L3 - Influenza II and PHA

Question 1

What 3 glycoproteins are encoded in the influenza virion?

Answer 1

Matrix protein - M1
Haemagglutinin
M2

Question 2

What is the haemagglutinin glycoprotein?

Answer 2

Binds to sialic acid residues
Activates PKC
Transient inhibition of ENaC

Question 3

What is the M2 glycoprotein?

Answer 3

Forms an acid activated, amantadine inhibited H+ channel
- Can change the pH  impacts on epithelial Na channel  ENaC
Inserted into apical membrane host cell during infection
Involved in long term regulation of ENaC during infection

Question 4

What happened when ENaC currents were recorded with over expression of M2?

Answer 4

Mimics what happens during infection 
M2 decreases ENaC
- Number in membrane
- Function/currents 
- Open probability 
Western blot shows ENaC protein/band reduced in presence of M2

Question 5

What two reasons are there for the reduction in ENaC?

Answer 5

ENaC not reaching membrane

ENaC being removed

Question 6

How did they test if endocytosis of ENaC was a response to the over expression of M2?

Answer 6

They exploited a Liddles mutant of ENaC
- It is resistant to endocytosis
If you now overexpress M2 and endocytosis is
- Promoted by M2, you wont see such a big drop in the size of currents
- Not promoted by M2 you would still see the same drop in currents as with WT ENaC

Question 7

What did the endocytosis results of ENaC with a Liddle’s mutant show?

Answer 7

Reduction in currents but not as big a drop as in wild-type

• A significant drop in ENaC currents in the presence of M2 is because endocytosis is promoted
• If you inhibit endocytosis you can reduce the inhibition seen with M2

Question 8

Overall what does M2 expression in response to infection do? - ENaC

Answer 8

M2 expression in response to infection is promoting the endocytosis of ENaC from the apical membrane
- Removed from membrane faster than they would in the absence of M2

Question 9

How are ROS and M2 linked?

Answer 9

In the absence of M2 – very little ROS
When M2 overexpressed – much more ROS
- Co localisation shows same cells express M2 and ROS

Question 10

Overall what does M2 expression in response to infection do? - ROS

Answer 10

M2 increases ROS expression

Question 11

What happens when cell over expressing M2 and exposed to glutathione - an anti-oxidant? - If we reduce ROS do we see a recovery of ENaC currents?

Answer 11

ENaC currents recovery nearly fully

Tells us ROS plays a role in ENaC inhibition

Question 12

What happens when cell over expressing M2 and exposed to PKC inhibitors? - If we reduce PKC do we see a recovery of ENaC currents?

Answer 12

ENaC currents recovery nearly fully

Tells us PKC plays a role in ENaC inhibition

Question 13

Overall what is the role of M2, ROS and PKC during infection?

Answer 13

Overexpression of M2 reduces open probability and number of channels and some of this is mediated by an increase in ROS and PKC activation

Question 14

Does infection with influenza virus increase or decrease the ASL?

Answer 14

Conflict in literature

Question 15

What could the differences in the height of the ASL and whether it increases or decreases during infection be due to?

Answer 15

Cell source
Strain of mice 
Influenza virus properties
Lab conditions
- Different experimental solutions
Could this reflect what happens in vivo
- Whole range of symptoms could be seen
- Genetic background – predisposition 
- Where virus spreads to

Question 16

What two channels are inhibited by influenza infection?

Answer 16

CFTR and ENaC

Question 17

What experiment was used to show infection inhibited CFTR?

Answer 17

Stimulate cells with Forskolin –> cAMP –> CFTR
Response to Forskolin is a measure of the function of CTFR
In infected cells almost no currents/response –> CFTR inhibited

Question 18

How are CFTR and M2 linked?

Answer 18

Overexpression of M2 reduces the amount of mature CFTR

• The CFTR that goes to the membrane
• Tested using western blot

Question 19

If you run a western blot and probe for CFTR you will get two different bands, why?

Answer 19

Band C – higher molecular weight – mature glycosylated version

Question 20

How did they test what cells would do if they were infected but could not make M2?

Answer 20

M2 knockdown using an siRNA experiment

Question 21

What were the results of the siRNA M2 knockdown experiment?

Answer 21

In uninfected cells – no significant different between the two bars
- The M2 is coming from the virus
In infected cells
- In control – inhibition –> reduced mature CFTR
- In M2 knockdown –> WT levels of mature CFTR
Overall - cells infected with influenza –> inhibit amount of mature CFTR –> reliant of M2 protein

Question 22

What were the results of the siRNA M2 knockdown experiment when looking at CFTR whole cell currents?

