A novel, DNA-based alphavirus gene expression system for rapid recombinant protein purification and virus-based gene delivery to retina and retinoblastoma tumor cells

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. I 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 (pSCA) alone generated 20–30-fold more protein than obtained from a simple CMV-driven expression vector. Expression from pSCA 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 106 infectious particles per ml. A single electroporation, plated on one 10 cm plate, generated enough virus (107 particles) to produce >500 μg of protein. Wild type, replication proficient virus was not detected in three tests utilizing almost 108 viral particles, a distinct advantage over a DNA Sindbis-based system in which over half the virus particles generated are fully infectious. I also present additional enhancements to the DNA-based SFV vector system, pSMART, for use in protein structure and function studies, vaccine development and gene therapy. To simplify and enhance viral and protein production, I characterized an optimal producer/target cell combination, generated a packaging “helper” cell line that amplifies viral yield 100-fold and improved the DNA-based replicon with an expanded multiple cloning site, viral-based translational enhancer and FLAG and HIS₁₀ epitope/affinity tags. The translation enhancer improved expression 3-8 fold from infected and transfected cells, respectively. The presence of affinity tags facilitated purification of nuclear retinoblastoma protein (pRB) and, in a single step, cytoplasmic β-galactosidase. Full-length, functional pRB was produced in mammalian cell culture without the truncated products characteristic of bacterially produced pRB. In addition, pSMART, and its predecessor pSCA1, efficiently infected developing mouse retinal explants and hard-to-transduce retinoblastoma tumor lines. The new SFV vectors significantly enhance the utility of this expression system and will be useful in both molecular studies and novel applications in health care.