Resistance to a Nucleoside Analog Antiviral Drug from More Rapid Extension of Drug-Containing Primers

Nucleoside analogs are mainstays of antiviral therapy. Although resistance to these drugs hinders their use, understanding resistance can illuminate mechanisms of the drugs and their targets. Certain nucleoside analogs, such as ganciclovir (GCV), a leading therapy for human cytomegalovirus (HCMV), contain the equivalent of a 3′-hydoxyl moiety, yet their triphosphates can terminate genome synthesis (nonobligate chain termination). For ganciclovir, chain termination is delayed until incorporation of the subsequent nucleotide, after which viral polymerase idling (repeated addition and removal of incorporated nucleotides) prevents extension. Here, we investigated how an alanine-to-glycine substitution at residue 987 (A987G), in conserved motif V in the thumb subdomain of the catalytic subunit (Pol) of HCMV DNA polymerase, affects polymerase function to overcome delayed chain termination and confer ganciclovir resistance. Steady-state enzyme kinetic studies revealed no effects of this substitution on incorporation of ganciclovir-triphosphate into DNA that could explain resistance. We also found no effects of the substitution on Pol’s exonuclease activity, and the mutant enzyme still exhibited idling after incorporation of GCV and the subsequent nucleotide. However, despite extending normal DNA primers similarly to wild-type enzyme, A987G Pol more rapidly extended ganciclovir-containing DNA primers, thereby overcoming chain termination. The mutant Pol also more rapidly extended RNA primers, a previously unreported activity for HCMV Pol. Structural analysis of related Pols bound to primer-templates provides a rationale for these results. These studies uncover a new drug resistance mechanism, potentially applicable to other nonobligate chain-terminating nucleoside analogs, and shed light on polymerase functions.