Alterations that Influence dNTP Substrate Utilization by the Adenovirus Type 5 DNA Polymerase Result in Selective Modification of Adenovirus Replication

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2011.Viral DNA polymerases that are catalytically active only at high dNTP concentrations have been found to contain mutations that alter their ability to efficiently utilize the dNTP substrate. As a result, such polymerases are functionally impaired in normal cells, which contain low dNTP concentrations, but are active in cancer cells, which contain high dNTP concentrations. We therefore set out to determine if such polymerases could be developed and used in the context of a full-length replication competent adenovirus as a means of creating a virus that replicates selectively in cancerous cells. To do this we performed a mutational analysis on the adenovirus type-5 DNA polymerase, focusing on conserved motifs that are essential in dNTP substrate binding and utilization. We found that even conservative mutations in these motifs had a dramatic effect on viral replication, with the majority of our mutants being replication defective. Of the five replicationcompetent molecular clones recovered, four reduced biochemical dNTP utilization by the polymerase by 1.7-4 fold, and were greatly impaired in their ability to replicate in both A549 cancer epithelial cells and Wi-38 normal fibroblast cells. The addition of exogenous dNs as a means of increasing the intracellular dNTP concentration available for virus replication was unable to rescue these mutants. One of these four grossly attenuated clones (M689V), was found to mediate high level, stable transgene expression in slowly dividing fibroblasts. Lastly, a fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold, but was able to replicate to wildtype levels in A549 cancer cells. This virus was significantly impaired in Wi-38 normal cells, which contain 10-fold lower dNTP concentrations than A549 cells, but its replication could be rescued in Wi-38 cells by administration of exogenous dNs. In conclusion, these data reveal that (i) the adenovirus DNA polymerase is unexpectedly sensitive to even minor conservative mutations, resulting in either grossly attenuated or replication incompetent viral molecular clones, (ii) mutations to the adenovirus DNA polymerase can have unexpected effects on virally vectored gene expression, and (iii) conditional-replication can be achieved by selectively mutating regions of the polymerase protein that affect dNTP substrate utilization