3. Interferon Flashcards by Urooj Fatima

Errors in which genes can lead to increased fever and/or death?

Increased fever and/or death due to errors in:

- IRF-1 genes – results in increased interferon production meaning they feel very ill.
- IFNAR2 gene – this is an interferon synthesis receptor gene that is faulty so they don’t make interferon.
- IRF-3 gene – important in the stimulatory pathway to create interferon.

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What is the most common cause of sporadic encephalitis in the Western World?

In who is this most common?

Herpes simplex encephalitis

Most common in childhood – affecting previously healthy individuals on primary infection with HSV-1

Inborn gene errors implicated in HSE – TLR3, UNC93B1, TRIF, TRAF3, TBK1, IRF3.*
People that get infected with HSE normally have an effective INF response but sick HSE people may have inborn errors in the activation of this pathway.*

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What is interferon?

What is the effect of interferon binding to interferon receptors on cells?

“A transferable factor produced by exposure of eggs to a heat-inactivated influenza virus that protected new cells from infection”

“A soluble cytokine secreted from infected cells, which binds to specific receptors to stimulate transcription of interferon-stimulated gene”

Binds to specific receptors and signals activation of de novo transcription of hundreds of Interferon Stimulated Genes, ISGs

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What are type 1 interferons and what are their functions?

Polypeptides secreted from infected cells

Three major functions:

Induce antimicrobial state in infected and neighboring cells
modulate innate response to promote Ag presentation and NK
Activate the adaptive immune response

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What are the types of Type 1 interferons?

Type 1 - IFNa (IRF-7) and IFNb (IRF-3)

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Which interferons is produced first in a viral infection and what is this induced by?

IFNβ is secreted first and by ALL cells (IFNAR is the receptor) and production is induced by IRF-3

IFNβ is secreted by ALL CELLS and the IFNAR receptor is present on ALL tissues
IFNβ induction is triggered by IRF-3

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Name a cell type that is specialised for producing IFN alpha.

What do these cells express high levels of?

Plasmacytoid dendritic cells (pDC) are specialist IFNa-secreting cells and express high levels of IRF-7 constitutively

(pDC and IRF-7)

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How many genes are there for IFN alpha and IFN beta?

*Alpha** – 13/14 isotypes of genes
*Beta** – one

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What are type 2 interferons?

What are they produced by and what receptor do they use?

Type II Interferon = IFN γ

This is a much more specialist immune signalling molecule
It is produced by immune cells - activated T cells and NK cells
It signals through a different receptor - IFNGR (interferon-gamma receptor)

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What are type 3 interferons?

What receptors do they use and where are these present?

Type III Interferon = IFN λ

Signals through receptors: IL28R receptor and IL10β
These are mainly present on epithelial surfaces (e.g. respiratory epithelium and gut)
normally expressed during respiratory tract infections and liver infections.*

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What is the role of IFNlamba in the liver and what is the result of a polymorphism in the gene?

Important in the liver/at epithelial surfaces

- Polymorphisms in IFNlamba associated with the improved outcome from HCV and HBC both spontaneous clearance and response to antiviral therapy

Polymorphisms in the IFN lambda gene are associated with quite different outcomes from liver viruses (e.g. Hep B and Hep C) in terms of some people being able to spontaneously clear the virus if they have good IFN lambda responses
It is also important in terms of response to antiviral therapy

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How does the innate immune system differentiate self from non-self?

PRRs (pattern recognition receptors) on innate immune cells/ sit inside cells and detect PAMPs

PAMPs (pathogen-associated molecular patterns) – often sense foreign nucleic acids (e.g. dsRNA)

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Name receptors that are involved in detecting the presence of viruses and state where they are found.

- RLRs – RIG-I-like Receptors (cytoplasmic) - bind to Mavs (found on mitochondria) and stimulate signalling and IFN-b production - recognise viruses that uncoat within the cell

- TLRs – Toll-Like Receptors (endosomal) - found in endosomes and make IFNa - membrane proteins so recognise things coming into the cell

- NLRs – NOD (Nucleotide Oligomerisation Domain) Like Receptors (cytoplasmic)

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Describe RIG-I signalling.

