Expert: Florence M Rollwagen - 4/22/2009

Question
I have a question at the end of the chapter that states the following:
Borna disease virus produces a protein that localizes to the mitochondria and inhibits apoptosis.  
a) How would you show that the protein localizes to the mitochondria?  I think it would involve binding to a luciferase to observe the lumination of the Luc,which would be bound to the protein.  I'm unsure if this is correct- or how/what portion of the protein it should bind to.
b) How would you show which protein in the mitochondria the specific protein binds to?
(Would this be the same procedure- using the luciferase?)
c) How can you demonstrate that apoptosis is inhibited by the protein? How can you show what part of the protein is responsible for the anti-apoptotic activity?


Answer
Hi Jasmine:  This one I can answer!

Luc is not the only label that can be used to localize proteins.  I would use in situ PCR on the slide to find the protein attached to the mitochondrion.

For detecting which protein in the mitochondrion, first isolate the mitochondria, digest to get the mito proteins, run a gel,then incubate with labeled viral protein.

Apoptosis is easily demonstrated on a slide of viral infected cells by the TUNEL technique.  To determine which part of the protein is responsible, controlled cleavage or synthesis of short protein transcripts would be the way to go.

Hope this helps, write back (if) there are any questions I can answer!

FM Rollwagen, PhD