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 39-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. IMPORTANCE While resistance to antiviral drugs can hinder their clinical use, understanding resistance mechanisms can illuminate how these drugs and their targets act. We studied a substitution in the human cytomegalovirus (HCMV) DNA polymerase that confers resistance to a leading anti-HCMV drug, ganciclovir. Ganciclovir is a nucleoside analog that terminates DNA replication after its triphosphate and the subsequent nucleotide are incorporated. We found that the substitution studied here results in an increased rate of extension of drug-containing DNA primers, thereby overcoming termination, which is a new mechanism of drug resistance. The substitution also induces more rapid extension of RNA primers, a function that had not previously been reported for HCMV polymerase. Thus, these results provide a novel resistance mechanism with potential implications for related nucleoside analogs that act against established and emerging viruses, and shed light on DNA polymerase functions.

Automated summary

Nucleoside analogs are crucial in antiviral therapy, but drug resistance remains a challenge. A study on HCMV DNA polymerase revealed that an alanine-to-glycine substitution enhances resistance to ganciclovir by accelerating DNA primer extension, while also facilitating RNA primer extension, offering insights into novel drug resistance mechanisms and polymerase functions.