Interferon

Question 1

How does the immune system respond to virus?

Answer 1

At start of an infection you have an onslaught of viruses, first barrier is mucous membrane, mucus, skin = Intrinsic immunity.
2nd barrier would be innate immunity, no specificity, phagocytes, cytokines, NK, will suppress virus replication may not feel negative effects of response.
If virus replication is uncontrolled in first few hours, you need acquired immune response, B T cells.

Question 2

What are viruses?

Answer 2

Masters of evolution

L0000000000L

Question 3

What are CpG and ZAP?

Answer 3

Many pathogens have a different ratio of nucleotides in their genomes than we do. Too much/high load CpG is recognised foreign.
ZAP detects it while virus trying to affect your cells and transports the genome to a RNA exosome where it’s digested.
Non-specific recognition of generic difference
(CpG is just how many CG sequences there are in the genome)

Question 4

What are interferons?

Answer 4

A soluble protein that spreads and binds to specific receptors on neighbouring cells and signals activation of de novo transcription of hundreds of Interferon stimulated genes that are fairly toxic.
Makes these cells into an antiviral state.
Produced by fibroblasts and monocytes

Question 5

What are type 1 interferons?

Answer 5

Switch on first and earliest

Polypeptides secreted from infected cells and there are type 1 interferon receptors on all cells in the body.

Question 6

What are the 3 major functions of interferon 1?

Answer 6

Induce antimicrobial state in infected and neighbouring cells, interferon stimulated genes
(Can signal on cell secreted by)
Modulate innate response to promote Ag presentation and NK cells (upregulation)
Activate the adaptive immune response, better antibody response
Primary role is first point.

Question 7

What is the primary interferon?

Answer 7

Interferon beta

Any cell in your body can make it and respond to it.

Question 8

What is the primary interferon secreted by macrophages and dendritic cells?

Answer 8

Interferon alpha

Question 9

What receptor does interferon beta signal through?

Answer 9

IFNAR receptor which is present on all tissues

Question 10

What is primary signalling molecule that switches on production of interferon beta?

Answer 10

IRF-3

Question 11

What are plasmacytoid dendritic cells?

Answer 11

Specialist IFN alpha secreting cells as they express high levels of transcription factor IRF-7

Question 12

What are the genetic differences between interferon beta and alpha?

Answer 12

One gene for beta

There are 13 to 14 interferon alphas which are all subtly different.

Question 13

What is type 2 interferon?

Answer 13

IFN gamma
Produced by activated T cells and NK cells
Signals through a different receptor IFNGR

Question 14

What is type 3 interferon?

Answer 14

IFN lamda
Recently described 15 years
Signals through IL28R and IL10B that are mainly present on epithelial surfaces.
Important in early events of viral infections that go through resp. tract and hepatic viruses
The receptor is present on the basolateral surface of epithelial cells in resp. and liver.
Imporved outcomes in Hep have been determined by polymorphisms in IFN lamda/receptor with both spontaneous clearance and response to anti-viral therapy.

Question 15

How do we differentiate non-self and self?

Answer 15

PAMPs - Pathogen associated molecular pattern - usually a piece of foreign nucleic acid
No DNA in cytoplasm so this existence may indicate invasion
PRRs find DNA in wrong place and elicit a response

Cytoplasmic RIG-I like receptors RLRs
Toll like receptors TLRs
Cytoplasmic nucleotides oligomerizatoin domain receptors NLRs

Sensors that see RNA, DNA, RNA in endosomes

Question 16

How does interferon induction work?

Answer 16

Cell just infected, virus replicating and producing single stranded RNA in cytoplasm (no CAP polyA tail). RNA is sensed by RIG-I and/or mda-5, they bind the RNA and change conformation to allow binding to a downstream molecule MAVS(mitochondrial activator of virus signalling)stuck on mitochondrial membrane.
Complex formed from PRR, signalling molecule and mitochondrial membrane leads to conformation change in MAVS which signals to downstream cascades leading to phosphorylation in IRF-3 causing dimerisation and movement into nucleus, it’s a TF and now can attach to promoter region of INF-beta to transcribe some and upregulate INF-beta

Question 17

How do TLRs induce a response?

Answer 17

TLRs sit in endosomes, lots of RNA viruses use endosomes to uncoat and get into cells. Exposed viral RNA in an endosome can be picked up by TLR3/7/8.
Once bound to RNA ligands they signal downstream pathways, TLR3 is same as normal interferon induction.
However, TLR7/8 pathway is unique to plasmacytoid dendritic cells signal through Myd88, triggers phosphorylation of IRF7 which is constitutively expressed in plasmactyoid dendritic cells and switches on both IFN alpha and beta, burst of very strong response due to large production of IFNs.

Question 18

How is DNA sensed to induce a interferon response?

Answer 18

Sensed by cGAS that signals through STING.
DNA viruses - herpes, pox viruses.
Need to pick up DNA in the cytoplasm of the cell.
cGAS is an enzyme which is activated by binding of dsDNA in the cytoplasm and makes a second messenger cGAMP (dinucleotide of G and A).
STING picks up the cGAMP on ER and acts as a signalling platform, switches on same phosphorylation cascade.

Question 19

What is IFN beta?

Answer 19

Small polypeptide which is synthesised by infected cells and secreted to bind to IFNalphaR on neigbouring cells.
Autocrine and paracrine signalling.
Soluble cytokine.
Switching on hundreds of genes in neighbouring cells unlike the infected cell where one gene activated.

