Novel mutant Semliki Forest virus vectors: gene expression and localization studies in neuronal cells

Semliki Forest virus vectors (SFV) are suitable for high-level transgene ex pression in neuronal tissue, both in vitro and ill vivo. Cortical and hippo campal primary neurons in culture are efficiently infected resulting in 75- 95% of GFP-positive cells, and injection of SFV vectors into hippocampal sl ice cultures revealed a highly neuron-specific expression pattern with more than 90% of the infected cells being neurons. Here, we present novel SFV v ector mutants and describe their infection patterns obtained in cultures of baby hamster kidney (BI IK) cells, dissociated hippocampal neurons, and or ganotypic hippocampal slices. A less cytotoxic vector SFV(PD), carrying two point mutations in the nsP2 gene, showed much higher GFP expression levels in primary hippocampal neurons compared to the wild-type SFV vector. A tri ple mutant vector SFV(PDE153) demonstrated a temperature-sensitive phenotyp e in both BHK cells and primary neurons. In hippocampal slices cultured at 36 degreesC, SFV(PDE153) showed a remarkably higher (ca 250-fold) preferenc e for expression in interneurons rather than in pyramidal cells as compared to wild-type SFV. The quadruple mutant SFV(PDTE) led to substantially incr eased and prolonged GFP expression in primary neurons. Relative to SFV(PDE1 53), a more pronounced temperature-sensitive phenotype was found resulting in no virus production and no GFP expression at the non-permissive temperat ure (36-37 degreesC) in BHK cells, in dissociated neurons, and in organotyp ic hippocampal slices. The described novel SFV vectors will be useful for s everal specific applications in neurobiology.