Topic 5: Viruses

Question 1

Dimitri Ivanowski

Answer 1

• Russian botanist
• Studied the tobacco mosaic virus
• one of the founders of virology

Question 2

Felix d’Herelle

Answer 2

• first discovered viruses that affect bacteria
• called them bacteriophages (means bacteria eaters)
• discovered and named plaques, circular clearings where viruses killed

Question 3

Walter Reed

Answer 3

• identified yellow fever and how it spread

Question 4

Structure of Virus: size

Answer 4

• smaller then bacteria, between 10 nm and 100 nm
• Genome typically a few thousand to 200 000 nucleotides long

Question 5

Exceptions to small size of viruses

Answer 5

Megavirus chilensis (dsDNA virus of amoebas):
- Has a genome greater than 1.2 megabase pairs
- encodes 1,200 proteins

Mimivirus (dsDNA virus of amoebas):
- 400 nm in diameter
- 1.2 megabase pair genome coding for 979 proteins
- mimics bacteria cell size
- resembles gram-positive bacteria

Pandoravirus (2013; dsDNA virus of amoebas):
- 2.5 megabase genome
- 1 um in diameter

Question 6

Structure of Virus: genome

Answer 6

• Typically a few thousand to 200,000 nucleotides long
• Single or double-stranded DNA or RNA (linear or circular)
• Protein shell (capsid) around the genome

Question 7

Structure of Virus: capsule

Answer 7

• all viruses have capsids, they protect RNA of virus
• Protein shell (capsid) around genome composed of many capsomere proteins
• Capsid and genome together = nucleocapsid
• Capsids structure can be helical, icosahedral, or complex

Question 8

Structure of Virus: envelope

Answer 8

• Enveloped virus: a membrane surrounds the nucleocapsid
• Naked virus (non-enveloped virus): no membrane

Question 9

Steps of viral replication

Answer 9

1. adhere (stick to host cells by protein/sugar receptors)
2. enter cell (this is the most important part)
3. uncoat (release genome)
4. synthesis (express and replicate genome)
5. assembly (create new virus particles within the host cell)
6. exit (new particles exit the host cell, differs between naked and enveloped)

Question 10

Viral cell entry of enveloped viruses (gaining entry to animals)

Answer 10

Membrane fusion
1. virus attatches to cell receptor
2. conformational change in the attachment protein and bound receptor initiates membrane fusion
3. the viral envelope fuses with plasma membrane
4. the nucleocapsid enters the cytoplasm and uncoats to release genome

Endocytosis
1. virus attaches to cell receptor
2. endocytosis is initiated
3. endosome forms with the virus inside
4. the low pH of the endosome initiates fusion of the viral envelope with the endosome membrane. the nucleocapsids are released

Question 11

Viral cell entry of non enveloped viruses (gaining entry to animals)

Answer 11

Endocytosis
1. virus attaches to the cell receptor
2. endocytosis is initiated
3. endosome forms with virus inside
4. the nucleocapsid escapes to the cytoplasm and uncoats to release genome

Question 12

Virus gaining access to plants

Answer 12

• enclosed in a wall of cellulose, no virus can recognize any type of receptor or gain access through thick cellulose
  – Requires damage to plant tissue to open the cell wall:
  —- Insects feeding on plants
  —- Wind, hail, rain or fire damage
  —- Human-induced damage

Question 13

Virus gaining access to bacterial cells

Answer 13

1. tail fibers attach to receptors
2. conformational change in tail fibers brings base of the tail in contact with host cell surface
3. rearrangement of tail proteins allows inner core tube proteins to extend down into the cell wall
4. contact with plasma membrane initiates transfer of DNA through pore formed in lipid bilayer

Question 14

Lytic replication cycle

Answer 14

• Phage enters, replicates and lyses host cell
• Lytic (or virulent) phage can only undergo lytic cycle

Question 15

Lysogenic replication cycle

Answer 15

• Phage integrates its genome into host cell genome, becoming a “prophage”
• prophage is stable and can be in virus for many generations
• Phage genome replicated along with the host cell’s until… stress
• Temperate phage can undergo both lytic and lysogenic cycles

Question 16

Hypotheses for the origin of viruses

Answer 16

Coevolution hypothesis:
viruses originated about the same time as other microbes and have been coevolving with them.

Regressive hypothesis:
viruses originated from living organisms that regressed into host-dependent particles.

Progressive hypothesis:
viruses originated from genetic material that gained the ability to replicate and be transmitted semi-autonomously.

