Engineered viruses to select genes encoding secreted and membrane-bound proteins in mammalian cells.
We have developed a functional genomics tool to identify the subset of cDNAs encoding secreted and membrane-bound proteins within a library (the 'secretome'). A Sindbis virus replicon was engineered such that the envelope protein precursor no longer enters the secretory pathway. cDNA fragments were fused to the mutant precursor and expression screened for their ability to restore membrane localization of envelope proteins. In this way, recombinant replicons were released within infectious viral particles only if the cDNA fragment they contain encodes a secretory signal. By using engineered viral replicons to selectively export cDNAs of interest in the culture medium, the methodology reported here efficiently filters genetic information in mammalian cells without the need to select individual clones. This adaptation of the 'signal trap' strategy is highly sensitive (1/200 000) and efficient. Indeed, of the 2546 inserts that were retrieved after screening various libraries, more than 97% contained a putative signal peptide. These 2473 clones encoded 419 unique cDNAs, of which 77% were previously annotated. Of the 94 cDNAs encoding proteins of unknown function, 24% either had no match in databases or contained a secretory signal that could not be predicted from electronic data.
D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D005818:Genetic Engineering; D016679:Genome, Viral; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics; D012093:Replicon / Q000235:genetics; D012845:Sindbis Virus / Q000235:genetics* / Q000254:growth & development; D014162:Transfection
D018076:DNA, Complementary; D008565:Membrane Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals