Week 5 - Viruses

Ivanovski and Beijerinck

Isolated tobacco mosaic virus in the later 1800s, beginning viruses as a science

Walter Reed

Showed that yellow fever was caused by a virus transmitted by mosquitoes

Virus + size

Small intracellular obligate parasites between 10-100 nm

Size variation of viruses

Some viruses like CroV, Megavirus chilensis, and mimivirus are huge

Structure of viruses

• Use ssRNA or dsDNA

• A capsid shell protects the virus

• Some have an envelope of plasma membrane around the capsid shell

Nucleocapsid

Includes both the shell of capsomere proteins and the virus genome it surrounds

Shapes of capsids

Can be helical, icosahedral (20 faced polygon), irregular, or complex

Bacteriophages + replication cycle

Tail fibers stick to cell surfaces, inducing a conformational change. Inner core tube proteins are injected into the cell wall.

Hemagglutinin and neuraminidase

Viral proteins on the surface of many nucleocapsids

Enveloped viruses vs naked viruses

Presence of a plasma membrane surrounding the nucleocapsid

Replication cycle of every virus (4 steps)

1. Attachment to host cell

2. Entry and uncoating of the viral genome

3. Gene expression and protein production

4. Assembly and exit from the host cell

Entry into animal cells of non-enveloped and enveloped viruses

Non-enveloped: via endocytosis

Enveloped: via membrane fusion or endocytosis to fusion

Entry into plant cells

Often requires some type of damage to plant tissues since there is a protective cell wall

Entry into bacteria

Uses bacteriophages, injects the inner core tube proteins into the cell

Why are viruses considered acellular?

They have no ability to replicate independently

Coevolution hypothesis

• Viruses evolved along with their host cells

• Could explain RNA viruses, but there is overall little evidence

Regressive hypothesis

• Viruses are cells that lost their replication and metabolism

• Although examples of this exist, it doesn’t explain RNA viruses

Progressive hypothesis

• Genetic material gained the ability to replicate semi-autonomously and move from cell to cell

• Transposons and retrotransposons are evidence of this

Transposons vs retrotransposons

• DNA transposons use a "cut-and-paste" method and can insert copies of themselves into the host cell

• Retrotransposons use a "copy-and-paste" method via copying their genome into RNA, then converting it to DNA, and then integrating it into the host genome

Using the progressive hypothesis, how might bacteriophages and animal viruses evolved?

May have been genetic components that escaped bacteria/animal cells and moved between cells

Lytic cycle

Replicate in and lyse the host cell, killing it

Lysogenic cycles

Bacteriophages integrate their genome into the host cell genome, which is replicated each time the host cell replicates

Key properties of viruses

• Cannot replicate independently

• Use host cell processes (like transcription and translation) to make new virus particles

• Can have DNA or RNA

Helical vs icosahedral viruses

Helical capsids tightly associate with the viral genome and form a cylinder-like structure. Icosahedral capsids are like balls that contain the viral genome

Contrast bacteriophages with animal and plant viruses

Bacteriophages typically do not enter the host cells and inject their genetic material in instead

What evidence does not support the regressive evolution of viruses?

Sequences of viral genomes do not seem like they were once cells that lost most of their genetic material. They resemble the genomes of their hosts, and share no features indicating their ancestors were bacteria or archaea

Plaques in a bacterial lawn

Zones of clearing where a phage has killed an infected cell and its adjacent cells. Can be counted to determine the concentration of phages in the solution applied to the agar plate.

Cultivating animal viruses

Must use a tissue culture of host cells to grow targets for the virus

HeLa cells

The first human cell line, isolated from Henrietta Lacks. Immortal due to them being cancerous

Cytopathic effects (CPE)

Changes in cells caused by viral infection, ranging from morphology to cell death

Syncytia

A cytopathic effect where cells fuse into a large, multi-nucleated cell structure

Viral purification

Filtering out large cells and cellular debris via differential centrifugation or gradient centrifugation

Differential centrifugation

A method of viral purification.

1. Low speed centrifugation, pellet of whole and broken cells forms at the bottom

2. Medium speed centrifugation, pellet of nuclei and other large organelles

3. High speed centrifugation, pellet of virus

Gradient centrifugation

Viral component and particles of different density settle into different bands, forming a density gradient

Methods of viral quanitification (4)

Direct count, hemagglutination assay, plaque assay, endpoint assays

Direct count + cons

• Electron microscope is used to see a known volume of material, count the viruses, and scale it up to determine titer

• Not easy and doesn’t differentiate between viable and nonviable particles

Titer

Concentration of a virus preparation

Hemagglutination assay + pros and cons

• Engineer viruses to stick to RBCs. Lower number of viral particles forms a button, while high numbers form a shield.

• Pros: cheap, fast, easy

• Cons: not all viruses can do it, doesn’t differentiate live/dead viruses, doesn’t give a virus number

Plaque assay

Virus is diluted and placed on target cells, then plaques are counted to determine the number of viral particles.

Endpoint assays

Determine either TCID50 (amount of virus to induce CPE in 50% of the cells) or LD50 (amount of virus to kill 50% of the test animal subjects)

Explain how filtration and centrifugation can be used to purify viruses

Viral particles are very small and can be filtered out of larger cell counterparts. Centrifugation causes the largest particles to form a pellet, leaving viral particles only

Strategies used to name viruses

Location, disease they cause, physical characteristics, organism they infect, simple letter/number combinations

ICTV classification scheme (5)

Classify viruses into order, family, subfamily, genus, and species, based on morphology, genome structure, replication strategy, host range, and pathogenicity

Baltimore classification scheme

Separates viruses into seven classes based on how they generate mRNA

Electron microscopy in virus identification

• First step in virus identification

• Allows for visual observations of viral morphology

• Some viruses are indistinguishable under a microscope

Nucleic acid analysis in virus identification

• PCR and RT-PCR are used to identify viruses by genome sequence

• Can study viral evolution patterns

RT-PCR + uses for viruses

Converts RNA to DNA, which can then be amplified via PCR. Can study RNA produced by infected cells

RT-PCR process (7)

1. Isolate RNA

2. Add primers, reverse transcriptase, and nucleotides

3. RT synthesizes DNA copy of RNA (now you have a DNA copy, proceed with normal PCR)

4. Heat sample to denature strands and deactivate RT

5. Add primers, Taq polymerase, and nucleotides

6. Taq makes a second DNA strand

7. Repeat PCR cycles to amplify DNA

How can viruses be used as molecular biology tools?

Can be engineered to deliver working copies of genes to replace damaged versions (gene therapy)

Oncolytic viruses

Replicate in and destroy cancerous cells while having limited effects on normal cells

Oncoviruses

Viruses that cause cancer