Question 17

Cultivating viruses

Answer 17

Cultivating Bacteriophages
- Requires a suitable host population for infection, often on solid growth medium
- Virus particles can be purified from zones of lysed bacterial cells

Cultivating Animal Viruses
- Tissue culture of host cells used to grow targets for the viruses (or in embryonated chicken or duck eggs for less money)
- Cultures must be kept sterile and bacteria-free
- Methods have only been in place since the 1950s
- Modern virology couldn’t exist without these tools. Many developed from first human cell line, known as HeLa cells.

Question 18

Purifying viruses

Answer 18

• usually begins with simple filtration to remove large cells and cellular debris
• viruses then purified and concentrated with centrifugation

– Differential centrifugation
—- involves three different centrifugations at three different speeds

– Gradient centrifugation
—- depends on different densities of viral components and particles
—– The tube is successively filled with layers of decreasing concentrations of sucrose
The suspension containing the virus is layered on top
The preparation is centrifuged and as it spins the particles at the top move down each layer until they reach the density that matches the contents

Question 19

Quantifying viruses

Answer 19

• Not easy or straightforward
• Usually measured as a titer, or concentration of virus preparation
• Methods are: direct count, hemagglutination assay, plaque assay, endpoint assays

Question 20

Quantifying: Direct Count

Answer 20

• Electron microscope used to visualize a known volume of material
• Viruses within the material are counted and scaled up to determine the titer
• Requires an expensive, specialized microscope
• Doesn’t differentiate between infectious and non-infectious viral particles

Question 21

Quantifying: Hemagglutination assay:

Answer 21

• Exploits trait of some viruses to stick to red blood cells, causing them to form a gel mat
• Cheap, easy, fast, no microscope needed
• Some viruses won’t do this, only works for animal cells that recognize colic acid sugar residues in the membrane, animal viruses can bind to those residues. The blood cells get networked together by the sticking viruses and then the network floats at the top of the wells
• When high numbers of viral particles are present, the virions bind to the RBCs and form a diffuse mesh, or shield
• When fewer viral particles are present, the RBCs settle to the bottom of the well, forming a button.
• This doesn’t differentiate between viable/non-viable viruses, can’t tell if they are infectious, and doesn’t get a number of viruses, just gives a comparison

Question 22

Quantifying: Plaque assay

Answer 22

• Virus diluted and placed on target cells
• Plaques are counted to determine plaque-forming units (PFU) titer of original suspension
• Good measure for the infectious viruses
• Useful for phages and plant viruses

Question 23

Quantifying: Endpoint assays

Answer 23

Tissue culture infectious dose 50 (TCID50):
- The amount of virus needed to induce a cytopathic effect (CPE) in 50% of the cells it’s exposed to

Lethal dose 50 (LD50)
- Amount of virus needed to kill 50% of test animal subjects

TCID50 is about the amount of virus needed to infect half of the cells in a lab setting, while LD50 is about the amount of a substance that can kill half of the organisms in a study.

Question 24

Methods of viral classification

Answer 24

Historically, the naming of viruses was varied.

• Simple letter/number combinations (T4 phage)
• Organism(s) they infect (tobacco mosaic virus)
• Location of discovery (Ebola virus for Ebola River, Zaire)
• Appearance (coronavirus, “crown”)
• Disease caused (hepatitis viruses)

International Committee on Taxonomy of Viruses (ICTV)
- Classifies viruses based on Order, Family, Subfamily, Genus, and Species
- Incorporates several characteristics related to virion morphology, genome structure, and biological feature

Baltimore Classification system
- All viruses rely on host for translation (ribosomes)
- 7-class system based around mRNA production methods

Question 25

Viroids

Answer 25

• Consist only of naked RNA; extremely small (less than 400 nucleotides)
• High degree of internal complementarity
• Resistant to ribonucleases; so far, only observed to cause disease in plants

Question 26

Prions

Answer 26

• Proteinaceous infectious particles; problematic proteins that misfold
• Replication method still unclear
• Thought to revolve around conversion of protein conformations from normal to abnormal form over time

fold weird and make other proteins fold weird leading to mad cow disease

Question 27

Explain the basic mechanism of the CRISPR/Cas system and its potential for genome engineering

Answer 27

Gene editing technology that is faster, cheaper and more efficient

Mechanism:

1a. Transcription and translation of cas genes forming cas proteins
1b. Transcription of CRISPR locus, forming the pre-crRNA transcript
2. Cas proteins cleave the pre-crRNA forming mature crRNA subunits
3. Other Cas proteins interact with mature crRNA subunits forming CRISPR-Cas surveillance complexes
4. CRISPR-Cas surveillance complex surveys cells for complementary phage DNA. Once detected, the complementary sequences are aligned and the phage DNA is targeted for destruction