The study of a viral protein, or any protein, for that matter, depends on the ability to visualize and purify that protein, which typically relies on the availability of an antibody that specifically recognizes that protein. Such antibodies facilitate the study of protein levels within a cell through various methods (such as Western blotting, protein-protein interactions by immunoprecipitation, and protein localization and immunofluorescence and confocal microscopy). Unfortunately, it is sometimes difficult to generate a “good” antibody for a protein of interest. To circumvent this issue, recombinant versions of proteins can be engineered containing various tags, such as green fluorescent protein (GFP), hemagglutinin (HA) or polyhistidine (His). Although such methods are powerful and have facilitated the discovery of relevant findings for many proteins, the structural impact of such tags can affect the function and topology of a protein, resulting in the appearance of artefactual results.
Over the past two decades there has been a push to expand the human genome through the use of unnatural amino acids (Uaa). To date over 100 Uaa containing side chains with diverse physiochemical and biological properties are in use. At this point Uaa technology has been applied to the study of bacteria, yeast, mammalian cells, stem cells, neurons, multicellular organisms, insects, plants, and very recently, in mice. The application of Uaa technology to the study of viruses is an exciting possibility that allows us to perform analyses of viral proteins in ways that we have not been able to in the past.
The goal of this proposal is to establish a program for the study of viral proteins through the use of unnatural amino acid technology. Our lab has been studying the molecular biology of the hepatitis C virus (HCV) for ten years, and to establish and validate Uaa technology in the context of a virus, we have tested whether the fluorescent Uaa Anap can be incorporated into the HCV Core protein. Preliminary results showed that Core proteins efficiently incorporated Anap, and that these Uaa-containing proteins displayed the typical cellular distribution of unmodified Core proteins. With this system now established, we will carry out the following objectives:-
In summary, the establishment of unnatural amino acid technology in the context of viruses will enable fluorescence-based imaging of virus proteins without modification by peptides or large protein tags. More importantly, this strategy can be applied to other proteins of any virus, and given that numerous different Uaa with various side chains can be incorporated in this manner, we will be able to perform a broad spectrum of Click-Chemistry-based analyses on proteins of interest.