In vivo selection of protease cleavage sites by using chimeric Sindbis virus libraries

Identifying protease cleavage sites contributes to our understanding of the ir specificity and biochemical properties and can help in designing specifi c inhibitors. One route to this end is the generation and screening of rand om libraries of cleavage sites. Both synthetic and phage-displayed librarie s have been extensively used in vitro. We describe a novel system based on recombinant Sindbis virus which can be used to identify cleavage sites in v ivo, thus eliminating the need for a purified enzyme and overcoming the pro blem of choosing the correct in vitro conditions. As a model we used the se rine protease of the hepatitis C virus (HCV). We engineered the gene coding for this enzyme and two specific cleavage sites in the Sindbis virus struc tural gene and constructed libraries of viral genomes with a random sequenc e at either of the cleavage sites. The system was designed so that only vir al genomes coding for sequences cleaved by the protease would produce viabl e viruses. With this system we selected viruses containing sequences mirror ing those of the natural HCV protease substrates which were cleaved with co mparable efficiencies.