3 - Interferon (05.03.2020) Flashcards
What are the barriers to viral infection?
- Intrinsic immunity (barriers)
- innate immunity (not specific, this is what makes you feel sick)
- acquired immunity (needed for clearance)
Intrinsic immunity: CpG and ZAP
- if a virus has high levels of CpG this is detected by ZAP which activates RNA exosome to degrade viral RNA and prevent replication
- could be used to make attenuated live vaccines! (engineer viruses to have too much CpG and then the immune system would catch them more)
- not specific, just recognises something very generic
ZAP
zinc-finger antiviral protein
CpG
C followed by G in the genome
What is interferon?
- a substance that is produced by cells infected with a virus and has the ability to inhibit viral growth
- Binds to specific receptors and signals activation of de novo transcription of hundreds of Interferon Stimulated Genes, ISGs (warns the cells that there is a problem in the surrounding and that they should prepare for a virus)
- active against many different viruses
ISG
interferon stimulated genes
Type 1 interferon
- switch on earliest/first
- T1 interferon receptors are found on all cells int he body
- Polypeptides secreted from infected cells
- Three major functions:
- Induce antimicrobial state in infected and neighbouring cells
- modulate innate response to promote Ag presentation and NK
- Activate the adaptive immune response
= antiviral state + recruit troops
- IFN beta is secreted by all cells and IFNAR receptor is present on all tissues. IFNb induction is triggered by IRF-3.
- Plasmacytoid dendritic cells pdcs are specialist IFN a secreting cells. They express high levels of IRF-7 constitutuvely.
- One gene for IFN b, 13/14 isotypes of IFNa.
- primary T1 interferon is interferon beta
Which interferon do dendritic cells and macrophages mainly secrete?
interferon alpha (macrophages also secrete interferon beta)
What are the types of interferon?
Type I IFNs are IFN alpha and IFN beta
Type II IFN is IFN gamma
Type III IFN is IFN lambda
What receptor does type 1 IFN bind to?
IFNAR (present on all tissues)
Type 2 IFN
Type II IFN is IFN gamma
- Produced by activated T cells and NK cells
- Signals through a different receptor IFNGR.
Type 3 IFN
Type III IFN is IFN lambda
- Signals through receptors IL28R and IL10b that are mainly present on epithelial surfaces -> doesn’t affect immune cells
- important in the early events of viral infection going through respiratory surfaces and the liver
- e.g. outcome of HepB/C is influenced bu IFN-lambda polymorphism
How many IFN alpha / betas are there?
1 beta
around 13/14 isotypes for IF alpha
How do cells differentiate self from non-self?
- Pathogen Associated Molecular Patterns, PAMPs
- Pattern Recognition Receptors, PRRs
- Often sense foreign nucleic acid
- cytoplasmic RIG-I like receptors RLRs, endosomal Toll like receptors TLRs
- Cytoplasmic nucleotide oligomerization domain receptors NLRs
Pathogen sensors detect viral nucleic acid
=> PRRs
Examples:
- TLR - endosomal
- RLR (Rig-1 like Receptor) - RIG1, MDA5, LGP2 - cytoplasmic
- NLR - cytoplasmic nucleotide oligomerization domain receptors
- DNA sensors (cGAS)
Interferon induction via the RIG pathway
- RIG1 and/or MDA5 are in the cytoplasm
- if they see RNA that is wrong (e.g. no cap or poly A tail) they bind to it and change conformation
- then binds to MAVS (mitochondrial activator of viral signal), this is stuck on the mitochondrial membrane
- downstream signals and cascades follow
- phosphorylation of IRF-3 -> dimerises and moves to the nucleus where it is the TF for IFN-beta
What pathways can induce interferon production?
