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

Abstract

Semliki Forest virus vectors (SFV) are suitable for high-level transgene expression in neuronal tissue, both in vitro and in vivo. Cortical and hippocampal primary neurons in culture are efficiently infected resulting in 75-95% of GFP-positive cells, and injection of SFV vectors into hippocampal slice cultures revealed a highly neuron-specific expression pattern with more than 90% of the infected cells being neurons. Here, we present novel SFV vector mutants and describe their infection patterns obtained in cultures of baby hamster kidney (BHK) cells, dissociated hippocampal neurons, and organotypic 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 triple mutant vector SFV(PDE153) demonstrated a temperature-sensitive phenotype in both BHK cells and primary neurons. In hippocampal slices cultured at 36 degrees C, SFV(PDE153) showed a remarkably higher (ca 250-fold) preference for expression in interneurons rather than in pyramidal cells as compared to wild-type SFV. The quadruple mutant SFV(PDTE) led to substantially increased and prolonged GFP expression in primary neurons. Relative to SFV(PDE153), a more pronounced temperature-sensitive phenotype was found resulting in no virus production and no GFP expression at the non-permissive temperature (36-37 degrees C) in BHK cells, in dissociated neurons, and in organotypic hippocampal slices. The described novel SFV vectors will be useful for several specific applications in neurobiology.

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

“…2). In agreement with these results, it has been reported that BHK cells infected or transfected with SFV cytopathic and non-cytopathic vectors are able to express higher levels of recombinant proteins when cultured at 31-33 °C compared to 37 °C [14,15,34].…”

“…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].…”

K+-dependent Cerebellar Granule Neuron Apoptosis

“…7). We used a non-cytotoxic Semliki Forest Virus mutant (SFV(PD)) to introduce rTASK channel subunits into cultured neurons (53). Hippocampal neurons (7 DIV) infected with control virus, bearing only EGFP, were highly fluorescent as early as 5-h postinfection.…”

Generation of Functional Inhibitory Neurons in the Adult Rat Hippocampus

“…To obtain nontoxic virus vectors with high infection rate, we generated a mutant form of pSFV1 vectors, as reported previously (Lundstrom et al, 1999(Lundstrom et al, , 2001). The SacI-XbaI fragment from pSFV1 (Invitrogen, Carlsbad, CA) was subcloned into the pGEM7Zf ϩ vector (Promega, Madison, WI).…”