Lecture 4- Early Steps of Infection

Question 1

Steps of infection

Answer 1

1) Adsorption (attachment)
2) Penetration (entry)
3) Uncoating
4) Component production (genomes plus viral proteins)
5) Assembly (maturation, encapsidation)
6) Release

Question 2

For what type of virus does penetration and uncoating occur at the same time?

Answer 2

T4 phage and most other bacteriophages

Question 3

Virion

Answer 3

Virus particle

Question 4

Virion attachment proteins

Answer 4

Antireceptor- part on outside of virion that bind to receptor

Question 5

Receptor

Answer 5

Part on outside of host cell that virion attachment proteins bind to

Question 6

What is the nature of the interaction between the virion attachment protein and the receptor?

Answer 6

Complementary shapes. Specific and strong because of shape, multiple bonds, and multiple receptors. Types of bonds involved are H bonds, ionic, hydrophobic interactions (all weak, but there are many of them). Typically no covalent bonds. Once associated, typically does not dissociate.

Question 7

Tropism of Poliovirus

Answer 7

Exhibits a very narrow host range.

1) Infects only human cells and some non human primate cell types. Will not infect mouse or other rodent cells.
2) Only infects a subset of human cells (intestinal mucosa, lymphoid tissues, and certain neuronal cells (paralysis)).

Question 8

What is special about the polio RNA genome?

Answer 8

Purified polio RNA is infectious (just RNA will still mount an infection)

Question 9

Why can’t mice become infected by polio virus?

Answer 9

Host range is determined at level of adsorption. It is determined by interaction with receptor, and mice do not have a receptor. Unless naked RNA genome makes it into the mice, and then will infect because it bypasses the receptor problem and the RNA genome is infectious with polioviruses.

Question 10

What do we learn from the direct plaque assay vs one step growth experiment with polio virions vs RNA in humans and mice?

Answer 10

RNA genome is infectious. Also, works for one step growth experiment but not direct plaque assay because direct plaque assay requires many cycles of infection, so would get virions after the first cycle which can’t infect mouse cells.

Question 11

Cloning of poliovirus receptor gene- Importance of monoclonal antibodies

Answer 11

Monoclonal antibody (Mab) directed against polio receptor.  Make antibodies that are specific for polio virus receptor.  Add virus after Mab, virus can't bind.  Then you can clone the gene for the receptor.  
Human cells -> high molecular DNA -> mouse cells on petri dish -> monoclonal antibody against receptor -> remove these mouse cells from the plate.  Next, identify gene from mouse genome

Question 12

What are the two types of entry by enveloped viruses?

Answer 12

Direct fusion and receptor-mediated endocytosis. Both involve fusion of viral envelope with cell membrane.

Question 13

What are some examples of viruses that use direct fusion?

Answer 13

Paramyxoviruses (sendai, measles), some herpes viruses, HIV-1 (other lentiviruses)

Question 14

What are some examples of receptor-mediated endocytosis?

Answer 14

Toga viruses (sindbis, SFV), rhabdoviruses (VSV, rabies), some herpes viruses, orthomiyxoviruses (influenza A), some retroviruses

Question 15

Direct fusion

Answer 15

Envelope hits receptors, adsorption occurs (tight interaction), fusion of envelope with plasma membrane (like melting), nucleocapsid ends up inside the cell in cytoplasm, envelope stays behind and becomes part of membrane. Can fuse with another cell that isn’t infected.

Question 16

Receptor-mediated endocytosis (viropexis)

Answer 16

Lipoproteins, polypeptide hormones, growth factors
Envelope hits receptors, clatherin coated pit forms (shallow indent in plasma membrane). Virus is exploiting a path that already exists. Plasma membrane engulfs particle. Now have an endosomal vesicle/lysosome inside cell (2 membranes)
Acidification of vesicle occurs: protons pumped inside, internal environment becomes acidic. Vesicle and envelope fuse and dump nucleocapsid directly into cytoplasm.

Question 17

Acidification of endosomal vesicle and entry

Answer 17

Methylamine, a lipophilic amine inhibits entry. Will pass through membranes.
Methyl amine protonated on inside, can’t pass back out. Acts as a buffer and sucks up protons and prevents acidification
This demonstrates the importance of acidification of entry