Development of a new noncytopathic Semliki Forest virus vector providing high expression levels and stability

Abstract

Alphavirus vectors express high levels of recombinant proteins in mammalian cells, but their cytopathic nature makes this expression transient. In order to generate a Semliki Forest virus (SFV) noncytopathic vector we introduced mutations previously described to turn Sindbis virus noncytopathic into a conserved position in an SFV vector expressing LacZ. Interestingly, mutant P718T in replicase nsp2 subunit was able to replicate in only a small percentage of BHK cells, producing beta-gal-expressing colonies without selection. Puromycin N-acetyl-transferase (pac) gene was used to replace LacZ in this mutant allowing selection of an SFV noncytopathic replicon containing a second mutation in nsp2 nuclear localization signal (R649H). This latter mutation did not confer a noncytopathic phenotype by itself and did not alter nsp2 nuclear translocation. Replicase synthesis was diminished in the SFV double mutant, leading to genomic and subgenomic RNA levels that were 125-fold and 66-fold lower than in wild-type vector, respectively. Interestingly, this mutant expressed beta-gal levels similar to parental vector. By coexpressing pac and LacZ from independent subgenomic promoters this vector was able to generate stable cell lines maintaining high expression levels during at least 10 passages, indicating that it could be used as a powerful system for protein production in mammalian cells.

A Semliki Forest virus vector engineered to express IFNα induces efficient elimination of established tumors

“…37 In addition, BHK cells can support the replication of noncytopathic alphavirus RNA mutant replicons, in contrast to cell lines with an intact type I IFN response where those replicons are rapidly lost. [38][39][40] Not only is the production of SFV-IFN feasible but IFNa expression could also be achieved in infected cells independently of endogenous type I responses. We have observed that on infection with SFV-IFN, similar levels of IFNa are expressed in TC-1 and L929 cells, in spite of the fact that TC-1, the main tumor target cells used in our study did not seem to develop a type I IFN response after infection with SFV-LacZ control virus (Figure 1f).…”

“…49 A rabbit polyclonal antiserum specific for the nsp2 subunit of SFV replicase was used as primary antibody. 39 Analysis of SFV infectivity, replication and expression in vitro BHK-21, L929 or TC1 cell lines were infected in six-well plates with serial dilutions of SFV-LacZ or SFV-IFN vp. After 24 h, infectivity was measured by indirect immunofluorescence against SFV replicase as described above.…”

A quick and efficient method to generate mammalian stable cell lines based on a novel inducible alphavirus DNA/RNA layered system

“…This PCR product was digested with NsiI and EcoRV and the obtained 796-bp fragment was subcloned into pGEM-A1 vector digested with the same enzymes, generating a plasmid in which tetO7-Palb-5´UTR-SFV was linked to the neo-polyA-ori sequence (pGEM-A2). This plasmid was digested with SfiI and EcoRV and the 2,874-bp fragment was subcloned into the same sites of pBK-T-ncSFV-1, which derives from pBK-T-SFV-1 [7] and contains P718T and R649H mutations in the nsp2 subunit of the Rep [13]. In this way, we generated i-ncSFV in which the sequence of the full ncSFV replicon is downstream of the inducible tetO7-Palb promoter and is followed by Neo R downstream of the SV40 promoter.…”

“…These mutant vectors have been developed from alphaviruses that include Sindbis virus (SIN) [11,12], Semliki Forest virus (SFV) [12][13][14][15][16], and Venezuelan Encephalitis virus (VEE) [17]. Some of these mutant vectors can be used to generate RNA-based stable mammalian cell lines if a selection gene is included in the replicon [18].…”

“…Some of these mutant vectors can be used to generate RNA-based stable mammalian cell lines if a selection gene is included in the replicon [18]. We have previously shown that a noncytopathic mutant derived from SFV (ncSFV) containing mutations P718T and R649H in the nsp2 subunit of Rep and a gene conferring resistance to puromycin (pac) can be used to select cells that maintain the RNA replicon after many passages [13]. Using this strategy, we were able to generate stable cell lines producing high levels of human therapeutic proteins, like insulinlike growth factor I (IGF-I) and cardiotrophin-1 (CT-1), by including their genes after a second subgenomic promoter or by fusing them at the carboxy-terminal end of pac, using as a linker the sequence coding for foot-and-mouth disease virus (FMDV) 2A autoprotease [19].…”