Conditionally replicating adenoviruses (CRADs) have the potential to selectively eliminate cancer cells as a result of tumor-specific virus replication and resultant cytolysis. We propose a novel approach to obtaining selective viral replication by mutating key residues within highly conserved motifs of the Adenovirus (Ad5) DNA polymerase important for dNTP binding. In closely related viruses, mutations within the I/YxGG and Kx3NSxYG motif have resulted in polymerases that were only active in the presence of high concentrations of dNTPs, such as those found in cancer cells. Therefore, we reasoned that analogous mutations to the Ad5 DNA polymerase might produce similar results. To investigate this hypothesis, PCR-based mutagenesis and standard molecular cloning methods were used to introduce desired mutations into the polymerase gene of a molecular clone of human adenovirus type-5 (Ad5). The replication competence of the resulting panel of virus mutants was evaluated by transfection of the cloned Ad5 genomes into human HEK 293A cells. A panel of eight mutant viruses was constructed in this manner, bearing the following mutations in the polymerase gene: (1) mutations in the I/YxGG polymerase motif: R665K; (2-5): mutations in Motif A: C687S; G688S; M689V; M689I; (6-8) mutations in the Kx3NSxYG polymerase motif: N841E; N841Y; and Y884F. Of these clones, 5 were found to be replication-incompetent (even in the presence of high levels of exogenous dNTPs), while 3 were determined to be viable (C689S, M689V and R665K). These results show that conservative polymerase mutations preserved the ability of the virus to replicate while non-conservative mutations sufficiently resulted in non-viable viruses.
|Presenter:||Michael-John Beltejar (Undergraduate Student)|
|Time:||3:10 pm (Session IV)|