Question 20

What are some genetic errors that can affect interferon response?

Answer 20

Monogenic inborn error of IRF-7
mutation in IFRNAR2 gene
mutation in IRF-3 gene - Herpes Simplex Encephalitis : Inborn errors in at least 6 genes in HSE impaired CNS intrinsic interferon alpha/beta response to HSV infection leading to uncontrolled virus replication in brain.

Question 21

How important is interferon?

Answer 21

Absolutely essential

Need it to deal with simple viruses we would otherwise be at large risk to.

Question 22

What happens after IFNs have been secreted?

Answer 22

Secreted by infected cell
Float around on outside cells until it lands on heterodimer IFNAR 1/2.
Another signalling cascade that involves JAK and STAT. Phosphorylation of STAT and dimerisation allowing entry of STAT into nucleus where it sits on promoter of hundreds of different genes, transcribed and translation of mRNA in cytoplasm, spectrum of 2-300 proteins.

Question 23

Give some examples of proteins produced by neighbouring cells.

Answer 23

Protein Kinase R - inhibits translation
2’5’Oligoadenylate synthase - activates RNAase L that destroys ssRNA
Mx - inhibits incoming viral genomes
ADAR - induces errors in viral replication
Serpine - Activates proteases
Viperin - inhibits viral budding

virus is dependent on YOUR machinery
transient shut down of protein synthesis may save the cell.
Local response

Question 24

What are 3 things that STAT activates?

Answer 24

a. Antiviral response (ISRE).
b. Inflammatory response (GAS).
c. Repressors of the inflammatory pathways (GAS).

Question 25

What is IFITM3?

Answer 25

Restrictrs virus entry through endosomes, fuses with endosome membrane, prevents endosome changing. Stops virus escaping so broken down by acidic pH
E.g. influenza can’t do it
People lacking IFITM3 have more severe influenza
It’s an interferon stimulated gene

Question 26

What are Mx1 and 2?

Answer 26

Mx1 and Mx2 – GTPases with a homology to dynamin:
Forms multimers to wrap around nucleocapsids of viruses.
Mx1 – inhibits influenza.
Mx2 – inhibits HIV.
All reliant on initial interferon response

Question 27

How is damage limited during antiviral state?

Answer 27

IFN response only maintained for several hours.
Ability to respond to IFN lost due to negative regulation
SOCS (suppressor of cytokine signalling) genes turn off response
Antiviral state is a harmful necessary response.

Question 28

What are different strategies that viruses use to evade IFN response

Answer 28

Hide PAMP
Interfere globally with host cell gene expression/protein synthesis
Block IFN induction cascade by destroying or binding
Inhibit IFN signalling
Block action of individual IFN induced antiviral enzymes
Activate SOCS
Replication that is insensitive to IFN

Question 29

Give some examples of interferon control by viruses

Answer 29

Hep C: NS3/4 protease acts as an antagonist to interferon induction by cleaving MAVS.
Influenza virus: NS1 protein acts as an antagonist to interferon induction by binding RIG-I/TRIM25/RNA complex and preventing activation of signalling pathway, prevents nuclear processing of newly induced genes by entering the nucleus and bind to TF to prevent transcription.

Question 30

How do Pox viruses prevent signal getting through?

Answer 30

DNA viruses have lots of space to deal with IFN and immune response.
They are called accessory genes - they don’t need these for replication just to combat our IS.
More than half of Pox genome is compromised of accessory genes that modify immune response.
An example: Pox encodes a soluble cytokine receptor binding to any interferon produced by infected cells.
Being developed for future autoimmue therapies e.g. RA where you have to many cytokines in order to mop it up

Question 31

What are general mechanisms of ebola to evade immune system?

Answer 31

Endoces proteins that stop it being seen/sensed reducing interferon production.
In neighbouring cells VP24 stops signalling in neighbouring cells

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.
Only has a small genome 7 genes 3 of which are dedicated to evasion L00000L

Question 32

Why do we get symptoms?

Answer 32

Consequence of innate immunity

Combination of damage of infected cells by virus and damage/infected and bystander cells by immune responses

Question 33

How does interferon interference skew immune response?

Answer 33

Immune response modulated for virus own replication and transmission.
Inadvertent pathology can result.
Proportions are all wrong too much IL-6 fever/lethargy
100 times more IFN needed for IL6 induction compared to Mx.

Question 34

What is a cytokine storm?

Answer 34

Cytokine storm – virus replication than induces high IFN accompanied by a massive surge in TNF-alpha and others.
o Differences in the clinical outcome may vary with age and health and can reflect vigour of innate immune system.
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.
The interferons and other factors are not successfully mediating the viral replication.

Question 35

Can IFN be used as an anti-viral treatment?

Answer 35

Associated with unpleasant side effects, feel worse than normal.

Question 36

Can IFN lambda be used as an influenza therapeutic?

Answer 36

IFN - influenza therapeutic drug:
IFN only stimulates an anti-viral state and
NOT an immune response and immunopathology.
Only stimulates response at epithelial surface to protect it.

Question 37

How can viruses be used as a new generation of live attenuated viruses?

Answer 37

Viruses deficient in control of IFN are attenuated in IFN competent cells.
The high IFN levels they induce can also recruit useful immune cells, IFN acting as an adjuvant.
E.g. removal of NS1

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.

Question 38

What is relationship between IFN system and cancer?

Answer 38

Cancer cells don’t make good IFN response. Replicate and kill cancer cells but not healthy cells. Opportunity to genetically engineer cancer oncolytic viruses.