A New Generation of Animal Cell Expression Vectors Based on the Semliki Forest Virus Replicon

Abstract

We have developed a novel DNA expression system, based on the Semliki Forest virus (SFV) replicon, which combines a wide choice of animal cell hosts, high efficiency and ease of use. DNA of interest is cloned into SFV plasmid vectors that serve as templates for in vitro synthesis of recombinant RNA. The RNA is transfected with virtually 100% efficiency into animal tissue culture cells by means of electroporation. Within the cell, the recombinant RNA drives its own replication and capping and leads to massive production of the heterologous protein while competing out the host protein synthesis. The expression system also includes an in vivo packaging procedure whereby recombinant RNA is packaged into infectious virus particles using cotransfection with packaging-deficient helper RNA molecules. The resulting high titer recombinant virus stock can be used to infect a wide range of animal cells with subsequent high expression of the heterologous gene product, but without expression of any structural proteins of the helper. The infected cells produce protein for up to 75 hours post infection after which the heterologous product can constitute as much as 25% of the total cell protein. The general utility of the system is demonstrated through the expression of human transferrin receptor, mouse dihydrofolate reductase, chick lysozyme and Escherichia coli beta-galactosidase.

Replication-defective recombinant Semliki Forest virus encoding GM-CSF as a vector system for rapid and facile generation of autologous human tumor cell vaccines

“…[15][16][17][18] SFV is a positive-strand RNA virus belonging to the genus Alphaviruses of the family Togaviridae. 19 Alphaviruses replicate in the host cell cytosol without integration of the viral genetic material in the cellular genome.…”

“…To permit insertion of heterologous genes, the subgenomic region of the SFV genome, encoding the viral structural proteins, has been deleted from pSP6-SFV4 and replaced by a polylinker site to produce the vector pSFV1. 16 This vector retains the nonstructural region of the viral genome encoding the RNA replicase, thus preserving the self-replicating ability of the SFV replicon. Insertion of a heterologous gene in the polylinker site of SFV1 now allows in vitro production of RNA transcripts containing the foreign gene in the context of the SFV replicon.…”

“…When introduced into cells these RNA transcripts will direct extensive synthesis of the heterologous protein with simultaneous down-regulation of host-cell protein synthesis. 16 Packaging of the recombinant RNA containing the heterologous gene into SFV particles can be achieved by the introduction of two independent RNAs into cells. The first RNA contains the replicase, the subgenomic promotor and the heterologous gene, and the second RNA (the 'helper') contains the subgenomic promotor and the genes encoding the viral structural proteins.…”

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

“…It was reported that recombinant RNA vectors based on SFV were transfected with high efficiency into animal tissue culture cells by means of electroporation. 4 Although this system can be used, the preparation of capped RNA vectors by in vitro transcription is necessary before transfection and the RNA molecules are unstable in general. Therefore, it is of great use to construct a DNA vector based on self-amplifying systems of SFV, especially in consideration of in vivo expression.…”

“…Some of these self-amplifying systems use RNA genomes of alphaviruses, such as Sindbis virus [1][2][3] and Semliki Forest virus (SFV). 4,5 SFV, a member of the Alphavirus genus, is a smallenveloped virus with a single-stranded RNA genome of positive polarity. The 5′ two-thirds of the viral genome encode nonstructural (replication) proteins (nsPs1-4) and the 3′ one-third encodes structural proteins.…”