Molecular Cell Biology II Viruses

Key Terms

Bacteriophage / phage – A virus that infects bacteria

Capsid / coat protein / CP – Protein coat of virion
Capsomere – Single protein unit of a capsid
Copy number / burst size – the number of virions released from host cell on lysis
Early protein – A protein produced in viral replication that is expressed immediately
Host range – The collection of hosts that a virus can infect and reproduce in
Late protein – A protein produced in viral replication that is expressed at a latter stage
Prophage – A phage genome inserted as part of the DNA chromosome of a bacterium
RDRP – RNA-dependent RNA Polymerase
RT – Reverse Transcriptase
Virion / nucleocapsid – A mature viral particle

Introduction
Viruses are parasites of the cellular genetic system
Viruses cannot replicate on their own – they require a host to carry out replication for them
The simplest viruses can replicate using 4 proteins, more complex viruses may have up to 300

Most viruses have a narrow host range – they will only infect closely related species
e.g. Polio can only affect closely related primates
Sometimes, viruses can move from one species to another
e.g. Avian flu (Influenza A, H5N1)
Rarely, insect viruses can infect plants too
e.g. Tobacco Mosaic Virus

The genetic information within a virus can be DNA/RNA, ss/ds, sense/anti-sense

Most viruses have a specific receptor that they attach to on a cell, which is a factor that limits the
number of cells that virions can infect. This is what restricts the host range.
Plant viruses are less specific – they have no specific targets to attach to for infection

DNA viruses
Replication usually occurs in the nucleus

RNA viruses
Replication usually occurs in the cytoplasm

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Most plant viruses are RNA viruses

Structure

Almost all viruses are encapsidated in protein, known as the coat protein or capsid
The capsid is often formed from many repeated copies of the same protein
Each of the capsid proteins are encoded by single genes
Viruses have evolved so as to use the minimum amount of information on their genome to encode
a gene. Efficient use of genetic information.

Capsid proteins and the viral genome that they enclose often form helical structure
The helices can encapsidate RNA / DNA
Nucleic acids is laid down within a helical groove within the helix
Helices assume a rod-like shape, which can be flexuous or rigid

Tobacco Mosaic Virus is rigid

Potato virus is flexuous
The helices can form quasi-spherical (icosahedral) structures
 Spherical, with triangular faces
 In simple viruses, a single capsomere is repeated in each face
 In complex viruses, different capsomeres will come together

Rigid rod-like helices

Flexuous rod-like
helices

Bacteriophage

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In some viruses there are many extensions on the surface of the virion e.g. adenovirus
Some viruses are surrounded by an envelope, which is gained from the last cell that was infected
In the case of influenza, there are two different types of extension…
 Neuraminidase – a tetrameric protein
 Hemagglutinin – a trimeric protein

These extension, or ‘spikes’ are involved in host cell recognition

Bacteriophages

T4 is a well-studied phage
dsDNA is contained within its head region

1. The tail region attaches to the surface of bacteria, normally

recognising lipopolysaccharides (LPS)
2. The outer tail region contracts, an energetic change that
forces the DNA from the head region into the target cell,
resulting in infection

The virus can be cloned and grown in a synchronised fashion – all of the bacteria can be
inoculated simultaneously, allowing demonstration of one-step growth curves

This is not possible in other systems, if animals or plants are infected the virus will be at different
replication stages in different parts of the organism

1. Generate a confluent layer of cells that covers the agar plate e.g. E. coli
2. Add dilute viral suspension
3. Incubate

After some time there will be a development of clearing zones, also known as plaques / lesions
 These plaques indicate where cells have become lysed
 The termination step of most phages is cell lysis

The agar restricts viral movement, and only cells in the confluent layer will be infected
Each lesion represents a single initial virus that has subsequently been cloned

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Animal viruses are now grown in this way too – using cell lines such as HeLa

More plaques = more viruses  an indicator of virus concentration

These plaque assays are used to determine viral contamination of water (water quality)
Lytic growth cycle – Bacteria:

1. Adsorption – Viruses interact with host cell by binding onto a receptor

2. Replication – Virus enters cell and starts to replicate

 Production of virus-specific mRNAs
 Use of RDRP in RNA viruses (often encoded by the virus)
 Multiple copies of viral genome produced
 Multiple copies of capsomeres produced

3. Maturation – Assembly of virions

 When a cell is full of virions it is said to heave reached burst size
 Cell lysis

The lytic process involves an enzyme that is manufactured in the replication phase, but is inactive
until triggered at an appropriate time. It is a so called late protein. Enzymes also degrade the
hosts nucleic acids to supply nucleotides for the synthesis of more viral DNA.

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Lytic growth cycle – Animals:

The lytic cycle in animal viruses is more complex and involves more processing e.g. Rabies virus

1. Virion adsorbs to host cell membrane

2. Cell engulfs virion as an endosome

3. Proton pump acidifies the endosome, decreasing pH

 Conformational change occurs in the viral glycoprotein
 Viral envelope fuses with the endosomal bilayer

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4. Nucleocapsid is released into the cytosol

5. Viral RNApol uses rNTPs in the cytosol to replicate the viral RNA genome

6. Viral RNApol also synthesises viral mRNAs

7. One viral mRNA encodes the viral transmembrane glycoprotein, which is synthesised on
ribosomes that are bound to the endoplasmic reticulum. Carbohydrate is added in the ER lumen.

8. The folded protein is modified as it passes through the Golgi apparatus

9. Vesicles containing the mature glycoprotein fuse with the plasma membrane of the host cell,
depositing the viral glycoprotein on the cell surface. Large, receptor-binding domains project
outwards from the cell surface.

