INTERFERONS

Interferons

• Interferons are proteins, immunologist prefer to call them cytokines– They are glycosylated

• The name originates from the fact that they interfere with viral infection• 2 types exist

– Type I (IFN-, 13 different alleles exist in an individual and IFN-)– Type II (IFN-)

• Cells producing IFNs– Plasmacytoid DCs (major producers of IFN- and IFN- )– Fibroblasts and epithelial cells– Macrophages and Th1 Cells (predominantly IFN- )

• Type I are stable at pH=2• Type II are unstable at pH=2 they are referred to as labile

How They Were Discovered (1957)

Interferon Antiviral Activity

• Currently recombinant pegylated interferons used in HCV and HBV infections

– Pegintron (IFN--2b, Schering Plough) and Pegasys (IFN--2a, Roche) used in conjuction with Ribavirin (nucleoside analog similar to D-ribose)

– Pharma claims 50% effectiveness in either lowering or pushing viral load below detection (in conjuction with Ribavirin)

– They cause serious side effects such as depression and anemia

– Risk of antibody production against pegylated interferon always a possibility

– Effectiveness of interferons is more evident before viral infection has spread

Interferons As Therapeutic Agents

• 13 different IFN- alleles– Similar in a/a sequence

– A lot of variation in glycosylation

– Same a/a number

– Produced a variety of cells, champion though is pDC

• IFN- one gene– Produced by fibroblasts and epithelial cells

– Also pDCs

• IFN- (Type II)– Produced by Th1 and M– More of an immunomodulator rather than anti-viral agent

– 2 versions secreted, differ only in glycosylation

Interferon Variation

• Best inducer is viral infection

• dsRNA viruses are exceptionally good at inducing IFN production

• Synthetic dsRNA can do the same thing suggesting that viral infection and subsequent exposure to dsRNA is what triggers IFN production

• You do not have to be dsRNA virus to produce dsRNA

Interferon Transcription Induction

Interferon Transcription Induction

The receptors that detect bacterial and viral antigens are called TLR (toll like receptors)

TLR3 detects dsRNA

• Uninfected cells do not express IFNs– Strong suppression of promoters and enhancers

• IFN/ relies on positive regulatory domains (PRDs)– These domains are binding sites for transcription factors

– These domains are 200 nt upstream transcription start site

– A well studied enhancer is the IFN- (enhancer is made up of a number of PRDs)

Transcription Factors That Induce IFN Expression

All the proteins together with TFIID and Pol II form the enchanceosome

9 IRFs are known

Recently HSV was found to produce IRF look alike avoiding expression of IFNs

• Interferon signaling mediated thru JAKs and STATS– JAKs (Janus Kinases) or just another kinase!– STATS (Signal transducers and activators of transcription)

• The JAK/STAT pathway utilized by other cytokines as well• Binding of Interferon to receptor causes receptor dimerization• JAKs associate to receptor before interferon binding

– Binding causes them to get activated and phosphorylate receptor and collaborating JAKs

• Phosphorylated receptors behave as docking sites for STATs• JAKs phosphorylate STATs once they dock to receptors• Phosphorylated STATs dimerize and translocate to nucleus• JAK-STAT pathway is extremely rapid

– STAT binding to DNA can be detected within minutes of interferon receptor binding

• Over 100 genes can be induced via interferon signaling • Rapidity is needed to respond to danger

Interferon Signaling

Interferon Signaling

JAK JAKJAK

• Interferon release is part of innate immunity

• Adaptive immunity kicks in at a later point

• IFN has been shown to block entry and uncoating of viruses

• However its best understood anti-viral mechanism is:

– 1. Block viral mRNA synthesis

– 2. Block translation of viral mRNA

• Three anti-viral systems are being studied and party understood at this point

– Mx proteins

– 2',5' oligo(A) synthetase and ribonuclease L

– PKR, double stranded RNA dependent protein kinase

• Mx proteins (myxovirus proteins) are induced by interferon

– They can hydrolyze GTP

– Block viral RNA polymerase

– Block transport of viral nucleoproteins (influenza virus) into nucleus

• How it is done is unclear at this point

• 2',5' oligo(A) synthetase and ribonuclease L

– This enzyme gets activated by dsRNA

– Unique ability to synthesize oligos of A in the 2'- 5' linkage, norm is 3'-5' linkage

– Poly(A) oligos bind ribonuclease L and activate it mRNA is destroyed

– Both cellular and viral

– Cell may die due to this activity though

Interferons Provide First Line of Defense Against Viral Infection

2',5' oligo(A) synthetase and ribonuclease L

• PKR (dsRNA dependent protein kinase)

– PKR expression increases after IFN treatment

– PKR activated by dsRNA

• 2 PKRs bind to same dsRNA and phosphorylate each other

• PKR kinase activity increases and targets proteins

– The best known target protein is eIF2

– Phosphorylated eIF2 cannot initiate translation

– Both cellular and viral translation is inhibited

– PKR also causes apoptosis

Anti-Viral Mechanisms

• Viruses adapt to the interferon response by making proteins that neutralize PKR

• Adenoviruses Evasion Mechanisms– They produce VI A RNA (160 nt long) that binds PKR

– This RNA takes double stranded form

– However PKR does not get activated

– No eIF2 phosphorylation

– Translation proceeds as expected

– Adenoviruses also produce E1A which sequesters p300/CPB• These are needed for interferon expression, they are co-factors

• Epstein Barr Virus encodes for RNA that acts in similar manner

Viruses Evade Interferon Response