Interferon

Question 1

What is the most common cause of sporadic encephalitis worldwide and describe the epidemiology of this?

Answer 1

Herpes simplex encephalitis

• prevelance is 1/10,000

Question 2

Which subset of the population is herpes encephalitis most common in?

What are the implications of this

Answer 2

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

Inborn errors in at least 6 genes are implicated in HSE: TLR3, UNC93B1, TRIF,

TRAF3, TBK1 and IRF3

It impairs the CNS’ intrinsic interferon alpha/beta response to HSV infection

The virus replicates to a much higher extent than it would otherwise have done

Question 3

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

Answer 3

Transferrable factor produced when the cells are exposed to virus.

It binds to specific receptors and signals the de novo transcription of hundreds of interferon stimulated genes (ISG)

Question 4

What are the three functions of type I interferons?

Answer 4

1. Induce antimicrobial state in infected and neighbouring cells
2. Modulate innate immune response to promote antigen presentation and NK cells but inhibit proinflammation
3. Activate the adaptive immune response

Question 5

What are the type I interferons ?

Answer 5

Polypeptides secretted from infected cells

IFN alpha and IFN beta

Question 6

What is the first interferon to be produced in a viral infection and what does this lead to?

Answer 6

Cells will sense a viral infection and make an interferon response that will result in the synthesis of new copies of IFN-beta (the first interferon to be made)
The IFN beta is secreted from these cells, from where it can diffuse and interact with receptors on neighbouring cells

This will lead to the switching on of genes in neighbouring cells to switch them into an anti-viral state
Plasmacytoid dendritic cells (PDCs) are specialised cells that are very good at making interferon (in particular IFN-alpha)

The secretion of type I interferon will recruit APCs and adaptive immune cells such that you amount an adaptive immune response

Question 7

Which cells produce IFN beta?

Answer 7

All cells produce IFN beta and all tissues have IFNAR receptors

Question 8

What is IFN beta induction triggered by?

Answer 8

IFNβ induction is triggered by IRF-3

Question 9

Name a cell type that is specialised for producing IFN alpha. What do these cells express high levels of?

Answer 9

Plasmacytoid dendritic cells are specialised IFNα secreting cells - they express high levels of IRF-7 constitutively

Question 10

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

Answer 10

Alpha – 13/14 isotypes

Beta – ONE

Question 11

Which IFN comes under type II interferon?

Which cell types produce this IFN?

Which receptor do these IFNs signal through?

Answer 11

IFN-gamma - specialist immune signalling molecule

Produced by activated T cells and NK cells

It signals through a different receptor - IFNGR (interferon gamma receptor)

Question 12

Which IFN falls under type III IFN?

Answer 12

IFN-lambda

Question 13

Which receptors do type III IFNs signal through?

Where are these receptors mainly present?

Answer 13

Signals through receptors: IL28 receptor and IL10β

Epithelial surfaces E.g. respiratory epithelium and gut.

This is thought to the be the IFN that protects the barriers of your body e.g. respiratory epithelium and gut

Question 14

Which organ is IFN lambda very important in and what is the evidence for this?

Answer 14

Liver

We know that it is important because 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

Question 15

How does the innate immune system recognise non-self?

Answer 15

PRRs (pattern recognition receptors) on innate immune cells recognise

PAMPs (pathogen-associated molecular patterns) NOTE: they often sense nucleic acids

Question 16

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

Answer 16

RIG-I like receptor (RLRs) – cytoplasmic

Toll-like receptors (TLRs) – plasma membrane + endosomal membrane

There are also cytoplasmic nucleotide oligomerisation domain receptors (NLRs)

Question 17

Describe RIG-I signalling.

Answer 17

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

The PRRs (e.g. RIG-I like receptors) will detect PAMPs such as single stranded RNA in the cytoplasm of the cell

RIG-I will then signal through Mavs (located on the mitochondrion), which will then trigger signalling through various different pathways that result in the translocation of molecules from the cytoplasm to the nucleus

These transcription factors will become phosphorylated, they will bind to the promoter regions of target genes (in this case IFN beta) and it will generate IFN beta transcripts

The IFN beta will then be released from these cells and it will travel to neighbouring cells to induce an anti-viral state

This is a way of the host controlling the amount of virus in the body

Question 18

Describe TLR signalling.

