Semliki Forest virus-based DNA expression vector: transient protein production followed by cell death.

Abstract

We have constructed a novel DNA expression vector based on Semliki Forest virus (SFV). SFV produces non-structural proteins (nsPs) which replicate genomic RNA and amplify the mRNA encoding the structural proteins of SFV. A recombinant cDNA genome of SFV, in which the SFV structural genes were replaced by a polylinker cassette to allow for insertion of heterologous DNA, was placed under the control of a cytomegalovirus immediate-early enhancer/promoter with a polyadenylation signal. Transfection of mammalian cells with this SFV-based plasmid vector, pSFV3-CMV-lacZ-pA, resulted in transient high-level expression of a beta-galactosidase reporter gene. The expression level of beta-galactosidase from pSFV3-CMVlacZ-pA was more than 20-fold higher than that obtained from the plasmid with deleted nsPs genes, pSFV3A5976-lacZ, demonstrating that the nsPs genes were essential for the high level of expression. Substantial beta-galactosidase activity was detected in the medium of pSFV3-CMV-lacZ-pA-transfected cells, suggesting that the overproduction of beta-galactosidase caused cell death and release of the protein into the medium. We have demonstrated a high-level expression of the exogenous beta-galactosidase gene from pSFV3-CMV-lacZ-pA constructed using an SFV replication system.