RIG-I like receptors will recognise single-stranded RNA in the cytoplasm of the cell and it will signal through MAVS (mitochondrial)
This will signal further downstream, leading to generation of IFN-beta transcripts

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Describe TLR signaling.

TLR detects nucleic acids in the endosome (this isn’t normal)
It will signal to molecules outside the endosome (MyD88) and send various transcription factors to the nucleus
It will result in the switching on of expression of IFN alpha

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Describe DNA sensing.

Mainly done by cGAS

- This is an enzyme that binds to dsDNA in the cytoplasm and synthesises cGAMP (second messenger)

cGAMP diffuses to STING (found on endoplasmic reticulum)
This triggers phosphorylation of the same sets of transcription factors and signalling molecules the RNA viruses were triggering

Describe the structure of IFN receptors for IFN alpha and IFN beta

IFN receptors when activated are heterodimers of IFNAR1 and IFNAR2

and the IFNs are SOLUBLE cytokines and so can signal in a paracrine manner.

Describe the signalling from IFNAR receptors

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

1. IFNAR1 and AR2 dimerise and JAK1 and TYK2 cross-phosphorylate.
2. STAT proteins are activated and this activates:
- a. Antiviral response (ISRE).
- b. Inflammatory response (GAS).
- c. Repressors of the inflammatory pathways (GAS).

Give examples of interferon-stimulated genes

Interferons induce transcription of HUNDREDS of antiviral mediators – hence why you get a fever and feel sick.

Interferon stimulated genes include –

- PKR - protein kinase R: inhibits translation
- 2’5’OAS: activates RNAse L that destroys ssRNA
- Mx: inhibits incoming viral genomes
- ADAR: induces errors during viral replication
- Serpine: activates proteases
- Viperin: inhibits viral budding

What is 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

These sit on the membrane of endosomes, in cells that have been previously stimulated by IFN
It prevents fusion of the virus membrane with the endosomal membrane so the virus gets trapped in the endosome
NOTE: mice and people lacking IFITM3 get more severe influenza*

What are Mx1 and Mx2?

GTPases with a homology to dynamin
Mx can form multimers that wrap around nucleocapsids of incoming viruses – this nullifies the viral genomes

Mx1 – inhibits influenza
Mx2 – inhibits HIV

Describe the actions of Protein Kinase R.

When is PKR activated by cells?

It phosphorylates the alpha subunit of eIF2 (initiation factor) that is important in translation
This prevents ribosomes from binding to mRNA so no new genes will be translated
It also phosphorylates NFkB, which is an important transcription factor that is part of the interferon and inflammatory response

When is PKR activated by cells? - It is an extreme measure and a last resort – only activated when the cell has no other option - if they don’t switch on these genes, the cells will be infected by the virus and the virus could kill the cell

Name a family of genes that suppress the cytokine signalling and turn off the response.

SOCS

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.
SOCS genes suppress the cytokine signalling and turn off the response - if SOCS genes are switched on, even if the IFN is bound to the receptor the signals won’t get through and no new PKR will be made

What is viral evasion mediated by?

State some mechanisms of viral evasion of the IFN response.

Viral evasion is mediated by:

- Virus avoid detection by hiding the PAMPS – e.g. inside vesicles.
- Interfere globally with host cell gene expression (or protein synthesis).
- Block IFN induction cascades (by destroying or binding) → NS3/4 protease (HCV)
- Inhibit IFN signalling directly → NS1 protein (influenza)
- Block action of individual IFN-induced antiviral enzymes.
- Activate SOCS.
- Replication strategy that is insensitive to IFN.

Give examples of viral evasion

* **- Hepatitis C** – NS3/4 proteases – cleave Mavs – Interferes with the induction of IFN. * **- Influenza** – NS1 protein – binds to RIG-I/TRIM25/RNA complex and prevents activation of the 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) → 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 signaling.

Explain how hepatitis C controls the interferon response.

