Semliki Forest Viren (SFV)
- Infection of dividing and non-dividing cells
- transient transgene expression, not integrating into the genome
- very fast gene expression (within a few hours)
- 7.5 kb DNA uptake capacity
- ss (+) RNA genome
- icosaedric shell structure. about 50 nm
- Security level S1
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Semliki Forest Virus - vectors and production
Semliki Forest viruses (SFV) belong to the family Togoviridae, genus alphavirus. They have an icosaedric nucleocapsid surrounded by a lipid membrane with viral envelope proteins. The genome of SFV is a single-stranded RNA of positive polarity. It has a CAP structure at the 5 'end and is polyadenylated at the 3' end. The RNA contains two reading frames, one for the replication proteins and one for the structural proteins. The packaging signal Psi lies within the reading frame of the replication genes and a subgenomic promoter lies between replicase and structural genes.
We use two vectors to produce SFV viral particles. In the first vector pSFV-1, the structural proteins have been replaced by the transgene. The structural proteins contain the second vector pSFV helper. Although recombinant RNA from pSFV-1 can thus replicate, no new viral particles are formed. This can only happen in the presence of the viral structural proteins by pSFV helper.
Both vectors include an SP6 promoter and a single restriction site (SpeI) at the 3 'end of the reading frame to form single-stranded RNA by in vitro transcription. Purified RNA is then transfected into BHK-21 cells by electroporation. In order to prevent the development of infectious and replication-competent virus particles (recombinations between the vector RNAs is possible), the proteolytic cleavage site between two structural proteins in the pSFV helper was mutated so that initially non-infectious particles are generated. Only subsequent treatment with alpha-chymotrypsin converts the viral vector particles into an infectious state. The particles thus obtained are subject to genetic engineering safety level S1.