- RLR (RNA ins cytoplasm, all cells have this)
- TLR (RNA in endoscope, mainly in plasmacytoid dendritic cells)
- cGAS (DNA)
Interferon induction via TLR
- TLRs sit in endosomes
- if there is viral RNA in an endosome this will be picked up by TLRs (TLR3,7,8)
TLR3:
- joins the sam pathway as RIG-1, causes phosphorylation of IRF-3 and the production of IFN-beta
TLR7 and TLR8
- signal through Myd88
- cause phosphorylation of IRF-7 (constitutively expressed in plasmacytoid dendritic cells)
- TF for production of IFN-alpha and IFN-beta
=> strong response burst with those 2 IFNs
This occurs mainly in more specialised cells (plasmacytoid dendritic cells
cGAS - STING pathway induction of interferon
- DNA is sensed by cGAS that signals through STING
- cGAS is am enzyme and it is activated by binding to dsDNA in the cytoplasm
- cGAMP acts as a second messenger binding to STING which sits on the membrane of the ER
- via TBK-1 there is increased phosphorylation of IRF-3 and increased production of Type-1 IFN
STING
stimulator of interferon genes
What kind of signalling does interferon beta do?
autocrine
paracrine
-> secreted by an infected cell and then binds to INFAR on that cell or on neighbouring cells
What triggers INF-beta induction?
phosphorylation of IRF-3 which is a TF for the IFN-beta gene
Summarise the effects of interferon beta production
- IFN-beta is produced and secreted by infected cells
- binds to INFAR (present on all cells)
- > autocrine and paracrine signalling
- de novo synthesis of around 300 or 400 INF stimulated genes
- this costs a lot of energy and can be quite toxic in terms of normal cell function so they have to be turned on and off quite rapidly.
Proof for the importance of IFN
- in mutations in genes in the pathway (e.g. IRF-7) you may not be able to fight off a viral infection
- might have a reaction to an attenuated vaccine
Herpes simplex encephalitis
- HSE is the most common cause of sporadic encephalitis in the Western world.
- Prevalence of 1/10,000.
- Most common in childhood, affecting previously healthy individuals on primary infection with HSV-1
Inborn errors in at least 6 genes implicated in HSE
- TLR3,UNC93B1,TRIF, TRAF3,TBK1, and IRF3.
- Impair CNS intrinsic interferon a/b response to HSV infection.
- uncontrolled viral replication in the brain -> encephalitis
Structure of INF R
heterodimer (e.g. IFNAR1 and IFNAR2)
What happens when INF binds to a receptor?
- IFN binds to IFN-R (e.g. INFAR)
- Jak and STAT are on the inside of the cell
- STAT phosphorylation and dimerisation
- STAT dimer enters the nucleus and binds to the promoter region of 100s genes that respond to the stat dimer
- transcription and translation occurs
- 300-400 proteins made
Which proteins are made in response to IFN binding?
- PKR Protein Kinase R: inhibits translation (viruses are dependant on the host machinery)
- 2’5’OAS: activates RNAse L that destroys ss RNA
- Mx: inhibits incoming viral genomes
- ADAR : induces errors during viral replication
- Serpine: activates proteases
- Viperin: inhibits viral budding
- IFITM3 (IFN induced transmembrane protein 3) restricts virus entry through endosomes, sits on endosomes and restricts them from fusing e.g. in flu.
=> very drastic response (better to kill the cell and block the virus)
IFITM3
=IFN induced transmembrane protein 3
- sits on endosomes and restricts them from fusing e.g. in flu
- restricts virus entry through endosomes
- mutation makes you more susceptible to influenza
- 25% Chinese and 50% Japanese carry it making them more susceptible to influenza (compared to <1 not so severe affected and around 6% hospitalised europeans)
- remember: this is an interferon stimulated gene
Antiviral mediators: Mx1 and Mx2
- GTPase with homology to dynamin
- Mx can form multimers which wrap around the nucleocapsids of incoming viruses.
- Mx1 inhibits influenza
- Mx2 inhibits HIV
- stops viruses from entering the nucleus
Self-regulation of antiviral response
- to limit damage
- IFN response may only be maintained for several hours
- Subsequently the ability to response to IFN is lost due to negative regulation
- SOCS suppressor of cytokine signalling genes turn off the response.
