Viral Infections: Interferons

Question 1

Summarize the principles of immunity and define interferons

Answer 1

The immune response to virus:

1. Intrinsic: physical barriers, mucus, skin
2. Innate immunity
   
   • sometimes related to symptoms: fever
   • sometimes not even felt
   • acquired immunity: B cells and T cells

Intrinsic immunity:

• Pathogens: different ratio of nucleotides in their genomes - CpG and ZAP
• Sometimes for a pathogen, CpG is virulent but we sense it as normal

Interferons

• Discovered more than 50 years ago
• A transferable factor produced by exposure of eggs to heat inactivates influenxa virus that protected new cells from infection
• Binds to specific receptors and signals activation of de novo transcription of hundreds of interferon stimulates genes - makes these cells antiviral

Question 2

Interferon: list the types of interferon, explain how interferon synthesis is regulated recall the cell types that produce them

Answer 2

Types of Interferons

Type 1 interferons – polypeptides with 3 functions:

1. Induce antimicrobial state (local and adjacent cells).
2. Modulate innate response: promote Ag presentation (and NK) but inhibit pro-inflammation.
3. Activate adaptive immune response.

Primary interferon: interferon-beta is secreted and goes to neighboring cells and to fibroblasts

Dendritic cells - secrete interferon-alpha

• Type 1 - IFNa (IRF-7) and IFNb (IRF-3):
  
  • IFNb is secreted first and by ALL cells (IFNAR is the receptor) and production is induced by IRF-3.
    
    • One gene for IFNb.
  • Plasmacytoid dendritic cells (pDC) = IFNa-secreting cells and express IRF-7 constitutively.
    
    • 13-14 isotypes of genes for IFNa.
• Type 2 - IFNg:
  
  • controls immune response (communicates with NK cells)
  • Produced by activated T-cells and NK cells.
    
    • Signals through a different receptor called IFNGR.
• Type 3 - IFNl:
  
  • Signals through receptors IL28R and IL10b (mainly present on epithelial surfaces).
    
    • So normally expressed during respiratory tract infections and liver infections.
    • Polymorphisms in IFNl are associated with IMPROVED outcomes from HCV and HBV with both spontaneous clearance and response to antiviral therapy.

Question 3

Outline the process of differentiation of self from non-self

Answer 3

Differentiation of Self from Non-Self

• PAMPs – Pathogen Associated Molecular Patterns – often sense foreign nucleic acids (e.g. dsRNA).
  
  • ​piece of foreign nucleic acid
• PRRs – Pattern Recognition Receptors – these sit inside cells and detect PAMPs.
  
  • RLRs – RIG-I-like Receptors (cytoplasmic).
    
    • Bind to Mavs (found on mitochondria) and stimulate signalling and IFN-b production.
  • TLRs – Toll-Like Receptors (endosomal).
    
    • Found in endosomes and makes IFNa.
  • NLRs – NOD (Nucleotide Oligomerisation Domain) Like Receptors (cytoplasmic).

some senses DNA or RNA or DNA in endosomes

Mechanisms

1.

1. When a viral RNA is sensed by sensors RIG-I OR mda-5 -> they bind to it -> they change shape
2. complex: pattern recognition receptor and pathogen cause MAVS to change shape -> ends up to the phosphorylation of one kinase - RF3
3. RF3 dimerises and goes onto the nucleus
4. RF3 is a promoter and switches on the production of interferon beta

2. Toll-like receptors in endosomes:

1. IF RNA virus exposes RNA in endosome. TLRs pick that up (TLR 3 and TRL 7 or 8)
2. They signal downstream pathway (TLR 3 activates MAVS etc)
3. TRL 7 or 8 pathways are linked to the dendritic cells - they signal to a different molecule - activate IRS7 response - interferon a and b
4. DNA is sensed by a cGAS (enzyme) that signals through STING (double stranded binds to it and activates it and activates enzyme c gamp and activates STING which sits on membrane of ER - (similar to mabs at mitochondria) and then turns on a cascade - swithces on Type 1 interferons
5. Interferon B then binds to neighbouring cells

Question 4

Explain the role of type I interferon-stimulated genes,

Answer 4

• Interferon B then binds to neighbouring cells
• Cells turns on hundreds of genes to make the cell stop working

IFN Type 1 Signalling

This could be an example of an adjacent cell responding to IFN.

