Cytoplasmic membranes

Question 1

What are the 2 positive sense RNA virus families?

Answer 1

Picornaviruses + Flaviviruses

Question 2

Picornavirus genome structure?

Answer 2

Polyprotein split into 3 
P1= VP1-4 = Structrual proteins 
P2= 2A-C = 2A is protease + additonal subunit ontop of 2B + 2C called 2BC (important)
P3= 3A-D = 3A = tail anchor
                    3B= VPg
                    3C = Protease 
                    3D = Polymerase

Question 3

Hepatitis C virus genome structure?

Answer 3

Polyprotein split into structural + non-structural

Structural
CORE - Capsid
E1 + E2 - Envelope glycoproteins

Non-structural
NS2, NS3, NS4A, NS4B, NS5A, NS5B

p7 in both structural + non

Question 4

The HCV replication cycle vs Picornavirus replication cycle

Answer 4

HCV :
Binding
Receptor mediated endocytosis
Fusion anduncoating
Translation and polyprotein processing
RNA replication
Virus budding into intracellular vesicles
Transport 
Fusion + release 

                                   Picornavirus : Virus binding Genome translocated across plasma membrane Translation of viral proteins RNA replication Virus assembly  Cell lysis and virus release

Question 5

What is Autophagy?

Answer 5

• self-eating
• Degradation of cytoplasmic proteins and organelles
• Removal of damaged or unwanted cellular components
• Provides energy under metabolic stress

Question 6

What is a marker for autophagy?

Answer 6

LC3 good marker for autophagy as it is released in the early stages of the process

Question 7

Where does picornavirus replication occur and how?

Answer 7

On autophagosomes

• 2BC and 3A proteins accumulate on ER and induce autophagy
• Replication complex on outside of autophagosome (?)
• Autophagy also important for release of virus from infected cells- see next lecture on viruses and apoptosis!

Question 8

What are the early stages in generation of autophagic membranes?

Answer 8

Vps34 – produces PI3P on ER membrane
Recruits DFCP1
Omegasome formation
Acts as a platform for production of autophagosomes

Question 9

HCV does not use a premature version of autophages called?

Answer 9

Omegasomes - sites from which phagophores form.

Phagophores are sack-like structures that mature into autophagosomes that fuse with lysosomes in order to degrade the contents of the autophagosomes

Question 10

Proteins responsible for double membrane vesicles in HCV

Answer 10

Replication complex
Vps34
PI3P
DFCP1

Question 11

What is Phosphatidylinositol-4 kinase (PI4K)?

Answer 11

Membrane structure in double membrane vesicles
PI4K drives PIP4/cholesterol trafficking
Protein 3A recruits PI4K to Golgi/TGN - where vesicle is made

Question 12

How does picornavirus recruitment of PI4K?

Answer 12

1. 3A protein is associated with Golgi/TGN membranes
2. Binds to and recruits (and activates?) PI4KIIIb
3. Arf1 retained but other trafficking proteins displaced (eg COPI, clathrin)
4. 6-fold increase in PI4P lipids, concentration at Golgi/TGN
5. Golgi disassembled – membranes become ‘replication organelles’

Question 13

How does HCV recruit PI4K?

Answer 13

NS5A – key component of replication complex
Binds to PI4KIIIa
Stimulates kinase activity
Increased PI4P levels

Question 14

Example of flavivirus?

Answer 14

Dengue virus, HCV, etc.

Question 15

How does Dengue virus replicate + assemble RNA?

Answer 15

Dengue virus induces membrane rearrangements to provide sites for RNA replication and assembly

Question 16

How does dengue virus induces lipophagy to deplete cellular lipids?

Lipophagy – selective autophagy targeted to lipid droplets

Answer 16

Fusion with lysosomes and lipid degradation

Release of fatty acids = more fatty acid = more energy production