SOCS
- interferon regulated genes
- suppressors of cytokine signalling
How do viruses evade IFN response?
- Avoid detection by hiding the PAMP (e.g. replicate within membrane bound compartments)
- Interfere globally with host cell gene expression and/or protein synthesis (e.g. blocks transcribing)
- Block IFN induction cascades by destroying or binding
- Inhibit IFN signalling (e.g. STAT proteins)
- Block the action of individual IFN induced antiviral enzymes
- Activate SOCS prematurely
- Replication strategy that is insensitive to IFN (e.g. so fast before the cell has mounted the response)
Examples of interferon control by viruses: stopping activation
Hepatitis C virus:
- NS3/4 protease acts as antagonist to IFN induction by cleaving MAVS.
Influenza virus:
- NS1 protein acts as antagonist to IFN induction by binding to RIG-I /TRIM25/RNA complex and preventing activation of signalling pathway
- also prevents nuclear processing of newly induced genes.
accessory genes
- genes that the virus does not necessarily need to replicate inside us
- if it has them it is good at shutting the cell down and blocking the immune repsonse
- makes it more successful
- half of the pox virus genome is genes that modify the host immune repsonse
Pox virus - why is it successfu?
- 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 e.g. for AI disease
- this soluble receptor mops up signalling molecules so that they don’t bind to cells.
Ebola virus cycle and immune evasion mechanisms
- has ways of stopping being sensed.
- encodes VP35 and VP30
- > cell makes less INF
- VP24 stops INF from having its action in the nucleus and INF induced genes are not transcribed
- 3 of 7 or 8 genes in the ebola genome are dedicated to blocking the INF system
How many genes does the Ebola virus have?
- 3 of them are dedicated to blocking the INF system
- 7 or 8 (I think 7 according to a website with the. genome)
Consequences of innate immunity: Viral pathology
- A combination of damage of infected cells by virus and
- damage of infected and bystander cells by the immune response (saving whole body by local inflammatory response)
How do viruses skew the immune response by interfering with interferons?
- 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 IFN is made- 100 times more IFN is required for IL-6 induction than for Mx.
- IL-6 might make you feel sick and have a fever and may cause cytokine storm
Cytokine storm
- innate immunopathology of virus infections
- Virus replicates, induces high IFN accompanied by massive TNFa and other cytokines.
- Differences in clinical outcome may reflect vigour of innate immune system, which may vary with age.
- > more INF, more IL-6 does more damage to the cell in many organs than the virus would have done in the patient.
- This is typical of Dengue haemorrhagic fever, severe influenza infections and Ebola.
What do mediators of cytokine storm cause?
- leaky endothelium / endothelial dysfunction
- inappropriate inflammatory responses
- can cause pulmonary fibrosis after respiratory infections
Consequences of the balance between viruses and interferons
Host range barriers
Therapeutics
Vaccines
oncolytic viruses
IFN as an antiviral treatment
- IFN as a treatment (HCV, pegylated IFN often used with ribavirin -> now not used anymore because we have quite good directly acting antivirals)
- Associated with unpleasant side effects (you feel like you have flu)
IFN lambda as an influenza therapeutic?
- IFN-lambda only signals to receptors on epithelial cells
- by giving local IFN-lambda you protect the cells from viral infection
- smaller amount of cytokines etc. because not immune cells
- may be a better therapeutic in the longer run
Viruses that lack ability to control interferon as a new generation of live attenuated vaccines
- 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’.
- you would have to grow the flu cells lacking NS1 in an environment without INF (otherwise they don’t survive) and use them as a live attenuated vaccine)
- Cells naturally or engineered to be deficient in IFN response can be used to grow these attenuated virus strains.
IFN and cancer
- cancer cells don’t make a good IFN response
- genetically engineer viruses that kill cancer cells but don’t infect healthy cells
‘healthy cells make IFN and can control viruses. Cancer cells can’t make interferon and the viruses kill them’
