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InterferonComments

Interferon

Interferon (IFN) is secreted by a virally infected cell, and induces an anti-viral state in surrounded cells. It is too late for the infected cell, which dies, but the surrounding cells might be saved. It is not a single protein, but rather a group of stable (acid pH) proteins of 17,000 d secreted by different cells types. Very potent, only a few molecules bound to surface of cell is sufficient. This initially made it very difficult to isoalte and purify. There are three categories:

Alpha (α) InterferonAlpha interferons (IFNα) include 14 closely related small proteins synthesized by granulocites (phagocytic white blood cells). MW: 17,000 daltons
Beta (β) InterferonBeta interferons (IFNβ), related to IFNα, include 2 known proteins and are synthesized by fibroblasts most types of cells in the body. IFNβs are produced in response to double-stranded RNA (a typical indicator that a virus is present). IFNβ causes: activation of endoribonuclease, which cleaves viral RNA; and induction of proteins inhibiting translation, thus stopping viral replication.
Gamma (γ) InterferonGamma interferons (IFNγ) is a glycoprotein secreted by T-lymphocytes in response to viral infection.

Signalling pathway for IFNγ:

  1. Infected cell secretes IFNγ
  2. Binds to neighboring cell expressing IFN receptor (most cells express them with very high affinity)
  3. Binding results in dimerizaion and activation of Jak kinase, which binds to cytoplasmic side of receptor
  4. Activated Jak phosphorylates STAT1, which dimerizes and translocated to nucleus
  5. Transcription factor which activates genes via gamma activated sequence.
  6. Genes expressed by STAT1 act to block replication of incoming virus

There are 2 mechanisms by which STAT1 genes block replication of incoming virus:

Mechanism 1: dsRNA-dependent eIF2 kinase (PKR)

  • First gene is PKR
  • Blocks viral replicaiton by inactivating cellular translation factor called PKR, thereby inhibited viral replicatoin at protein synthesis level.
  • eIF2 is important for translation
  • PKR phosorylates eIF2, which brings first tRNA-methiionine to reibosome to initiate protien translation
  • PKR phosphorylates eIF2, blocks recycling of eIF2-GDP to eIFS-GTP by GNEF, eIF2-GDP cannot bind tRNA-methionine.
  • In IFN-treated uninected cells IFN → PKR (inactive)
  • What activates it?
  • In IFN-treated cells infected with RNA virus....dsRMA forms during course of replication especially in an RNA virus like influenza and these are necessary intermediates and these nucleic acids activate PKR and then it phosphorylates eiF2 and blocks protein synthesis.
  • Most dsRNA appear in cell in high concentrations as a result of viral replication...cell doesn't use dsRNA. ONly viral infections will activate PKR.
  • But blocking proteint translation also blocks host cell protein synthesis. This protective mechanism preotects cell but also harms cell. However cell uses another mechnism to specifically block viral genome replication

Mechanism 2: 2'-5' oligo A induced pathway

In this pathway IFN stimulates expression of 3 host cell enzymes:

  • 2'-5'2 oligo A synthase (activated by dsRNA)
  • Ribonuclease L (activated by 2'-5' oligo A)
  • 2'-5' olido A phosphodiesterase (degrades 2'-5' oligo A)
  • This mechanism expalins specific inhibition of viral protein synthesis as opposed to host cell protein synthesis.
  • oligo A synthase syntehsizes a few A's...only adenine residues with 2'5' linkage...if it were DNA, phosphate would be joined onto 3' carbon instead of 2' carbon...like PKR oligo A synthase activated by dsRNA expressed when cell receives signal from interferon but only actie when virus enters cell and starts to replicate
  • 2nd enzyme also expressed, sitting around in inactive form. RNAseL when bound to oligo A becomes active. Degrades ssRNA, including viral and cellular mRNA, rRNA, and tRNA. Of course this also effects cellular and viral RNA's.
  • The result of these 3 enzymes is that the RNAse is only activated in the microvicinity of dsRNA.

Concentrations of antibodies do not peak until long after the virus has cleared. Therefore, antibodies are probably not the reason for the fall in virus titre. The host factor which does seem responsible is called interferon. Conentrations of interferons much levels of virus very closely. Interferon is a protein which protects cells from infection. It is not specific for one particular virus. It protects the cell from a number of different viruses.

Discovering Interferon

Influenza InfectionMock Infection
  1. Infect egg with influenza
  2. Incubate 2 days
  3. Remove chorioallontoic fluid
  4. Lower to pH2 to inactive virus. Return to pH 7.4.
  5. Inject treated chorioallontoic fluid into fresh egg
  6. Infect egg with NDV
  7. Embryo resistant to infection
  1. Mock-infected egg
  2. Incubate 2days
  3. remove chorioallontoic fluid
  4. inject chorioallontoic fluid into fresh egg
  5. infect egg with NDV
  6. Embryo killed

Isaacs and Lindenman found in 1957 that: sometimes patients are infected by more than one type of bacteria; very rare for somebody to be infected by more than one type of virus; infection with one virus somehow protects body from infection by 2nd virus. Use embryonated chicken eggs as host. Infect embryos with infleunza virus (common way of growing virus before development of tissue culture techniques). When infected with flue, and something in fluid other than virus itself protects Embryonic cells were secreted something into the chorioallantoic fluid which protected the 2nd egg from viral infection. interfered with viral replication, interferon