Effects of Acyclovir, Foscarnet, and Ribonucleotides on Herpes Simplex Virus-1 DNA Polymerase: Mechanistic Insights and a Novel Mechanism for Preventing Stable Incorporation of Ribonucleotides into DNA

Abstract

We examined the impact of two clinically approved anti-herpes drugs, acyclovir and Forscarnet (phosphonoformate), on the exonuclease activity of the herpes simplex virus-1 DNA polymerase, UL30. Acyclovir triphosphate and Foscarnet, along with the closely related phosphonoacetic acid, did not affect exonuclease activity on single-stranded DNA. Furthermore, blocking the polymerase active site due to either binding of Foscarnet or phosphonoacetic acid to the E-DNA complex or polymerization of acyclovir onto the DNA also had a minimal effect on exonuclease activity. The inability of the exonuclease to excise acyclovir from the primer 3'-terminus results from the altered sugar structure directly impeding phosphodiester bond hydrolysis as opposed to inhibiting binding, unwinding of the DNA by the exonuclease, or transfer of the DNA from the polymerase to the exonuclease. Removing the 3'-hydroxyl or the 2'-carbon from the nucleotide at the 3'-terminus of the primer strongly inhibited exonuclease activity, although addition of a 2'-hydroxyl did not affect exonuclease activity. The biological consequences of these results are twofold. First, the ability of acyclovir and Foscarnet to block dNTP polymerization without impacting exonuclease activity raises the possibility that their effects on herpes replication may involve both direct inhibition of dNTP polymerization and exonuclease-mediated destruction of herpes DNA. Second, the ability of the exonuclease to rapidly remove a ribonucleotide at the primer 3'-terminus in combination with the polymerase not efficiently adding dNTPs onto this primer provides a novel mechanism by which the herpes replication machinery can prevent incorporation of ribonucleotides into newly synthesized DNA.

Figure 1

ACVTP does not affect exonuclease activity on DNA_35ss. UL30 was incubated with DNA (1 μM) and aliquots were taken out at various times. (A) Phosphorimages of the products of DNA_35ss degradation using UL30 at varying concentrations of ACVTP including 0, 1, 5, 20, and 200 μM at various time intervals: 0, 0.5, 1, 5, 10, and 20 min. (B) Plot of exonuclease products as a function of ACVTP concentration at various time intervals. Note: The gels shown are representative of experiments that were performed multiple times.

Figure 2

Effect of ACVTP on polymerase and exonuclease activities under processive conditions. UL30 was incubated with DNA_15C (1 μM) in the presence of 0 μM or 10 μM dNTPs and varying concentrations of acyclovir triphosphate (0–80 μM). Aliquots of each reaction were analyzed at various times after initiating the reaction. (A) Phosphorimages of the products of DNA_15C full extension and degradation using UL30. (B) Plot of exonuclease products as a function of ACVTP concentration at 6 min.

Figure 3

Effect of Foscarnet on polymerase and exonuclease activities under processive conditions. UL30 was incubated with DNA_15C (1 μM) in the presence of 0 μM or 10 μM dNTPs, and 0–80 μM Foscarnet Aliquots were analyzed at 6 min. (A) Phosphorimages of the products of DNA_15C full extension and degradation using UL30. (B) Plot of exonuclease products as a function of PFA concentration.

Figure 4

Formation of UL30-DNA_15C-PFA, UL30-DNA_15C-PAA, UL30-DNA_16ACV, and UL30-DNA_16ACV-dTTP complexes do not affect the exonuclease activity on a second DNA. Assays contained DNA_35ss and the additional DNAs and compounds as noted. All DNAs were present at 1 μM. (A) DNA_15C. (B) DNA_15C and 50 μM PFA. (C) DNA_15C and 50 μM PAA. (D) DNA_16ACV. (E) DNA_16ACV + 50 μM dTTP. The time points for legends A-C were: 0, 0.25, 0.75, 1, 2, 3, 5 and 7 min. The time points for legends D-E were: 0, 0.25, 0.5, 0.75, 1, 2 and 5 min. In Panels B-E, DNA_15C is omitted for clarity.

Figure 5

Nucleoside structures.

Figure 6

The UL30 exonuclease does not efficiently remove acyclovir or dideoxyguanosine from single-stranded DNA. UL30 was incubated with various DNAs and aliquots were taken out at the noted times. All DNAs were present at 1 μM. (A) DNA_15ss. (B) DNA_16ACVss. (C) DNA16_ddss. (D) Amount of exonuclease products generated for each DNA.

Figure 7

Time course for degradation of DNA16_riboss and DNA_16ribo. UL30 was incubated with DNA (1 μM) and aliquots were taken out at various times. (A) Phosphorimages of the products of DNA_16riboss degradation using UL30 (1 nM) after 0, 0.25, 0.5, 1, 2, 5, 7, 10, 15, 20, 25, and 30 min. (B) Phosphorimages of the products of DNA_16ribo degradation using UL30 (25 nM) after 0, 0.25, 0.5, 1, 2, 5, 7, 10, 15, 20, 25, and 30 min.

Figure 8

HSV pol does not efficiently polymerize dNTPs onto DNA containing a ribonucleotide at the 3′-terminus. UL30/UL42 (exo-) (50 nM) was incubated with DNA_ribo (1 μM) in the presence of 100 μM TTP and aliquots were taken out after 15 min. (A) Phosphorimages of the products. (B) Plot of products as a function of TTP concentration. Data were fit to the Michaelis–Menten equation and gave V_max = 4.7 ± 0.4 (nM/min.) and K_mdTTP = 163 ± 53 μM.

Figure 9

UL30 preferentially degrades DNA containing a ribonucleotide at the primer 3′-terminus rather than adding the next correct dNTP. UL30 (exo+) (70 nM) was incubated with DNA_ribo (1 μM) in the presence of 100 μM dTTP and aliquots were taken at various time intervals. Phosphorimages of the products of DNA extension and degradation using 100 μM dTTP which is the next correct incoming dNTP.