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. me have generated a syst em that is wholly DNA-based, Both the replicon vector, encoding SFV po lymerase and the protein of interest, and the helper vector, encoding viral structural proteins, were modified so that expression was RNA po lymerase II-dependent. Transfection of the modified replicon plasmid a lone generated 20-30-fold more protein than obtained from a simple exp ression 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 p articles/ml. A single electroporation, plated on one IO-cm plate, gene rated enough virus (10(7) particles) to produce >500 mu g of protein, mild type, replication proficient virus was not detected in three test s utilizing almost 10(8) viral particles, a distinct advantage over a DNA Sindbis-based system in which over half the virus particles genera ted are fully infectious. The new SFV vectors significantly enhance th e utility of this expression system.