Answer 18

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

The virus enters the cells and at some point in their life cycle they will find themselves inside an endosome and their nucleic acids will be exposed inside the endosome

In a normal healthy cell, there shouldn’t be any nucleic acids inside the endosomes

The TLRs can sense the nucleic acids in the endosome and it will signal to a molecule outside the endosome (MyD88) that will then send various transcription factors to the nucleus of the cell

It will result in the switching on of expression of IFNα

Question 19

Describe DNA sensing.

Answer 19

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.

Single stranded RNA is a PAMP

If the RNA doesn’t look like normal host RNA then it will be detected

cGAS is the main way that DNA viruses are sensed

cGAS is an enzyme that binds to dsDNA in the cytoplasm and it synthesises a

second messenger - cGAMP

This small dinucleotide then diffuses to a protein called STING, which is found

on the endoplasmic reticulum

This then triggers the phosphorylation of the same sets of signalling molecules

and transcription factors that the RNA viruses were triggering

STING is a central player in IFN induction through cGAS

NOTE: Mavs is found on the mitochondrion

Question 20

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

Answer 20

They are heterodimers of IFNAR 1 and IFNAR 2

The IFN receptors are heterodimers of IFNAR1 and IFNAR2

On binding to the cell surface receptor, the interferon signals to the nucleus to

switch on the transcription of a whole set of interferon stimulated signals

Question 21

Describe the signalling from IFNAR receptors

Answer 21

IFN binds and the IFN receptor activates Jak and Tyk, which goes on to phosphorylate the STAT molecules STAT molecules dimerise and combine with IRF-9 It then goes to the nucleus, binds to a promoter and regulates transcription

There is a heterodimeric receptor - composed of IFNAR1 and IFNAR2

This IFN receptor is present on the surface of ALL cells in the body

It is capable of sensing IFNα and IFNβ

If the IFN binds to the IFN receptor, it

will activate Jak and Tyk which then goes on to phosphorylate the STAT molecules (STAT1 and STAT2)

The STAT molecules dimerise and combine with IRF9 - it then goes to the nucleus and binds to a promoter region that is responsive to that transcription factor

NOTE: we don’t need to learn the names of all the molecules in the pathway

Question 22

What is IFITM3?

Answer 22

Interferon-induced transmembrane protein 3 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

IFITM3 = interferon induced transmembrane protein 3

These proteins sit on the membrane of endosomes, in cells that have been previously stimulated with interferon

If a flu virus or dengue virus tries to enter cells it will normally pass through the endosomal pathway and fuse its membrane with the endosomal membrane and release its contents into the cytoplasm

If IFITM3 is being expressed then the virus cannot do that

The virus gets trapped in the endosome because the IFITM3 modifies the membrane of the endosome and prevents the virus from being able to fuse with the endosomal membrane and release its genome into the cell

mIce and people lacking IFITM3 get more severe influenza

Question 23

What are Mx1 and Mx2?

Answer 23

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

Antiviral Mediators: Mx1 and Mx2

GTPase with a homology to dynamin

Mx can form multimers that wrap around the nucleocapsids of incoming

viruses

Mx1 = inhibits influenza

Mx2 = inhibits HIV

Question 24

Describe the actions of Protein Kinase R.

Answer 24

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.

It phosphorylates the alpha subunit of eIF2 which is important in translation

If you permanently phosphorylate the alpha subunit of eIF2 it means that the ribosomes can’t bind onto the mRNA and translate any new genes

So when PKR is activated in a cell, no new genes will be translated - this is a pretty extreme measure to take PKR also phosphorylates NFκB, which is an important transcription factor that is part of the interferon and inflammatory response PKR is an extremely toxic thing to switch on - it has such an intricate control mechanism because the cell can’t tolerate making these gene products all the time

The cells only express PKR when they have no choice - if they don’t switch on these genes, the cells will be infected by the virus and the virus could kill the cell

Question 25

When is PKR activated by cells?

Answer 25

It is an extreme measure and a last resort – only activated when the cell has no other option

Question 26

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

Answer 26

Antiviral state does not last

IFN response may only be maintained for several hours

Subsequently the ability to respond to IFN is lost due to negative regulation

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 wont get through and no new PKR will be made

Question 27

State some mechanisms of viral evasion of the IFN response.

