Attenuated and Protease-Profile Modified Sendai Virus Vectors as a New Tool for Virotherapy of Solid Tumors
Figure 1
Generation of recombinant Sendai virus variants.
(A) Schematic representation of SeV genomes and the protein sequence of the SeV-F protein cleavage-site of newly generated SeV virus variants. At the 3′-end all variants encode a reporter gene for enhanced green fluorescent protein (EGFP). In contrast to the P and F gene wild-type variant (D52), in all SeV Fmut variants the wild-type F protein cleavage-site (VPQSR) was replaced by the oligobasic cleavage-site of Newcastle disease virus F protein (RRQKR). The seven wild-type P gene encoded proteins are a result of multiple open reading frames (ORF; C′, C, Y1, Y2) and RNA editing, respectively, leading to a frame shift and thus to the V or W ORF's. For attenuation in non-malignant cell types, different mutations were introduced in C (ORFs; C′-, C-, Y1-, Y2) or V/W-ORFs. For the Vko variants (no V and no W proteins), mutations of the editing-site within the P frame were introduced without changing the amino acid sequence of the P protein. Thus, P protein but no truncated P protein variants (V and W proteins) can be synthesized. For the C protein deficient variants (Cko: no C′ and C proteins; Yko: no Y1 and Y2 proteins), inserted point mutations are marked by numbers from 1-5 (1: M1T, 2: L5stop, 3: L11stop, 4: M24T, 5: M30T; numbers refer to amino acid position in C/Y-frame whereas 1 refers to the initiator methionine of the C protein). (B) Schematic overview of functional ORFs from the P gene of different SeV variants. The SeV P-gene wild-type expression pattern is shown for D52 and Fmut (1.+2.).