Rapid, high level protein production using DNA-based Semliki Forest virus vectors.

Semliki Forest virus (SFV) vectors can be produced faster, and have a wider host range, than baculovirus vectors. However, the original SFV system requires in vitro manipulation of RNA. We have generated a system that is wholly DNA-based. Both the replicon vector, encoding SFV polymerase and the protein of interest, and the helper vector, encoding viral structural proteins, were modified so that expression was RNA polymerase II-dependent. Transfection of the modified replicon plasmid alone generated 20-30-fold more protein than obtained from a simple expression vector. Expression required the SFV replicase, which amplifies replicon RNA. The SFV-based vector generated 10-20-fold more protein than a plasmid based on Sindbis virus. Cotransfection of SFV replicon and helper vectors generated viral titers of around 10(6) infectious particles/ml. A single electroporation, plated on one 10-cm plate, generated enough virus (10(7) particles) to produce >500 microg of protein. Wild type, replication proficient virus was not detected in three tests utilizing almost 10(8) viral particles, a distinct advantage over a DNA Sindbis-based system in which over half the virus particles generated are fully infectious. The new SFV vectors significantly enhance the utility of this expression system.