NONCYTOPATHIC SINDBIS-VIRUS RNA VECTORS FOR HETEROLOGOUS GENE-EXPRESSION

Infection of vertebrate cells with alphaviruses normally leads to prod igious expression of virus-encoded genes and a dramatic inhibition of host protein synthesis. Recombinant Sindbis viruses and replicons have been useful as vectors for high level foreign gene expression, but th e cytopathic effects of viral replication have limited their use to tr ansient studies. We recently selected Sindbis replicons capable of per sistent, noncytopathic growth in BHK cells and describe here a new gen eration of Sindbis vectors useful for long-term foreign gene expressio n based on such replicons, Foreign genes of interest as well as the do minant selectable marker puromycin N-acteyltransferase, which confers resistance to the drug puromycin, were expressed as subgenomic transcr ipts of noncytopathic replicons or defective-interfering genomes compl emented in trans by a replicon, Based on these strategies, we develope d vectors that can be initiated via either RNA or DNA transfection and analyzed them for their level and stability of foreign gene expressio n. Noncytopathic Sindbis vectors express reasonably high levels of pro tein in nearly every cell. These vectors should prove to be flexible t ools for the rapid expression of heterologous genes under conditions i n which cellular metabolism is not perturbed, and we illustrate their utility with a number of foreign proteins.