1. IFNAR1 and AR2 dimerize and JAK1 and TYK2 cross-phosphorylate.
2. STAT (transcription promoter) proteins are activated and this activates…
   
   1. Antiviral response (ISRE).
   2. Inflammatory response (GAS).
   3. Repressors of the inflammatory pathways (GAS).

• Interferons induce transcription of HUNDREDS of antiviral mediators – hence why you get a fever and feel sick.
• Interferon stimulated genes include –
  
  • PKR - inhibits transcription (lethal)
  • Mx, IFITM3 (stops endosomes from being able to fuse -therefore influenza which enters through the endosomes cannot do that - people who don’t have that get more severe influenza), miRNAs, ADAR, apoptosis, cell cycle arrest, etc.

Interferon Response – E.G. IFITM3 and Mx1/Mx2:

• IFITM3 – Interferon Induced Transmembrane Protein 3:
  
  • Restricts virus entry through endosomes by stopping them escaping so the virus is broken down by the acidic pH.
• Mx1 and Mx2 – GTPases with a homology to dynamin:
  
  • Forms multimers to wrap around nucleocapsids of viruses.
    
    • Mx1 – inhibits influenza.
    • Mx2 – inhibits HIV.

Question 5

Recall examples of viruses controlling or evading type I interferon

Answer 5

Viral Evasion of IFN Response

• The IFN response cannot last (maintained for a few hours) and the ability to respond to IFN is lost gradually due to negative regulation – SOCS (Suppressor of Cytokine Signalling) genes turn OFF the IFN response.​

Influenza

Viral evasion is mediated by:

• H Virus hides the PAMPS – e.g. inside vesicles.
• I Interference with host cell gene expression (or protein synthesis).
• B Block IFN induction cascades.
• I Inhibit IFN signaling directly.
• B Block action of individual IFN-induced antiviral enzymes.
• A Activate SOCS.
• R Replication that is insensitive to IFN.
• Examples of viral evasion:
  
  • Hepatitis C – NS3/4 proteases – cleave Mavs – Interferes with induction of IFN.
  • Influenza – NS1 protein – binds to RIG-I/TRIM25/RNA complex and prevents activation of signalling pathway and prevents nuclear processing of newly induced genes.
  • POX (and herpes) viruses:
    
    • More than half of the pox virus genome is comprised of accessory genes that can modify the immune responses.
    • Pox viruses encode soluble cytokine receptors (vaccinia virus B18) à future immune therapies.
  • Ebola – evades the IFN response by VP35, VP24 and VP30:
    
    • VP35 – blocks RIG-I Like complexes and RNAi expression.
    • VP30 – blocks RNAi expression.
    • VP24 – directly blocks IFN signalling.

Answer 6

Hepatitis C virus: NS3/4 protease acts as an antagonist to interferon induction by cleaving MAVS away from mitochondria membrane

Influenza virus: NS1 protein acts as antagonist to interferon induction by binding to RIG-I /TRIM25/RNA complex and preventing activation of signalling pathway, and also prevents nuclear processing of newly induced genes.

Pox viruses prevent the signal getting through

• Pox viruses and herpes viruses are large DNA viruses
• More than half the pox virus genome is comprised of accessory gees that modify immune response.
• Pox viruses encode soluble cytokine receptors (vaccinia virus B18), that are being developed as possible future immune therapies

Ebola virus cycle and immune evasion mechanism - stops it from being seen and hence inadequate interferon is being produced

By interfering with interferons, viruses skew the immune response

• Many viruses modulate the immune response, presumably to increase their own replication and transmission.
• This can result in inadvertant pathology.
• The effects of interferon can vary from protective to immunopathologic. This may depend on how much interferon is made- 100 times more IFN is required for IL-6 induction than for Mx.

The cytokine storm: innate immunopathology of virus infections - histological signs of fibrosis

Question 7

Explain how this can direct new therapy development

Answer 7

IFN as an antiviral treatment

• IFN as a treatment (HCV, pegylated IFN often used with ribavirin)
• Associated with unpleasant side effects

1. Anti-viral treatments:
   
   • IFN can be used as a treatment (e.g. HCV) but has unpleasant side effects.
2. IFNl - influenza therapeutic drug:
   
   • ​protect epithelial cells
   • IFNl only stimulates an anti-viral state and NOT an immune response and immunopathology.
3. Live attenuated vaccines:
   
   • Creation – viruses deficient in control of IFN are attenuated in IFN competent cells.
     
     • Cells naturally or engineered to be deficient in IFN response can be used to grow these attenuated virus strains.
   • Use – high IFN levels they induce can recruit useful immune cells with IFN acting as an adjuvant.
4. Cancer treatment:
   
   • oncolytic viruses as cancer therapies because cancer cells do not make interferons
   • Cancer cells may be deficient in IFN and so if a cancer patient is given a novel virus, the virus can kill the cancer cells whilst the healthy cells produce interferon to combat the virus.

Question 8

summarise the canonical type I interferon signaling pathway