Answer 27

• Avoid detection by hiding the PAMP
• Interfere globally with host cell gene expression and/or protein synthesis
• Block IFN induction cascades by destroying or binding
• Inhibit IFN signalling
• Activate SOCS
• Replication strategy that is insensitive to IFN

Question 28

Explain how hepatitis C controls the interferon response.

Answer 28

NS3/4 This is a protease that cleaves MAVS MAVS is important in detecting Hep C through the RIG-I pathway So Hep C is not detected

Hepatitis C - NS3/4

This is a protease that acts as an antagonist to interferon induction by

cleaving MAVS

MAVS is important in detecting Hep C through the RIG-I pathway
It causes destruction of the sensor system
Normally, Hep C entering a cell will be detected by RIG-I receptors - this will then signal to MAVS, which will then switch on the IFN response
But Hep C rapidly synthesises NS3/4, which cleaves MAVS away from the mitochondrion and prevents the signal from getting through

Question 29

Explain how influenza controls the interferon response.

Answer 29

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

Influenza - NS1

Acts as an antagonist to interferon induction by binding to RIG- I/TRIM25/RNA complex and preventing activation of the signalling pathway
So it acts at an early stage in terms of detection of viral RNA

It also prevents nuclear processing of newly induced genes
Normally, influenza will be triggering RIG-I via the production of viral RNA in the cytoplasm, which would normally signal to MAVS but the NS1 protein in the influenza binds to the RIG/Trim25 complex, which is detecting the viral RNA, and stops it from triggering this pathway
The NS1 also migrates to the nucleus where is prevents the export of newly synthesised genes

Question 30

What type of virus are Pox and Herpes viruses?

Answer 30

Large DNA viruses

Question 31

What do Pox viruses encode that helps deal with the interferonresponse?

Answer 31

Pox viruses encode soluble cytokine receptors (vaccinia virus B18) which mops up IFN and prevents it from ever reaching its receptor

This could be useful because in some autoimmune and inflammatory conditions, IFN and other cytokines are produced in abundance and contribute to the pathology of the condition

So these naturally evolved virus modifiers are thought of as potential therapies for things like autoimmunity

Question 32

Describe a potential therapeutic use of this feature

Answer 32

This could be useful in autoimmune or inflammatory conditions where IFN and other cytokines are produced in abundance

Question 33

Name two proteins produced by HIV that helps deal with restriction factors and state what they target.

Answer 33

Vif – APOBEC

Vpu – Tetherin

Question 34

Describe the normal action of APOBEC.

Answer 34

APOBEC is involved in the innate immune resistance to retroviruses and hepadnaviruses

• APOBEC modifies some of the nucleotides in reverse transcription and makes them into the wrong version
• APOBEC deaminates dC to dU in the minus strand of viral cDNA during reverse transcription This leads to G to A hypermutation resulting in ERROR CATASTROPHE
• This results in so many mutations that the viral genome becomes nonsense and the virus can’t replicate

Question 35

What is the effect of Vif on APOBEC?

Answer 35

Vif counteracts the activity of APOBEC and targets it for degradation This removes the interference of APOBEC with reverse transcription

Question 36

Describe the normal action of tetherin.

Answer 36

Tetherin sits on the cell surface of infected cells and binds to viruses that try to escape the cell to go and infect other cells This limits the spread of viral infection

Tetherin sits on the cell surface of virus infected cells and as the virus tries to escape to go and infect other cells, the tetherin grabs hold of the virus and prevents it from being released from the infected cell

This is a mechanism by which viral infection can be limited
Vpu pulls tetherin back from the cell surface and targets it for degradation and gets rid of it

Question 37

What is the effect of Vpu on tetherin?

Answer 37

Vpu pulls tetherin back from the cell surface and targets it for degradation

Question 38

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

Answer 38

Ebola is very good at counteracting our immune response

It encodes TWO proteins that are particularly important:

• *VP35** - inhibits the RIG-I pathway
• *VP24** - stops the signal from getting through from the IFN beta receptor to the nucleus (it stops the STAT1 molecules from getting to the nucleus)

The production of these virus proteins means that the virus can continue to replicate unchecked and accumulate to incredibly high titres because the patient is unable to amount a proper immune response

Question 39

What do these proteins do?

