Inducible expression of foreign genes using dengue virus genomic terminal regions.

摘要

Replication of dengue virus is mediated by the viral replicase recognizing the terminal regions of the dengue genome. Conserved RNA sequences in the 5' and 3' terminal regions form secondary structures for recognition and initiation of (-) and (+) strand RNA synthesis by the viral replicase. Based on an understanding of dengue replication, the 5' and 3' terminal regions were used as promoters for replication and the inducible expression of a foreign gene, eGFP. A transient expression system used cells infected with Vaccinia virus-T7 and transfected with a T7 promoter driven dengue virus genomic plasmid and T7 promoter driven dengue terminal region constructs containing eGFP. The orientation of the dengue terminal region constructs placed the T7 promoter driving expression of the 3' UTR, eGFP, the 5' TR and finally the hepatitis delta virus ribozyme in the reverse orientation. This generated a negative strand RNA species that did not express eGFP by itself. The presence of dengue viral replicase complex proteins was required to transcribe the negative strand template to a positive strand species with a functional open reading frame for reporter gene expression. This transient expression system was used to determine the requirements for (+) strand RNA synthesis from (-) strand RNA dengue terminal region constructs by the viral replicase. As a result, the requirements for inducing expression of the foreign gene were determined using several variants of this system containing either the 5' UTR or larger fragments of 200 or 250bp of the dengue 5' terminal region.