Oncolytic virotherapy utilizing adenoviruses engineered to selectively replicate in and lyse cancer cells is being actively pursued as a cancer treatment modality (9
). Most previous approaches to the development of conditionally replicating adenoviruses have focused on the transcriptional regulation of the essential Ad5 E1 genes or have targeted the adenovirus replication proteins located in the E2 region (8
). Our study aimed at regulating adenovirus replication by the rational mutagenesis of its DNA polymerase. We based our approach on studies which showed that one can create DNA polymerase mutants that are catalytically active only at high dNTP concentrations, such as those found in cancer cells, by selectively mutagenizing regions of the protein that affect dNTP substrate utilization (7
We focused our efforts on Ad5 DNA polymerase motifs known to interact with the dNTP substrate either directly (motif A or B), through the polymerase active site, or indirectly ([I/Y]xGG), through the template DNA (2
). The mutagenesis of the selected residues had a dramatic effect on the replicative capacity of Ad5. None of the nonconservative mutations gave rise to viable virus, nor did any of the motif B mutants. This included mutations of motif B that targeted amino acid residues R833 and K837, which have been shown to directly bind and stabilize the incoming dNTP within the polymerase active site via their basic side chains (1
). The loss of these basic side chains (R833T and K837N mutants) was sufficient to abrogate virus replication (). Additional mutations in motif B, N841Y and Y844S, which were expected to impair the formation of the proper nascent base pair binding pocket, required for the stabilization of the nucleotide in the templating position, were also not tolerated (1
Mutations of the (I/Y)xGG motif gave rise to three of the five viable virus mutants that were obtained. This motif has been shown to be involved in stably binding the template DNA at the polymerization active site, in shuttling the primer terminus between the polymerase and exonuclease active sites, and in playing an essential role in the transition between initiation and elongation (7
). In particular, residue I664 has been implicated in having a direct interaction with the ribose functional group of the 3′ nucleotide that precedes the templating nucleotide, directly influencing template strand stability in the polymerase active site. Similar to previous studies, different site-directed mutations made at I664 gave rise to viruses with distinct phenotypes (i.e., I664V [which was replication competent], I664M [which was grossly attenuated], and I664S and I664Y [which were completely replication defective]). This is likely due to the fact that such mutants can lead to alterations in the polymerase/exonuclease equilibrium, with potentially deleterious effects on virus replication (7
). Interestingly, mutations that favor exonucleolysis have been found to require high dNTP concentrations to carry out efficient polymerization and exhibited altered dNTP binding affinities compared to the wild-type polymerase (7
). This is consistent with the phenotype exhibited by the I664V Ad5 mutant, which replicated to wild-type levels in all cancer cells in which it was tested but was significantly impaired in all normal cells (which are expected to have approximately 10-fold-lower levels of endogenous dNTPs than cancer cells ). Moreover, the replication of the I664V Ad5 mutant was significantly enhanced by the addition of exogenous dNs to primary MRC5 and Wi38 cells ().
Mutagenesis of the (I/Y)xGG region of the Ad5 DNA polymerase has also been shown to produce mutants that are impaired in their ability to transition from initiation to elongation. Such mutations reduce elongation activity even in the presence of high dNTP concentrations (7
). This may be the case for the I664M mutation, which showed only a modest defect in dNTP utilization (a 1.7-fold change compared to the wild-type polymerase) as well as impaired replication in all cell lines tested, regardless of the concentration of dNTPs present during infection.
The severe phenotype of the R665K mutant virus was unexpected, since this is a highly conservative substitution that has a minimal effect in the context of the closely related Phi29 DNA polymerase (28
). In Phi29, the residues of the (I/Y)xGG motif have been shown to interact with the templating nucleotide through a series of Van der Waals interactions and hydrogen bonds that are facilitated by a network of water molecules that mediate a large portion of the protein-nucleic acid interactions (1
). It is possible that while the lysine residue maintains the basic charge and size of the side-chain group, the removal of amine groups that are present in arginine may impair the hydrogen bonding that is critical for template DNA binding or I664 orientation.
The mutagenesis of motif A gave rise to two viable virus mutants. Residues in this motif have been shown to interact with both incoming dNTPs and two highly conserved aspartate residues important for the proper catalysis of the polymerase reaction. None of the mutations carried out on the “steric gate” tyrosine (Y690) gave rise to replication-competent virus mutants. Even the highly conservative Y690F mutant was nonviable, suggesting that both the phenolic ring and the hydroxyl group are essential for proper polymerase function.
In conclusion, our data show that even modest changes in polymerase activity, associated with dNTP substrate utilization, are sufficient to completely abrogate adenovirus replication. Despite this, one mutation (I664V) was found to replicate in a dNTP-dependent manner and to selectively induce the killing of tumor cells rather than primary cells. This mutant provides encouraging proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viral DNA polymerase utilizes dNTP substrates.