L5 - Viruses

History of virology - Dimitri Ivanoski (1892)

infectious tobacco mosaic virus isolated in filtered, bacteria-free fluid.

Felix D’Herelle

Discovered bacteriophages (coined term “plaque”)

Walter Reed (and colleagues)

Showed that yellow fever was a virus transmitted by mosquitoes in 1901.

Structure of virus

• what kind of parasites?

• dimensions?

• how long are genomes typically?

• intracellular obligate parasites

• 10-100nm

• genome typically a few thousand to 200,000 nucleotides long

Exceptions to small size of viruses

• Megavirus

• mimivirus

• pandoravirus

Megavirus chilensis

Exceptions to small size of viruses

dsDNA virus of amoebas, geom >1.2 megabase pairs, encodes 1200 proteins

Mimivirus

Exceptions to small size of viruses

dsDNA virus of amoebas, can be 400nm in diameter with a 1.2 megabase pair genome coding for 979 proteins

Pandoravirus

Exceptions to small size of viruses

2013; dsDNA virus of amoebas

~2.5 megabase genome, 1 micron in diameter

Viral structure

• DNA/RNA

• shell?

• envelope?

• single or double-stranded DNA or RNA (linear or circular)

• protein shell (capsid) around genome

• possible envelope (cell-derived membrane around capsid)

In viral structure, what is the protein shell (capsid) around genome?

Composed of many capsomere proteins. Capsid & genome together = nucleocapsid.

Poliovirus (picornavirus) - host

humans

Poliovirus (picornavirus) - structure

non-eneveloped; icosahedral

Poliovirus (picornavirus) - size (nm)

30 diameter

Poliovirus (picornavirus) - genome size (bp)

7700

Poliovirus (picornavirus) - genetic material

ssRNA (single stranded RNA)

Tobacco mosaic virus (TMV) - host

Tobacco & related plants

Tobacco mosaic virus (TMV) - structure

non-enveloped, helical

Tobacco mosaic virus (TMV) - size (nm)

300 × 18

Tobacco mosaic virus (TMV) - genome

6400

Tobacco mosaic virus (TMV) - genetic material

ssRNA

T2 (bacteriophage) - host

e-coli

T2 (bacteriophage) - structure

non-enveloped, complex

T2 (bacteriophage) - size (nm)

200 × 90

T2 (bacteriophage) - genome size (bp)

170,000

T2 (bacteriophage) - genetic material

dsDNA

Smallpox virus (poxvirus) - host

humans

Smallpox virus (poxvirus) - structure

enveloped, complex

Smallpox virus (poxvirus) - size (nm)

300 × 250

Smallpox virus (poxvirus) - size (bp)

186,000

Smallpox virus (poxvirus) - genetic material

dsDNA

Mimivirus - host

amoeba

Mimivirus - structure

enveloped, complex

Mimivirus - size (nm)

400 - diameter

Mimivirus - genome size (bp)

1,200,000

Mimivirus - genetic material

dsDNA

Capsids shape

Often exhibit either helical or icosahedral

Virus shape can be…?

Irregular or complex

Viral envelopes - enveloped virus

Plasma membrane surrounds nucleocapsid

Viral envelopes - unenveloped/naked virus

No plasma membrane

Virus replication methods

1. adhere: stick to host cell

2. enter

3. uncoat: release genome

4. synthesis: express & replicate genome

5. assembly: create new virus particles

6. exit: new particles leave cell

HIV & CD4 - How does virus enter a cell (replication cycle)?

HIV & CD4 receptors on T cells

How does virus enter a cell (replication cycle)?

Mechanisms vary on host cell. E.g., animal viruses don’t have to contend with a cell wall.

How do viruses enter animal cells?

Endocytosis, membrane fusion

How do viruses enter plant cells?

Often depends on damage to plant tissues to open a spot in cell wall. E.g., insects feeding on plants, wind/rain/hail/fire/human-induced damage

Virus entering bacterial cells

Bacteriophage replication - cycles/phases? (3)

1. lytic cycle

2. lyosgenic cycle (“temperate phage”)

3. temperate phage vs virulent phage

Bacteriophage replication - lytic cycle

Viruses enter, replicate, and lyse host cell

Bacteriophage replication - lysogenic cycle (“temperate phage”)

Phage integrate their genome into host (i.e., “lysogen”) cell’s genome, becoming a “prophage”. Prophage genome is replicated along with the host cell’s until stress. Can then enter lytic cycle

Bacteriophage replication - temperate phage vs virulent phage

Temperate phage can be both lytic or exist as a prophage (“lysogeny”). Virulent phage are always lytic.

bacteriophage replication

Where did viruses come from? (i.e. name 3 hypotheses)

Coevolution, regressive, progressive

Coevolution hypothesis

Viruses originated about the same time as other microbes and have been coevolving with them.

Regressive hypothesis

Viruses are previously alive organisms that have evolutionarily regressed into host-dependent particles.

Progressive hypothesis

Viruses originated from genetic material that gained the ability to replicate & be transmitted semi-autonomously

Are viruses alive?

No b/c they are subcellular parasites that have no metabolism & don’t replicate independently.

