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Interferons

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Modified on 2013/03/21 21:30 by Ben Fulton Categorized as Uncategorized
Type-I interferons (IFNs) are important for antiviral and autoimmune responses.

Production is triggered by retinoic acid induced gene I (RIG-I) and mitochondrial antiviral signaling (MAVS) proteins

They start the production process when cytosolic double-stranded RNA or single-stranded RNA containing 5′-triphosphate (5′-ppp) are nearby

Cytosolic B-form double-stranded DNA can also induce IFN-β. For example, a DNA sequence of repeating AT can induce it (It’s known as poly(dA-dT). But no one knew how. Here’s how:

In the cell, the poly(dA-DT) is converted into 5′-ppp.

But how? It turns out that an enzyme uses the poly(dA-dT) as a template to synthesizes 5′-ppp RNA. The enzyme is called DNA-dependent RNA polymerase III (Pol-III).

If you inhibit the working of Pol-III in a cell, and then introduce a bacteria like Legionella pneumophil, the bacteria grows in the cell. The implication is that Pol-III senses the DNA of the bacteria and triggers the IFN process.

How did they do it? In a cell, they attached a luciferase reporter to the IFN-β promoter, so if the cell creates IFN-B it would bioluminesce.

Then, they put different things in the cell. Of all the things tested, only poly(dA-dT) activated the IRF3.

To ensure that there wasn’t something going on at another step in the path, some other things were tried: A silencing RNA strand was introduced into the cell that would stop the production of RIG-I and MAVS. No IFN- β was produced. DNASE-I is an enzyme that breaks down DNA. When that was introduced, no IFN- β was produced. On the other hand, IFN- β was produced in the presence of RNASE-I, so breaking down RNA had no effect.

Nucleic acids from the poly(dA-dT) cells were able to induce IFN- β, even in the presence of DNase I, so it wasn’t DNA that was causing it. Production stopped in the presence of RNase I though, so it must have been RNA that was being produced.

Similar tests were done to determine the exact length of the poly. As few as 30 base pairs were able to trigger the IFN. But, longer sequences with G’s and C have failed to trigger anything.

RNA Characteristics Two enzymes, polynucleotide kinase (PNK) and shrimp alkaline phosphatase (SAP) are used by chemists: the former adds a phosphate group to a DNA or RNA molecule, the latter removes one. A third enzyme, Terminator Exonuclease, or Ter Ex, breaks apart RNA with exactly one phosphate at the 5’ end.

When the SAP was used to remove the phosphate, the RNA no longer induced IFN- β (the PNK had no effect). Even when the PNK was used to add back the phosphate that was removed, there was still no induction, implying that a single phosphate was inadequate. Similarly, treating the RNA with Ter Ex also made no difference.

Another pair of RNase enzymes break apart specifically single stranded RNA (ssRNA) or double stranded RNA (dsRNA). RNase III breaks apart dsRNA, while RNase T1 breaks apart ssRNA. RNase III turned out to inhibit the IFN- β, indicating that dsRNA was required.

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