Step 2: Entry and Fusion (Lecture 10)

Question 1

What is the first step in viral entry/fusion?

Answer 1

attachment of viral receptor binding protein to viral receptor molecule on cell surface

Question 2

What are the 2 roles that viral receptors play in uncoating?

Answer 2

initiate conformational changes to capsid and direct the virus into the endocytic pathway

Question 3

How are particles taken in via phagocytosis?

Answer 3

actin-mediated event

Question 4

What are the events in phagocytosis?

Answer 4

actin pushes cell membrane forward around whatever it’s going to take in &raquo_space;> taken to endosome = phagosome &raquo_space;> targeted to lysosome

Question 5

What is significant about lysosomes? What do they contain?

Answer 5

digestive enzymes and many protons = very acidic = degrades protein

Question 6

How are particles taken in via receptor-mediated endocytosis?

Answer 6

clathrin-coated vesicles (or calveolin)

Question 7

What are the events in receptor-mediated endocytosis with clathrin?

Answer 7

clathrin-coated vesicle takes in substance &raquo_space;> becomes early endosome &raquo_space;> H+ gets pumped inside = late endosome (at right pH) &raquo_space;> fuses with lysosome

Question 8

How do we distinguish between an early and late endosome?

Answer 8

level of pH, early endosome is more basic than late endosome as late endosome is more acidic

Question 9

What is the benefit/effect to the receptor of the differing levels of pH?

Answer 9

an increase in H+ = increase of removing the viral receptors off and recycle it back to plasma membrane for reuse

Question 10

What are the 4 triggering events that will cause the nucleocapsid to pop open?

Answer 10

receptor engagement, endosomal acidification, 2-entry method, receptor-mediated signaling event

Question 11

What is receptor engagement?

Answer 11

basic conformational changes will occur due to the receptor-ligand interaction

Question 12

What are the 2 ways that receptor engagement triggers viral entry/fusion?

Answer 12

1) fusion peptide is exposed and inserts itself onto host membrane and then delivers nucleocapsid into cytoplasm | 2) receptor-ligand interaction causes a pore formation on host cell membrane and injects nucleic acid into cytoplasm

Question 13

What is a fusion peptide and how is it exposed?

Answer 13

it is a second viral protein on the surface of the viral envelope; it is initially hidden but is exposed when virus binds to receptor on host cell membrane and causes a conformational change thus exposing the fusion peptide

Question 14

What is the triggering event in endosomal acidification?

Answer 14

acidity of endosome will expose viral fusion peptide = causes fusion

Question 15

At what pH does influenza pop open? What stage of endosome is this?

Answer 15

pH = 5 at the late endosome

Question 16

What are the events that take place in the entry/fusion of influenza?

Answer 16

receptor-ligand interaction causes virus to be uptaken by clathrin-coated vesicles &raquo_space;> then it begins to pump H+ in endosome &raquo_space;> turns into late endosome with acidic pH &raquo_space;> fusion peptide inserts into host membrane = fusion of membranes &raquo_space;> nucleic acids are released and directed to nucleus

Question 17

What allows influenza’s RNA genome to be directed to nucleus?

Answer 17

ribonucleo proteins (RNPs); genome is segmented

Question 18

At what pH does the fusion peptide get exposed in flaviviruses?

Answer 18

very very low pH (lysosome or late endosome)

Question 19

What are the events that take place in the entry/fusion of flaviviruses?

Answer 19

receptor-ligand interaction causes virus to be uptaken via fusion (endocytosis) &raquo_space;> early endosome turns into late endosome with the pumping of H+ &raquo_space;> fusion peptide is exposed and brings the membranes together allowing fusion &raquo_space;> RNA genome is released

Question 20

What is the 2-entry mechanism of viral entry and fusion?

Answer 20

two distinct and separate entry mechanisms that go hand-in-hand to get inside the host and help with uncoating and delivery; steps vary between viruses

Question 21

What are the 2 step entry mechanisms for Semliki Forest Viruses? What are they used for/do?

Answer 21

1) endosomal acidification induces membrane fusion but capsid is still bound to the cytoplasmic leaflet of the endosomal bilayer &raquo_space;> 2) ribosomes bind the capsid and disassemble it to release the nucleic acid into cytoplasm

Question 22

What are the 2 step entry mechanisms for Adenoviruses? What are they used for/do?

Answer 22

1) endosomal acidification destabilizes capsid and releases Protein IV = disrupt endosomal membrane to release capsid to cytoplasm &raquo_space;> 2) microtubules deliver and dock unstable capsid onto nuclear pore complex where the genome practically swims out into nucleus

Question 23

What are the events that take place in the entry/fusion of Ebola viruses? What are the 2-step entry mechanisms it uses?

Answer 23

Ebola gets taken up via CCV &raquo_space;> early endosome &raquo_space;> late endosome &raquo_space;> lysosome &raquo_space;> proteases cleave off gp protein off of ebola’s receptors = allows it to bind to NPC1 &raquo_space;> causes fusion event and releases nucleocapsid into cytoplasm ||| endosomal acidification and binding of cleaved gp with NPC1

Question 24

What is so unique about Ebola?

Answer 24

uses a co-receptor only found on the late endosome or lysosome

Question 25

What is the coreceptor that ebola uses?

Answer 25

NPC1

Question 26

What is the receptor-mediated signaling method of viral entry/fusion?

Answer 26

signaling cascade allows virus to enter in a unique way

Question 27

What are the 2 receptors that coxsackie virus requires?

Answer 27

DAF (decay-accelerating factor) and CAR (coxsackievirus-adenovirus receptor)

Question 28

What is the main receptor for Coxsackie viruses? What is it’s hurdle?

Answer 28

CAR receptor = buried between tight junctions making it unavailable for virus to bind to it

Question 29

How does Coxsackie virus get to it’s receptor?

Answer 29

Coxsackie does electrostatic interactions on cell surface and will eventually interact with DAF &raquo_space;> signaling cascade remodels actin causing membrane to ruffle = pushes virus to surf close to the tight junction proteins =CAR exposed &raquo_space;> virus binds to CAR and gets taken up via endocytic vesicle

Question 30

Why does the Coxsackie virus need the DAF receptor?

Answer 30

causes actin remodeling due to signaling event

Question 31

What are the 5 ways viruses regulate viral entry and fusion?

Answer 31

1) makes sure that genome gets into right cell (cytoplasm can easily degrade RNA) || 2) virus only fuses with right cell || 3) prevents aggregation of newly assembled virions by keeping genome delivery and virion assembly separate areas in cell || 4) maintains cell’s integrity by using cellular processes because it needs to survive || 5) prevents premature activation of fusion peptides during assembly in order to successfully deliver genome onto next cell

Question 32

What is the role of the nuclear pore complex in viruses?

Answer 32

ensures that viruses (especially DNA viruses) deliver their genome into the nucleus

Question 33

What are the 4 different strategies to delivering genome into nucleus?

Answer 33

viral ribonucleic proteins direct genome to nucleus, un-dismantled capsid docks onto NPC and injects genome, dismantled capsid docks onto NPC and genome flows through into nucleus, virus disrupts nuclear envelope to go into nucleus

Question 34

What do the RNPs need to have on them to get the nucleic acid into the nucleus?

Answer 34

specific signal sequence that other proteins recognize to bring into nucleus (ie: importin)