HCV NS5A protein

Question 1

Current treatment for HCV?

Answer 1

No vaccine, but effective new combination therapy

e.g. Direct acting antivirals (DAAs)

Question 2

What tools helped to study HCV in vitro?

Answer 2

Japanese Fulminant Hepatitis-1 (JFH-1) genotype 2a
Infectious clone = AN AGGRESSIVE HEPATITIS

Starts off as +ive strand –>transcribe –> transfect into Huh7 cells –> replicates to release virus particle – now have double stranded strain to manipulate and infect other cells

Question 3

NS5A structure?

Answer 3

Amphipathic helix
Domain 1 - binding site for zinc
Domain 2 - phosphorylated
Domain 3- phosphorylated

Question 4

NS5A role in viral RNA replication?

Answer 4

NS5A – RNA replication binding protein, hence a ‘hot-spot’ for mutations

Extra:
NS4A – targets
NS5B – not sure function

Question 5

Relationship between NS5A and cell signalling?

Answer 5

NS5A inhibit signals from interferon, growth factors + apoptotic signals

Question 6

How does NS5A help with interferon resistance?

Answer 6

Domain 2 - contains Interferon Sensitivity Determining Region (ISDR).
Binds to PKR
inhibition of the antiviral effects of eIFN-alpha

Question 7

Where does NS5A bind?

Answer 7

SH3 domain containing proteins - any sequence that looks like PxxPxR (amino acids)

Question 8

How does NS5A block growth factor signalling?

Answer 8

1. P2 motif in NS5A binds to SH3 domain of Grb2
2. Causes INHIBITION
3. Blocks downstream signalling to Ras
4. ERK1/2 is not phosphorylated
5. Blocks growth factor signalling

Question 9

How does NS5A effects on EGFR trafficking

Epidermal Growth Factor Receptor (EGFR) in plasma membrane

Answer 9

NS5A associates with several proteins involved in the endocytosis of the epidermal growth factor receptor (EGFR) and has been previously shown to inhibit epidermal growth factor (EGF)‐stimulated activation of the Ras–Erk pathway by a mechanism that remains unclear.

NS5A blocks late endosomal trafficking of EGFR

Question 10

Phosphatidylinositol-3 kinase (PI3K)

Answer 10

An anti-apoptotic factor. Recruits PH-domain proteins to plasma membrane

Question 11

The PI3K pathway?

Answer 11

PI3K activation inhibits apoptosis

EGF + Insulin activates PI3K complex –> PIP2 –> PIP3. Pip3 recuits Akt w/ PH domain –> Akt activation –> Phosphorylation and inactivation of Bad –> Mitochondrial permeability transition pore closed –> No apoptosis!!!

Question 12

How does NS5A interact with PI3K?

Answer 12

1. NS5A binds to p85
2. PI3K (p110) activation
3. Akt activation
4. Apoptosis blocked

Question 13

How does NS5A manipulate growth factor signalling?

Answer 13

GFS signals block Ras system but activates p85. Slows G1 phase but is unpleasant to cells – cells try to apoptosis. Akt blocks apoptosis