Date of Award
Daniel Brian Nichols, Ph.D.
Cosimo Antonacci, Ph.D.
Tin-Chun Chu, Ph.D.
Jessica Cottrell, Ph.D.
Vaccinia Virus, A7, D6, E9L, Transcription Factors, Poxvirus
Poxvirus early genes code for viral products involved in host interaction, immune evasion, replication, and transcription of the viral genome. These viral early genes contain a unique and conserved promoter sequence that is recognized by early transcription factors (ETFs). The working hypothesis is pharmacologically targeting the structure of the poxvirus early promoters can inhibit transcription of poxvirus early genes. Our group has identified unique secondary structure which deviates from a scrambled control near the Vaccinia Virus (VACV) E9L promoter. Targeting these unique structures, which most likely may be G-quadruplexes, with structure specific ligands may distort local DNA structures at the promoter. In turn, this may affect the ability of viral transcription factors to recognize the target sequence. As the E9L gene codes for the viral DNA polymerase, inhibition of E9L transcription would prevent replication of the VACV DNA during infection. To assess potential antiviral activity of G-quadruplex ligands, the central goal of this thesis is to develop an in-vitro system to identify compounds that prevent binding of ETFs to target promoters. Using VACV ETFs (VETFs) as a model system, VETFs, A7L and D6R, were cloned into a bacterial expression vector with a His epitope tag for purification. A7 and D6 have successfully been expressed and detected in bacterial cell lysates via immunoblotting with anti-His antibodies. Currently, protocols are being optimized for large-scale purification of viral proteins to use in an in vitro binding assay.
Park, Younghoon, "Development of a Reliable Method to Express and Purify Vaccinia Virus Transcription Factors A7 and D6 in Bacteria" (2018). Seton Hall University Dissertations and Theses (ETDs). 2586.