Protein Synthesis

Question 1

What the structure of eukaryotic mRNA?

Answer 1

5’ end –> m7g cap

• recognition signal to recruit ribosome + IFs
• protects RNA from degradation

3’ end –> polyA

• prevents degradation
• export from nucleus
• transcription + translation

Question 2

Give an overview of host cell protein synthesis

Answer 2

1. eIF4F recognises 5’ cap
2. eIF4F recruits the 43S ribosomal subunit
3. 43S scans the mRNA until AUG, then recruits 60S
4. Protein translated

Question 3

How is eIF4F involved in initation of protein synthesis?

Answer 3

1. Comprised of eIF4A, eIF4E + eIF4G
2. eIFG binds PABP + eIF3
3. eIF4E binds 5’ m7G cap
4. These interactions circularise the DNA making translation easier to perform multiple times over

Question 4

How is initiation negatively regulated?

Answer 4

By 4EBP1

• 4EPB1 normally bound to eIF4E
• when phosphorylated 4EB1 dissociates from eIF4E and eIF4E is free to interact with 5’ m7G cap
• translation is then initiated

Question 5

How is 4EPB1 regulated?

Answer 5

1. PI3K signalling
   - activates AKT
   - inhibits TSC
   - activates RHEB-GTP
   - activates mTORC1
   - phosphorylates 4EBP1
2. AMPK + hypoxia
   - activates TSC
3. Nutrient deprivation
   - activates AMPK

Question 6

How do viruses dysregulate 4EBP1?

Answer 6

1. Increase PI3K signalling
   - HCV, HPV16, EBV, HIV1, KSHV
2. Inhibit TSC
   - HSV1, CMV, HPV16
3. Increase 4EBP1 + eIF4E
   - VSV, PyV
4. Increase phosphorylated 4EBP1
   - EMCV

Question 7

How do viruses dysregulate eIF4F?

Answer 7

1. Cleavage of eIF4G –> separates ribosome complex from mRNA
   - Picornavirus 2A protease
   - FMDV leader protease
   - Calicivirus 3C protease
   - Retrovirus protease
2. Protein interactions with eIF4G
   - Influenza NS1
   - Adenovirus 100k
   - Rotavirus nsp3
3. Cleavage of PABP
   - Picornavirus 3C + 2A protease
   - Calicivirus 3C protease
   - Lentivirus protease
4. HSV vhs
   - mRNA-specific endoribonuclease degrades mRNA
5. Cap snatching –> cleave m7G cap off cellular mRNA + sequester it to translate viral mRNA
   - Vaccinia, Influenza, Hantavirus

Question 8

Explain regulation of translation by eIF2

Answer 8

1. critical role in re-cycling IFs
2. activated by eIF2B (guanosine nucleoside exchange factor)
3. eIF2-GDP –> eIF2-GTP
4. phosphorylation at Ser51 residue blocks interaction with eIF2B
   - GCN1 –> AA starvation + UV
   - HRIC –> haem deprivation
   - PERK –> ER stress
   - PKR –> dsRNA

Question 9

Go into more detail about PKR…

Answer 9

1. Protein kinase R
2. phosphorylates eIF2a
3. inhibits initiation of translation –> growth arrest
4. stimulated by IFN
5. activated by binding to dsRNA + dimerising (dont want it to be active all the time)
6. action can be inhibited by viruses
   - prevent dsRNA binding
   
   • Vaccinia E3L, Reovirus sigma3, Rotavirus nsp3, Influenza NS1
     - produce short dsRNA
   • Adenovirus, EBV, HIV, HCV (not RNA but NS5A)
     - acting as pseudosubstrate
   • Vaccinia K3L –> locks PKR in inacive formation
     - de-phosphorylating the inactive eIF2a
   • HSV gamma-34.5 –> recruits phosphatase

Question 10

How do viruses ensure translation of their own proteins?

Answer 10

Internal Ribosome Entry Site (IRES)

• Picornavirus, Flavivirus, Lentivirus
• mediate cap-independent translation
• reduced requirement for IFs
• 43S ribosome will bind directly to IRES

4 classes

• 1. poliovirus
• 3. HCV (no requirement for eIF4F)

Norovirus –> has 5’ VPg instead of IRES

• protein cap analogue
• recruits 43S ribosome complex
• no ribosome scanning

Question 11

What factors are involved in initiation of translation? (ie, summarise the lecture

Answer 11

1. eIF4F
   
   • viral cleaage of eIF4G
   • viral interaction with eIF4G
2. 4EBP1
   
   • regulation via cell signalling
   • viral interaction with cell signalling
3. cap snatching
4. eIF2
   
   • regulation
   • phosphorylation by PKR in particular
5. PKR
   
   • viral interaction
6. IRES + VPg