I work with a viral non structural protein, an integral ER membrane protein whose structure is unknown. During viral replication the protein goes to the ER membrane and supposedly interact with other non structural proteins. All the studies so far have been based on topology of this protein. I was wondering What different experiments can be designed to understand the role of this protein in viral replication? What can be done if I have this protein in purified form?
It's very difficult to get purified an integral membrane protein, but if you succeed you can study the interaction with the membrane, changes in the protein when interacts with membrane model systems, changes in the lipids, interactions with other non structural proteins...which protein is?
Yeast two-hybrid or bacterial two-hybrid screens are still an option even though this is a membrane protein. Depending on the structure of the protein, it may even make sense to use specific domains in PPI screens, whether they use two-hybrid methods or FRET like Daniel Ivanusic suggested.
Some aspects you might to look at is the induction of the ER stress response or autophagy. From there (depending on the results) the role of autophagy in terms of viral replication and/or antiviral signalling can be studied. I posted some blog posts on this topic which can be accessed at virologytibbits.blogspot.com .
You can certainly look for sequence similarity with other known virus non-structural proteins that localize to the ER (flaviviruses). You can examine if your protein has similar known restructuring of cellular membranes as these viruses' NS4B proteins. Do you have Ab to your protein?... Probably yes, since you have purified protein. You can try to co-immunoprecipitate other ER membrane proteins and identify or characterize which ones your protein might be interacting with. If you can delete or mutate your NS protein you can see if your virus has a changed phenotype, assuming you can mutate and/or culture your virus. (If not then you can certainly approach it the way Daniel Ivanusic suggests.)
You probably mutate this protein by site directed mutagenesis and see its function affecting in viral replication. May I know if you have the replicon of Virus like particles (VLP) or the non structural gene is cloned in a recombinant vector?
Thanks
yes, we do have a luciferase replicon system and the infectious clone. can you please be more clear on when you say nonstructural gene in recombinant vector, what does that mean? Is it something different than replicon system?
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