Characterization of Induced RNAi Silencing of Vaccinia Virus Essential Genes

Vaccinia virus (VACV) is a large double-stranded DNA virus and the prototypical member of the family Poxviridae and is most notable for its use as the vaccine that eradicated smallpox (variola virus). More recently, VACV has been used to develop recombinant vaccines and immunotherapies. However, many of these processes require VACV replication to be tightly controlled. RNA interference (RNAi) is a powerful tool for in vitro silencing of mRNAs that are complimentary to 19-21 base pairs (bp) of double-stranded RNA (dsRNA). This project outlines the design and preliminary analysis of two inducible RNAi silencing constructs targeting multiple VACV essential genes during VACV replication. The two designs include one multihairpin structure expressing multiple shRNAs separated by 6 bp spacers and one head-to-tail design with multiple shRNAs separated by 6 bp loops. Plasmids pKJ252 and pKJ253 (containing the two respective RNAi construct designs, designed such that their transcription can be controlled by tet operon elements) were transfected into BS-C-1 cells previously infected with VACV to generate two new recombinant VACVs. The newly generated recombinant VACVs were analyzed via plaque assays. Results suggest that one of the designed RNAi silencing constructs (multihairpin), when induced with tetracyclines, shows a statistically significant decrease in plaque size, indicating reduced VACV replication