Answer 22

In infected cell injected with Dsi-M2 (M2 KD) CFTR currents are returned to normal
If you knockdown M2 mature CFTR levels return to normal and these channels are fully functional

Question 23

What kind of channel is M2?

Answer 23

Amantadine inhibited H channel

Changes in pH mediate the inhibition of CFTR

Question 24

What is the importance of M2 being an Amantadine inhibited H channel?

Answer 24

Not all M2 that is made as a consequence of infection is amantadine inhibited
Udorn virus - makes a M2 amantadine sensitive protein
- If you add amantadine after infection with Udorn –> M2 is made but it can not transport H ions –> blocks H flux

Question 25

What experiments did they do to show the effect of Amantadine on M2?

Answer 25

No infection –> no M2 –> adding Amantadine does nothing
Udorn –> no amantadine –> inhibition of CFTR band C
- Infection with influenza reduces amount of CFTR
Udorn –> amantadine –> blocks M2 –> less inhibition of CFTR band C
- Stops protein movement
- So protein movement/pH changes must influence what happens to mature CFTR
PR8 –> no amantadine –> inhibition of CFTR band C
PR8 –> amantadine –>inhibition of CFTR band C
- No effect - M2 functions normally

Question 26

Overall what did the experiments with amantadine show?

Answer 26

Proton transport through M2 plays a role in what happens to CFTR

Question 27

What experiments did they do to show that infection and M2 leads to CFTR targeted to lysosomes for degradation?

Answer 27

Bafilomycin – prevents lysosomal acidification
Lactacystin – prevents proteosomal degradation
Uninfected
- No effect of lysosomal degradation blockers
Infected
- Vehicle - reduction in CFTR Band C
- Bagfiolmycin - reverses reduction in CFTR
- Lactacystin - does not reverse reduction in CFTR

Question 28

Overall what did the experiments with bafilomycin and lactacystin show?

Answer 28

M2 mediates movement of protons –> changes pH –> this change in pH targets mature CFTR to lysosomes where it is degraded –> preventing lysosomal degradation prevents degradation of mature CFTR

Question 29

What diseases can gain of function mutations in ENaC cause?

Answer 29

Liddles

• Hypertension
• Increased ENaC - resistant to endocytosis
• • Down to how ENaC interacts with Nedd4 – channel retrieval

Question 30

What diseases can loss of function mutations in ENaC cause?

Answer 30

PHA – pseudohypoaldosteroism

- Decreased ENac

Question 31

What are the symptoms of PHA1?

Answer 31

Salt wasting – lose Na in urine
Hypotension – water follows Na, low EXC fluid volume
Hyperkalaemia – if not reabsorbing enough Na - not secreting enough K – high plasma K
Metabolic acidosis – if not absorbing enough Na across collecting duct, not excreting enough H ion into urine
High renin and aldosterone – compensate for Na lose

Question 32

What are the characteristics of autosomal dominant PHA1?

Answer 32

Renal form
Problems localised kidney
Mutations in mineralocorticoid receptor gene – target for aldosterone

Question 33

What are the characteristics of autosomal recessive PHA1?

Answer 33

Systemic form
Multiple organs affected
Mutations in ENaC in all subunits
Frequent lower respiratory tract illness and runny nose

Question 34

Why does PHA1 lead to a runny nose?

Answer 34

ENaC inhibited more than CFTR - leads to runny nose
- Less Na reabsorption
Wet weight – liquid from the nose increased
- Excess Na in this nasal discharge

Shows loss of function mutations in ENaC in patients –> disruption of nasal surface liquid layer

Question 35

What is reduce in systemic PHA1 patients?

Answer 35

Nasal transepithelial potential

Question 36

What does amiloride do to the transepithelial potential - normal vs systemic PHA1 patient

Answer 36

% inhibition of the transepithelial potential by amiloride

• Normal – 60%
• Systemic patient – 5%

Question 37

Describe the impact of infection on the PCL and lung function. Explain the cellular mechanism that underpins the change in PCL.

Answer 37

Infection leads to an increase in the height of the PCL
It becomes more difficult for the cilia to remove mucous and therefore the lungs to clear infection
- Bigger volume that the cilia has to move
Causes
- Inhibition of CFTR and inhibition of ENaC
- Likely inhibition of ENaC predominated
Could also talk about mechanisms in a longer question