The 'infectious DNA' approach, which is based on in vivo transcription of (
+)RNA virus genome cDNA cassettes from eukaryotic promoters in transfected
cells, became a popular alternative to the classical scheme in the infectio
us clone methodology. Its use, however, is often limited by the instability
of plasmids due to a transcriptional activity of eukaryotic promoters in E
scherichia coli resulting in synthesis of products toxic for the bacterial
host. Using a highly unstable representative infectious clone of Japanese e
ncephalitis (JE) flavivirus, we tested a new approach in design of such pro
blematic 'infectious DNA' constructs, which is based on minimizing unwanted
transcription in the bacterial host. A plasmid containing full genome size
JE cDNA under control of the minimal cytomegalovirus (CMV) promoter can be
propagated in E. coli with growth and stability characteristics similar to
that of constructs controlled by the T7 promoter. Transfection of this pla
smid into susceptible cells leads to the establishment of a productive infe
ctious cycle. Reinsertion of the CMV enhancer at the 3'-end of the JE casse
tte substantially increased the specific infectivity without affecting the
stability and growth characteristics of the construct. This approach can be
useful when stabilization of infectious clones by modification of a viral
cDNA cassette is not the feasible or suitable alternative. (C) 2001 Elsevie
r Science B.V. All rights reserved.