy that

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Order, family, genus, species Increased phylogenetic studies contributing to this

Baltimore scheme (see next lecture) based on howthe viral nucleic acid replicates

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Bacteriophages

Bacteriophages are viruses that infect bacteria

mechanisms of viral replication were first workedout using bacteriophage (phage)

All species of bacteria probably susceptible tophage

Phages are highly host specific E Sta h aureus ha e will not attack a Sta h

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epidermidisbacterium

Many phages strain specific eg different phagesattack bovine strains of Staph aureusfrom those thatattack human strains

Phage

Most phage are tadpole-shaped ea s - con a n nuc e c ac

Tails attach the virus to the host cell

There are some simple icosahedral andhelically symmetrical phages

Phages vary in size

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Majority of phages ds DNA some smallicosahedral & helical phages have ss DNAor RNA

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Virus StructureBacteriophage

T4.

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Phages

Phages classified on the basis of the response theyproduce in the host cell

1. Virulent phage

As a result of infection of a bacterium with phage,phage particles replicate in the cell and the cell is lysedas the particles are released lytic response

2. Temperate phage

After infection the viral genome does not take over the

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host cell cellular activity; the cell survives; viral DNA isincorporated into host cell genome (prophage)

Cells carrying viral genes in this way lysogenic

Lysogenic infections can proceed to lytic infections

Replication of virulentbacteriophage

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Lysogeny

Infection of bacterial cells withtemperate bacteriophage

Integration of the prophage into the

each daughter cell will receive a setof viral genes each cell division

In normally growing cells the hostcells generally keep the prophagein a dormant state but in a fewcells multiplication of the virus andlysis of the cells will occur

Exposure of lysogenic cultures tocertain chemical & physical agents(eg H2O2, mitomycin C, UV light)leads to mass lysis & production of

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lots of phage ie a lyticresponse/cycle process calledinduction

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

Lysogeny is generally a very stable state but cells can lose theirprophage cured cells are once more susceptible to infection withthat phage

Lysogeny is a common phenomenon most bacteria carry one ormore prophages eg Staph aureus - some strains 4 or 5 prophages

Sometimes prophage can pick up some bacterial chromosomalgenes when they move out of the chromosome potential tospread bacterial genes from one bacterium to another

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Some phages carry virulence factors eg the -phage ofCorynebacterium diphtheriaeencodes for diphtheria toxin ie strainswithout the prophage are non-toxigenic.

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Lytic and lysogenic cycles

33Figure 13.12

Use of phages Studies on viral replication

Molecular biolo transduction transfer of enes fromone bacterium to another; transfer of virulence factors

Typing of bacteria strains of the same species ofbacteria can be differentiated by sensitivity to sets ofphages Staphylococcus aureus

Salmonellaspp

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age t erapy to treat n ect ons eg ,Pseudomonas aeruginosa-infected burns

Treatment of ready-to-eat food products to removepathogens eg Listeria monocytogenes, E coliO157