Answer 39

VP35 – inhibits the RIG-I pathway VP24 – stops the signal getting through from the IFN beta receptor to the nucleus (stops the STAT1 molecule from getting to the nucleus)

Question 40

What two techniques can be used to observe the skewing of the immune response by viruses?

Answer 40

Transcriptomimics – shows changes in mRNA production Proteomimics – shows changes in protein expression

Many viruses modulate the immune response, presumably to increase their own replication and transmission

This can result in inadvertent pathology

The effect of interferon can vary from protective to immunopathologic (e.g. if

you start making too much of it)

This may depend on how much interferon is made - 100 times more IFN is

required for IL-6 induction than for Mx

Transcriptomimics reveal the skewing of the IFN response by different viruses -

it is a way of measuring how much mRNAs get switched up

RED - switched up

GREEN - switched down

Proteomics reveals viral control of

protein expression - this is a method of
counting the proteins that get made from the various transcripts

Question 41

Describe how viral infections can cause cytokine storm.

Answer 41

Lots of virus propagation –> lots of interferon being produced –> massive release of TNF alpha and other cytokines

The virus replicates and induces high levels of IFN accompanied by massive release of TNF alpha and other cytokines

The differences in clinical outcome may reflect vigour of the innate immune system, which may vary with age

This is typical of dengue haemorrhagic fever, severe influenza infections and ebola

So in other words, there is a lot of virus and a lot of cytokines (which is very damaging)

The cytokine storm will lead to pulmonary fibrosis, which is caused by the accumulation of immune cells in the lung spaces

Eventually the patient will succumb to the immune pathology rather than from direct damage from the virus

Question 42

What is a serious consequence of cytokine storm?

Answer 42

Pulmonary fibrosis – due to accumulation of immune cells in the lungs

Question 43

Explain why viruses that cannot control the interferon can be used as the next generation of live attenuated vaccines.

Answer 43

They will be able to infect the cells and it will replicate sufficiently to be able to mount an immune response but it wont replicate to the extent where it causes disease

Viruses deficient in the control of IFN are attenuated in IFN competent cells

This will mean that they induce high levels of interferon when they enter these

cells and strongly switch on the antiviral response

This is what you would like in a vaccine - a live virus that can be injected into

the host and wont replicate enough to cause disease but will replicate sufficiently such that other parts of the immune system (e.g. aquired immune response) can recognise it as being foreign and mount an immune response that can generate immunological memory

There is a downside to engineering the virus so that it can’t control the host’s interferon system - it is no longer a fit virus so it’ll be difficult to propagate the virus sufficiently to produce enough virus for loads of vaccines

As the virus has been debilitated in its ability to control the IFN response, it will NOT grow very well in healthy cells

Solution - culture cells are genetically engineered to be deficient in the IFN response

This means that the deficient virus can replicate in the deficient cells

Question 44

The downside of this feature of the viruses is that these virus particles can’t be propagated in normal healthy cells. What is the solution to this issue?

Answer 44

Propagate the viruses in cells that are deficient in the IFN response

Question 45

Explain why interferons are not frequently used as an antiviral therapy.

Answer 45

They stimulate the production of several cytokines and this causes several unpleasant side effects

Question 46

What disease is IFN used to treat?

Answer 46

Hepatitis C (a combination of pegylated IFN is used with ribavirin

Question 47

Explain the reasoning behind using IFN-lambda as a treatment for influenza.

Answer 47

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

Question 48

Explain how oncolytic viruses would work.

Answer 48

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

MAIN CONCEPT: engineer a virus that can uniquely replicate inside cancer cells and kill them

As cancer cells accumulate mutations, they acquire mutations that lead to loss of pathways in their IFN system

So in general, cancer cells are deficient in their ability to mount a proper interferon response

So a virus that is UNABLE to overcome an interferon response will NOT be able to replicate in healthy cells but it WILL be able to replicate in the cancer cells

This virus can be injected into the tumour, it will replicate in the tumour cells

and kill these cells

It will be unable to replicate in surrounding cells because the healthy cells can

mount a powerful innate antiviral response against the virus

There are also attempts to make the virus even better (in the example above)

by making it express GM-CSF, which helps recruit killer cells to this tumour

Question 49

Answer 49