Figure 1.

Semliki Forest virus (SFV) vectors used in this study for replication-deficient expression system. All vectors comprised the prokaryotic SP6 RNA polymerase promoter for in vitro transcription of the recombinant SFV replicons. (A) Replication-deficient vectors encoding SFV non-structural protein genes (nsP1–4) and the heterologous genes (HGs) Tnfa or Ifng downstream of the SFV 26S subgenomic promoter. The mouse TNF-α gene (Tnfa) was followed by a FLAG-tag-encoding sequence. The mouse IFN-γ gene (Ifng) was inserted in frame with the minimal capsid translation enhancer sequence (Enh) using the 2A auto-protease sequence (2A) from foot and mouth disease virus as a linker. (B) Replication-deficient reporter construct consisting of SFV nsP1–4 and the fluorescent protein-encoding gene Ds-Red downstream of an SFV 26S subgenomic promoter. (C) The SFV helper vector SFV-Helper1, which encodes the full length of the structural protein ORF (i.e., C, capsid protein; p62, 6K and E1, and envelope proteins) downstream of an SFV 26S subgenomic promoter. (D) Production of recombinant particles and proteins by the replication-deficient SFV vector system: (1) in vitro transcription of RNA encoding the HG and the nsP1–4, and the transcription of helper RNA carrying SFV structural genes. The packaging signal (PS) for the selective encapsidation of the RNA encoding nsP1-4 and the HG is present in the nsP2 region of SFV-HG RNA. Both RNAs are capped at the 5-prime end and polyadenylated at the 3-prime end; (2) BHK-21 cells are transfected with SFV-HG and SFV-Helper RNAs by electroporation; (3) BHK-21 cells produce replication-deficient SFV particles carrying HGs; (4) target cells are infected with replication-deficient SFV particles; and (5) the heterologous protein is produced at high levels without production of new viral particles.