Generation of Constructs for DNA-Directed RNA Interference of Venezuelan Equine Encephalitis Virus Genes.

摘要

Venezuelan equine encephalitis (VEE) virus is an important human and veterinary pathogen with n6 effective treatment or prophiaxis. One strategy that has shown promise as an anti viral is a mechanism of gene silencing known as RNA interference. Although conventional RNA interference involves the use of dsRNA molecules, here we describe the generation of a panel of DNA cassettes which encode sIRNA sequences. Three different VEE virus genes encoding E2 glycoprotein, nucleocapsid, and non-structural protein 4 were selected as candidates for gene silencing. Using a PCR-based approach, we report here on the selection of the VEE targets, construction of these siRNA expression cassettes, and the cloning of these cassettes into siRNA expression plasmids. These DNA plasmids, once transfected into mammalian cells, are able to express putative small interfering RNA molecules targeting specific regions of the VEE viral genome.