A 'minimal' approach in design of flavivirus infectious DNA.
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 infectious clone methodology. Its use, however, is often limited by the instability of plasmids due to a transcriptional activity of eukaryotic promoters in Escherichia coli resulting in synthesis of products toxic for the bacterial host. Using a highly unstable representative infectious clone of Japanese encephalitis (JE) flavivirus, we tested a new approach in design of such problematic '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 plasmid into susceptible cells leads to the establishment of a productive infectious cycle. Reinsertion of the CMV enhancer at the 3'-end of the JE cassette 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.
Mishin
V P
VP
Department of Internal Medicine, P.O. Box 800708, University of Virginia, Charlottesville, VA 22908, USA.
Cominelli
F
F
Yamshchikov
V F
VF
D020121:5' Untranslated Regions / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D004664:Encephalitis Virus, Japanese / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D004742:Enhancer Elements, Genetic; D004926:Escherichia coli / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D020121:5' Untranslated Regions; D018076:DNA, Complementary; D004279:DNA, Viral; D012367:RNA, Viral