Generation of dsRNAs Targeting VP1 and VP3 Gene Regions of Coxsackievirus B1 Utilizing Bacteriophage ф6 Polymerase Complex

Abstract

Coxsackievirus infections can result in a wide variety of disease syndromes including upper respiratory tract symptoms, aseptic meningitis, pericarditis, pleurodynia, myocarditis, and encephalitis. No Coxsackievirus drugs are currently licensed and treatment is directed toward ameliorating symptoms. This places the need of new tools to control virus infections. RNA interference is a mechanism for silencing the transcriptional product of an activated gene. Its high conservancy and sequence-specificity are the basis for its huge potential for therapy against various infectious diseases, genetic disorders, and cancer. A way for high-quality and cost effective production of large quantities of double-stranded RNA is the use of virus-based systems targeting specific regions of the virus genome. T7 RNA polymerase and RNA-dependent RNA polymerase of bacteriophage ф6 were used to generate dsRNA molecules from 3’ part of VP3 and 5’ part of VP1 gene regions of Coxsackievirus B1. For large amounts of dsRNA production we utilized Pseudomonas syringae cells that constitutively express the bacteriophage ф6 complex, and plasmids with the target sequences placed under T7 promoter.