Bacteriophage

Used in the early 1900s to describe viruses that infect bacteria

Are viruses bigger or smaller than prokaryotes & eukaryotes?

Usually smaller - 10-100 nm.

Virus structure, morphology

1. small RNA or DNA genome protected by protein capsid

2. Vary in size & morphology. Sometimes exhibit helical or icosahedral symmetry 

Many bacteriophages have an ___ head and a ___ tail.

icosahedral, helical

Non-enveloped viruses

Capsid protecting genome

Enveloped viruses

Cell-derived lipid bilayer taken from cytoplasmic membrane of host cell, or occasionally derived from membranes of the golgi, endoplasmic reticulum or nucleus depending on virus

Are SARS-CoV-2 (covid-19) non-enveloped or enveloped viruses?

enveloped

How can most viruses enter a host cell?

Most viruses have an attachment protein that binds to a receptor protein on the surface of the host.

Do non-enveloped viruses & enveloped viruses have the same entry mechanism to enter a host cell?

No

How do bacteriophage enter host cell?

Use their tails to penetrate host cell’s plasma membrane & inject their genomes.

What happens when a virus enters a host cell?

All lytic viruses replicate their genome and produce new viral proteins. These particles assemble to form new viruses, which exist the host to infect more cells.

What are the hypothesis for the origin of viruses?

1. Coevolution

2. Regressive

3. Progressive

Coevolution hypothesis - origin of viruses

Viruses originated about the same time as other microbes and have been coevolving with them

Regressive hypothesis - origin of viruses

Viruses are previously alive organisms that have evolutionarily regressed into host-dependent particles.

Progressive hypothesis - origin of viruses

Viruses originated from genetic material that gained the ability to replicate and be transmitted semi-autonomously.

Can viruses be cultivated?

Yes, by inoculating the appropriate host cells and collecting the replicated viral particles. Particles can be purified by centrifugation or filtration.

Replication strategies for viruses

Lytic or lysogenic

Can viruses can be lysogenic?

not all

After a virus enters a cell or inserting their DNA into the cell in the case of ____, many viruses undergo a ___ cycle.

bacteriophages, lytic

Lytic viruses

Replicate & lyse infected host cells, and subsequently, surrounding cells. In isolated culture, all host cells eventually die and only viral particles remain.

Temperate viruses

Abstinence from lytic cycle. Can remain integrated thru many generations yet can enter the lytic cycle again.

For temperate viruses that infect bacteria, what is prophage?

Virus genome inserted within host genome. i.e. temperate bacteriophage

What results in reversion of prophage back into lytic cycle?

Environmental & cellular cues, usually related to some form of stress (e.g., exposure to UV light, DNA damage), and influx of nutrients (cells begin dividing & copying their own genomes).

Disadvantages for a temperate phage to go through lytic cycle?

Always kill host and lyse host’s cell after infection = may result in too few host cells for continued infection

Advantages for a temperate phage to go through lysogenic cycle?

Allowing virus to “hide and wait” for conditions to be right (e.g., more nutrients = more cells to infect) or just before a cell dies (e.g., DNA dies, virus moves).

Although there are eukaryotic cell viruses that can also undergo ____, the mechanisms for these are less well understood. E.g., ?

lysogeny, HIV (AIDS), herpes simplex (cold sores), human papilloma virus (HPV; warts)

How to quantify viral particles?

Direct count with an electron microscope or an assay such as a hemagglutination or plaque assay.

Viruses are named for many different reasons. Official virus classification system uses what kind of system in the following groups?

Order, family, sub-family, genus, species

Why are viruses usually grouped by physical & biological features?

no common ancestor

What is the Baltimore Classification System based on?

Physical & biological features of virus. E.g., nucleic acid characteristics of viruses

How can viruses be identified?

Through electron microscopy & nucleic acid analyses.

Viruses are the only sub-cellular particles to exist. T/F?

False - viroids, prions exist

Viroids

Naked RNA genome usually less than 400 nucleotides in length. Most likely replicated by host enzymes.

Prions

Proteinaceous infectious particles. Do not possess any sort of genome yet can replicate and infect host cells. Responsible for transmissible spongiform encephalopathies (TSEs), such as mad cow disease.

CRISPR

Clustered regularly interspaced short palindromic repeats.

Mechanism that bacteria use to prevent infection by bacteriophage. CRISPR-associated (Cas) proteins use snippets of DNA collected from previous (failed) virus infections to help identify & cut any identical incoming viruses entering the cell. Any new viral genomes entering the cell can also be cut by Cas proteins and added to CRISPR region of genome.

What can CRISPR/Cas system be used for?

• virus defence mechanism used by bacteria

• form of genome editing

Cultivating bacteriophage

What are the requirements for cultivating animal viruses?

1. Tissue culture of host cells used to grow targets for viruses (or in embryonated chicken or duck eggs for < $$).

2. cultures must be kept sterile & bacteria-free

HeLa cells

1st human cell line. Henrietta Lacks

Viral purification 

Usually begins with simple filtration to remove large cells and cellular debris. Viruses then purified and concentrated with centrifugation.

What are the types of centrifugation in viral purification?

differential, gradient