Abstract
9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a new antiviral compound with activity against herpes simplex virus (HSV) and retroviruses including human immunodeficiency virus. Although it has been suggested that the anti-HSV action of PMEA is through inhibition of the viral DNA polymerase via the diphosphorylated metabolite of PMEA (PMEApp), no conclusive evidence for this has been presented. We report that in cross-resistance studies, a PMEA-resistant HSV variant (PMEAr-1) was resistant to phosphonoformic acid, a compound which directly inhibits the HSV DNA polymerase. In addition, phosphonoformic acid-resistant HSV variants with defined drug resistance mutations within the HSV DNA polymerase gene were resistant to PMEA. Furthermore, the HSV DNA polymerase purified from PMEAr-1 was resistant to PMEApp in comparison with the enzyme from the parental virus. Moreover, PMEA inhibited HSV DNA synthesis in cell culture. These results provide strong evidence that HSV DNA polymerase is the major target for the anti-viral action of PMEA. Further studies showed that HSV DNA polymerase incorporated PMEApp into DNA in vitro, while the HSV polymerase-associated 3'-5' exonuclease was able to remove the incorporated PMEA. Thus, the inhibition of HSV DNA polymerase by PMEApp appears to involve chain termination after its incorporation into DNA.
Highlights
9-(2-Phosphonylmethoxyethyl)adenine(PMEA) is a new antiviral compound with activity against herpes simplex virus (HSV)and retroviruses including human immunodeficiency virus
These results provide strong evidence that the phosphonylmethoxyethy1)adenine;PMEAp, PMEA monophosphate; target for the antiviral action of PMEA is the HSVDNA
Because HSV DNA polymerase wascapable of incorporating PMEAppinto DNA, it was of interesttodetermine whether the enzyme could remove the PMEA from the end at leastone of which is resistant to degradation.A third possibility isthatproducts from the degradation of the PMEA-terminated primer are inhibitory to the reaction, alof the DNA by the polymerase-associated 3'-5' exonuclease which is thought to act asa proofreading activity
Summary
Compounds-PMEA, PMEAp, PMEApp, HPMPA, HPMPApp, and ACGTP were generously provided by A. The reaction mixture contained the same components as the HSV DNA polymerase assay in a volume of 0.2 ml except that thedeoxynucleotideswere omitted and the activated DNA was replaced with 10 pg/ml of 3' terminally labeled activated calf thymus DNA (6 X lo cpm/pg). The reaction mixture contained the same components as the HSV DNA polymerase assay except that the activated DNA was replaced by pg/ml labeled M13 template-primer. Ten units of Klenow enzyme (Boehringer Mannheim) was used as the DNA polymerase This resulted in the extension of the original 17base primer by 2 nucleotides with PMEA as the terminanl ucleotide. HSV DNA analysis, whole HSV-1 (K0S)-infectedcells were harvested and subjected to in situ lysis agarose gel electrophoresis as described under "Materials and Methods." After electrophoresis, the DNA was transferred to a nylon membrane andprobed with HSV-1EcoRI F fragment from plasmid.
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