**_NS3/4_** - This is a protease that cleaves MAVS - interferes with induction of IFN - MAVS is important in detecting Hep C through the RIG-I pathway - So Hep C is not detected

Explain how influenza controls the interferon response.

**_NS1_** - Acts an antagonist to interferon induction by binding to the RIG-I/TRIM25/RNA complex and preventing activation of the signalling pathway - It also prevents nuclear processing of newly induced genes - NS1 also migrates to the nucleus where it prevents the export of newly synthesised genes

What type of virus are Pox and Herpes viruses? What do Pox viruses encode that helps deal with the interferon response? Describe a potential therapeutic use of this feature

**Pox and herpes viruses:** large DNA viruses **Pox viruses:** they encode soluble cytokine receptors that mop up IFN and prevent it from reaching its receptors **Potential therapeutic use:** this could be useful in autoimmune or inflammatory conditions where IFN and other cytokines are produced in abundance

What are proteins produced by Ebola virus that are particularly important in dealing with the immune response? What do these proteins do?

**_VP35_** – inhibits the RIG-I pathway → blocks RIG-I Like complexes and RNAi expression **_VP30_** – blocks RNAi expression **_VP24_** – directly blocks IFN signalling → stops the signal getting through from the IFN beta receptor to the nucleus (stops the STAT1 molecule from getting to the nucleus)

What are the consequences on the body of viral evasion?

**The consequences on the body are damage of cells by the virus and by the immune response itself.** * - Viruses modulate the immune response to suit their own replication/transmission which results in pathology. * - The effects of IFN can vary from pathologic → immunopathology (depending on how much IFN is made). * - 100x more IFN is required for IL-6 induction (causes fever) than for Mx induction. * - Thus, more IFN stimulates a more severe response (up to a cytokine storm).

What is the result of a cytokine storm and which cytokines are involved? What does the clinical outcome depend on?

**Cytokine storm – virus replication than induces high IFN accompanied by a massive surge in TNFa and others.** _Differences in the clinical outcome_ * - may vary with age and health – so the less ill you get (healthier), paradoxically, the more severe the clinical outcome as you’re better at producing IFN. * - e.g. dengue haemorrhagic fever, Ebola and severe influenza. _Cytokines involved_ * – TNF, IFN(a/b), IFN(gamma), IL-1, IL-6, CCL2 * - result in endothelial dysfunction, inflammatory responses, and pulmonary fibrosis.

How are live attenuated vaccines created and what are their uses?

**_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.

What are the uses of anti-viral treatments and IFNlambda?

**_Anti-viral treatments:_** - IFN can be used as a treatment (e.g. HCV) but has unpleasant side effects **IFNlambda - influenza therapeutic drug:** - IFNlambda only stimulates an anti-viral state and NOT an immune response and immunopathology - Receptors for IFN lambda are only found on epithelial surfaces (the site of infection of influenza is respiratory epithelium - IFN lambda cannot signal through immune cells and cause immunopathology - It will only induce an antiviral state in the epithelial cells

How can viruses be used for cancer treatment? Explain how oncolytic viruses would work.

**_Cancer treatment:_** - 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. **_Oncolytic viruses:_** - Viruses are engineered that can uniquely replicate in tumour cells and kill them - Generally speaking, cancer cells are deficient in their ability to mount a proper interferon response - So, a virus that is unable to control the IFN response will NOT be able to replicate in normal healthy cells but they will be able to infect and replicate in cancer cells

**SBA: Which answer is _not_ true:** **Viruses that cannot control the innate immune system well might...** A. be useful as oncolytic agents B. be difficult to grow in standard cell culture systems C. be restricted at crossing the host range barrier and unlikely to spark outbreaks in other species D. be useful as live-attenuated vaccines E. be highly adapted to their host species

E. be highly adapted to their host species

**SBA: Which answer _is_ true?** **Viruses counteract the activation of the innate immune system by:** A. varying their coat protein sequences B. encoding proteins that cleave or target host immune factors for degradation C. preventing the loading of peptides by TAP D. inducing a cytokine storm E. encoding MHC homologues

B. encoding proteins that cleave or target host immune factors for degradation