Development of a new noncytopathic Semliki Forest virus vector providing high expression levels and stability.
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.
Casales
Erkuden
E
Division of Gene Therapy, School of Medicine, Center for Applied Medical Research (CIMA),University of Navarra, Av. Pio XII 55, 31008 Pamplona, Spain.
Rodriguez-Madoz
Juan R
JR
Ruiz-Guillen
Marta
M
Razquin
Nerea
N
Cuevas
Yolanda
Y
Prieto
Jesus
J
Smerdou
Cristian
C
D000123:Acetyltransferases / Q000096:biosynthesis / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D019677:Artificial Gene Fusion; D002460:Cell Line; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000235:genetics; D003588:Cytopathogenic Effect, Viral; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D008967:Molecular Biology / Q000379:methods*; D020125:Mutation, Missense; D019913:Nuclear Localization Signals / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012672:Semliki forest virus / Q000235:genetics* / Q000472:pathogenicity*; D012692:Serial Passage; D014773:Virology / Q000379:methods*; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D019913:Nuclear Localization Signals; D012367:RNA, Viral; D011994:Recombinant Proteins; D000123:Acetyltransferases; C045336:puromycin N-acetyltransferase; D012324:RNA-Dependent RNA Polymerase; D001616:beta-Galactosidase; D003546:Cysteine Endopeptidases; C078306:nsP2 proteinase