Engineering of a Chimeric RB69 DNA Polymerase Sensitive to Drugs Targeting the Cytomegalovirus Enzyme

Abstract

Detailed structural and biochemical studies with the human cytomegalovirus (HCMV UL54) DNA polymerase are hampered by difficulties to obtain this enzyme in large quantities. The crystal structure of the related RB69 DNA polymerase (gp43) is often used as a model system to explain mechanisms of inhibition of DNA synthesis and drug resistance. However, here we demonstrate that gp43 is ϳ400-fold less sensitive to the pyrophosphate analog foscarnet, when compared with UL54. The RB69 enzyme is also able to discriminate against the nucleotide analog inhibitor acyclovir. In contrast, the HCMV polymerase is able to incorporate this compound with similar efficiency as observed with its natural counterpart. In an attempt to identify major determinants for drug activity, we replaced critical regions of the nucleotide-binding site of gp43 with equivalent regions of the HCMV enzyme. We show that chimeric gp43-UL54 enzymes that contain residues of helix N and helix P of UL54 are resensitized against foscarnet and acyclovir. Changing a region of three amino acids of helix N showed the strongest effects, and changes of two segments of three amino acids in helix P further contributed to the reversal of the phenotype. The engineered chimeric enzyme can be produced in large quantities and may therefore be a valuable surrogate system in drug development efforts. This system may likewise be used for detailed structural and biochemical studies on mechanisms associated with drug action and resistance.Infection with the human cytomegalovirus (HCMV), 2 which belongs to the Herpesviridae, remains an important health problem in immunocompromised persons (1-7). Several drugs that target the viral DNA polymerase (UL54) have been developed to treat the infection (8 -12). Cidofovir (CDV), ganciclovir (GCV), or its prodrug valganciclovir are nucleotide or nucleoside analog inhibitors, respectively, that are intracellularly phosphorylated to their triphosphate form and compete with natural nucleotide pools for incorporation (13)(14)(15)(16)(17)(18)(19)(20). These compounds are characterized by an acyclic sugar moiety with the equivalent of a 3Ј-hydroxyl group that is required for the next nucleotide incorporation event (21). Thus, once incorporated, these compounds interfere with DNA synthesis at various positions downstream (18,22,23). In contrast, compounds that lack the 3Ј-hydroxyl group, such as the antiherpetic drug acyclovir (ACV) (Fig. 1), act as chain terminators (24,25). Although active against HCMV, ACV is not approved for treatment of HCMV infection, and its efficacy is inferior to GCV or CDV (8,26,27). The pyrophosphate analog foscarnet (phosphonoformic acid, PFA) is the third approved anti-HCMV drug that inhibits UL54 (Fig. 1) (28, 29). However, toxicity, problems with oral bioavailability, and the rapid development of resistance can limit the clinical utility of each of the approved drugs.PFA is a broad spectrum antiviral agent that was shown to inhibit various polymerases, including enzymes encoded by herpes simplex virus (HSV), ...

In Vitro -Selected Drug-Resistant Varicella-Zoster Virus Mutants in the Thymidine Kinase and DNA Polymerase Genes Yield Novel Phenotype-Genotype Associations and Highlight Differences between Antiherpesvirus Drugs

“…A recent study has highlighted the limitations of employing the crystal structures of related polymerases, such as that of the bacteriophage RB69 or Thermococcus gorgonariusas, as model systems to explain mechanisms of inhibition of DNA synthesis and drug resistance (63). It was demonstrated that the RB69 DNA polymerase (gp43) is ϳ400-fold less sensitive to the pyrophosphate analogue PFA than the HCMV DNA polymerase (UL54).…”

Phosphonoformic Acid Inhibits Viral Replication by Trapping the Closed Form of the DNA Polymerase

“…Secondary structure elements depicted at the top correspond to RB69 gp43, whereas helices shown at the bottom are predicted for HCMV UL54 from the HSV1 structure. The three HCMV blocks underlined comprise the nine chimeric mutations introduced into RB69 gp43 to induce PFA sensitivity (12). The arrows linking amino acids 365 and 478 represent the clashing Trp residues in the chimeric DNA pol.…”

“…The chimeric form of gp43 contains nine amino acid substitutions in helices N and P: V478W/F479V/N480S and I557M/N558A/R559L/L561V/ L562T/I563C, respectively (see Fig. 4) (12). D222A and D327A substitutions were introduced to eliminate the 3Ј-5Ј exonuclease activity of the polymerase for biochemical assays.…”

“…Based on the hypothesis that the intervening variable regions between these conserved side chains might influence PFA inhibition in herpesviridae, a chimeric DNA polymerase, derived from the parental sequence of RB69 gp43, was engineered to express several of these variable elements originating from the fingers domain of UL54 (12). These elements consisted specifically of three blocks of amino acids adjacent to the absolutely conserved basic residues, Arg-482 and Lys-560 in gp43, therefore allowing the chimeric mutations considerable sequential proximity to the proposed PP i binding site (see Fig.…”

Pronounced Inhibition Shift from HIV Reverse Transcriptase to Herpetic DNA Polymerases by Increasing the Flexibility of α-Carboxy Nucleoside Phosphonates

“…22,23 In all enzymes, the 3′,5′-exonuclease activity had been deleted to eliminate confounding effects of editing activities in the enzyme assays. When evaluated for their anti-DNA polymerase activity, the adenine (and also thymine)-butenyl-α-CNPs were highly inhibitory against HCMV-UL54 (IC 50 : ≤0.5 μM) but completely inactive against bacteriophage WT RB69 and the hybrid RB69/ABC5 (IC 50 : > 100 μM).…”