10. The other viral mRNAs from step 6. are translated on host-cell ribosomes to form
nucleocapsid protein, matrix protein and viral RNApol

11. These proteins are assembled with the replicated viral genomic
RNA to form progeny nucleocapsids

12. The progeny nucleocapsids associate with the cytosolic domain of

the viral TM glycoproteins in the plasma membrane.

13. The plasma membrane folds around the nucleocapsid forming a

bud, which is eventually released. The budding causing some lysis,
however the cell is not completely lysed.

Lysogenic growth cycle

It is not advantageous for a virus to kill all of its host’s cells
 It would no longer be able to replicate
Instead, viruses remain within cells as a potential infectious agent, without causing infection
Also known as temperate state

Has been shown to exist in HIV and phages (e.g. ) as well as many other viruses
Does not occur in some others e.g. T4, Ebola virus

1. The phage DNA integrates into the bacterial chromosome and becomes a prophage

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 This is a recombination event, that utilises the int protein

2. The chromosome with integrated prophage replicates
 This continues for many cellular divisions
3. A trigger will cause the cycle to switch to the lytic cycle
 UV light will trigger the virulence associated phenotype
 The XIS protein is involved in excision
The areas of integration on the chromosome are extremely A=T rich
 Less stable than GCm makes it easier for strands to separate

The lytic cycle is still the preferred cycle however

Recombination Mapping:

Before sequencing, methods could be used to map gene positions in bacteriophages

Certain viral genotypes produce easily recognisable phenotypes in lysed cells
Involves making crosses using viruses that differ in 2 or more different genes
Recombinant phages are produced, which can be identified then counted
Proportion of recombinants can be used to estimate the distance between the genes

Experiments were carried out on T2 phage

Host range differed between the two strains…

One strain could infect type B & B2 cells
The other could infect B cells only

Genotype h+ h- r+ r-
Phenotype Cloudy plaque Clear Plaque Small Plaque Large Plaque

Strain 1 = r - h + = cloudy, large Strain

2 = r + h - = clear, small
Cross: r - h +  r + h -

1. Within the bacterial cells, crossing over occurs between the two viral
chromosomes
2. Recombinant progeny are produced
 Some viral chromosomes do not cross over, resulting in non-
recombinants

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3. Progeny phages are plated on a mixture of E. coli B & B2 cells

4. All four progeny genotypes were identified
5. Percentage of recombinant progeny allowed mutants to be mapped

Recombinant 1 = r + h + = cloudy, small

Recombinant 2 = r - h - = clear, large

RF (recombination frequency) = recombinant plaques / total plaques

The RF indicates the distance between the genes
Viral genes may overlap
Many viruses have overlapping genes in order to maximise the potential of their genome

e.g. ΦX174
9 genes
Gene E is encoded completely within the sequence of another gene (D)
Within the sequence, but in a different reading frame an AUG (start codon) can be found
Bacterial cells have many different classes of ribosome, each with different degrees of fidelity
 Some high fidelity ribosomes will only recognise upstream start codons (at 5’)
 They wont recognise internal AUG codons
 Kozak’s rule = only the most 5’ AUG in mRNAs is recognised by ribosomes (most of the
time), otherwise there would be constant internal initiation

RNA Viruses
e.g. Rabies virus, HIV, rous sarcoma virus, influenza

Positive-strand virus = genome codes directly for coat proteins

Negative-strand virus = genome is copied into a complimentary form first

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RNA viruses that incorporate their genomes into a host chromosome are known as
retroviruses
The retrovirus needs to produce an enzyme to copy its RNA genome  cDNA
 As the host cell has a DNA genome
This enzyme is called reverse transcriptase (RT)
Once the ds cDNA has been produced, it integrates into the host chromosome as before
Again, when conditions are appropriate the provirus will undergo transcription and will produce
numerous copies of its genome and proteins

All known retroviruses have three genes in common, each encoding a precursor protein
 gag – produces 3 proteins that will make up the capsid
 pol – encodes for RT and integrase (used to insert cDNA into host chromosome)
 env – encodes for a glycoprotein in the viral envelope
The proteins are then proteolytically processed into two or more functional proteins

Many retroviruses encode oncogenes, which are involved in

stimulating cell division, and very often cause the production
of tumours.

An example of this is rous sarcoma virus, which was found to

cause tumours in chickens.

HIV infects T-lymphocytes and has a 10000 nucleotide genome

Its genome, like all RNA viruses is replicated by RDRP, which has no error correcting mechanism
DNA viruses have error correcting mechanisms, RNA viruses do not, so RNA viruses mutate at a
faster rate. This rapid evolution can even occur within a host cell
This makes it very difficult to produce a vaccine, as the viruses are constantly changing and new
strains are being produced

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Prions
A novel class of pathogens that cause a range of neuro-degenerative diseases
They appear to replicate without the presence of any genome

Scrapie is a disease in sheep, which destroys their brains The pathogen was found to be entirely
made of protein (PrP). The pathogenic protein is derived from a protein that is found across all
eukaryotes.

Normal form = PrPc, helical formation

Abnormal form = PrPsc, flattened -sheet form
The -sheet form can convert normal PrP into the abnormal protein

“Protein only hypothesis” – If abnormal protein is introduced into a cell, it will switch all normal
proteins in the cell into an abnormal form

“Mutant gene hypothesis” – A mutated gene causes the same thing to occur

Accumulation of PrPc causes the prion based diseases

Other prion diseases…

 Kuru (humans)
 Creutzfeldt-Jakob disease (humans)

List of Viruses

SV40 – Simian Virus 40

 Monkey
 DNA
 Small

Adenovirus
 Human
 DNA

Poliovirus
 Human

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 RNA

CPMV – Cowpea mosaic virus

 Plant
 RNA

T4 bacteriophage
 Phage (E. coli )
 Copy number = 250
 dsDNA
 Nonenveloped

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