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Journal of Virology, February 2003, p. 1868-1876, Vol. 77, No. 3
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.3.1868-1876.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Amino Acid Changes within Conserved Region III of the Herpes Simplex Virus and Human Cytomegalovirus DNA Polymerases Confer Resistance to 4-Oxo-Dihydroquinolines, a Novel Class of Herpesvirus Antiviral Agents

Darrell R. Thomsen, Nancee L. Oien, Todd A. Hopkins, Mary L. Knechtel, Roger J. Brideau, Michael W. Wathen, and Fred L. Homa*

Infectious Disease Biology, Pharmacia Corporation, Kalamazoo, Michigan 49001

Received 30 August 2002/ Accepted 31 October 2002


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ABSTRACT
 
The 4-oxo-dihydroquinolines (PNU-182171 and PNU-183792) are nonnucleoside inhibitors of herpesvirus polymerases (R. J. Brideau et al., Antiviral Res. 54:19-28, 2002; N. L. Oien et al., Antimicrob. Agents Chemother. 46:724-730, 2002). In cell culture these compounds inhibit herpes simplex virus type 1 (HSV-1), HSV-2, human cytomegalovirus (HCMV), varicella-zoster virus (VZV), and human herpesvirus 8 (HHV-8) replication. HSV-1 and HSV-2 mutants resistant to these drugs were isolated and the resistance mutation was mapped to the DNA polymerase gene. Drug resistance correlated with a point mutation in conserved domain III that resulted in a V823A change in the HSV-1 or the equivalent amino acid in the HSV-2 DNA polymerase. Resistance of HCMV was also found to correlate with amino acid changes in conserved domain III (V823A+V824L). V823 is conserved in the DNA polymerases of six (HSV-1, HSV-2, HCMV, VZV, Epstein-Barr virus, and HHV-8) of the eight human herpesviruses; the HHV-6 and HHV-7 polymerases contain an alanine at this amino acid. In vitro polymerase assays demonstrated that HSV-1, HSV-2, HCMV, VZV, and HHV-8 polymerases were inhibited by PNU-183792, whereas the HHV-6 polymerase was not. Changing this amino acid from valine to alanine in the HSV-1, HCMV, and HHV-8 polymerases alters the polymerase activity so that it is less sensitive to drug inhibition. In contrast, changing the equivalent amino acid in the HHV-6 polymerase from alanine to valine alters polymerase activity so that PNU-183792 inhibits this enzyme. The HSV-1, HSV-2, and HCMV drug-resistant mutants were not altered in their susceptibilities to nucleoside analogs; in fact, some of the mutants were hypersensitive to several of the drugs. These results support a mechanism where PNU-183792 inhibits herpesviruses by interacting with a binding determinant on the viral DNA polymerase that is less important for the binding of nucleoside analogs and deoxynucleoside triphosphates.


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INTRODUCTION
 
Eight herpesviruses are known to infect humans and several of these viruses are important human pathogens (28). These viruses cause a wide variety of diseases. All herpesviruses are capable of residing in a latent state within the host and recurring in response to environmental stimuli. Herpes simplex virus type 1 (HSV-1) and HSV-2 cause primary lytic infection in peripheral mucocutaneous tissue; the virus then sets up a lifelong latent infection in sensory ganglia where it can reactivate, leading to recurrent episodes of the disease. Human cytomegalovirus (HCMV) is a serious and often life-threatening pathogen of newborn and immunocompromised individuals, including transplant recipients and patients with AIDS. Varicella-zoster virus (VZV) is the causative agent of chicken pox upon primary infection and can recur in adults as herpes zoster (shingles) marked by prolonged pain which can last for 6 months or more. Infection with Epstein-Barr virus (EBV) results in approximately two million cases of infectious mononucleosis in the United States each year.

A key enzyme in the replication of all herpesviruses is the virus-coded DNA polymerase. Several drugs are available to treat herpesvirus infections (5, 9-11, 33). Most of these drugs target the viral DNA polymerase. Drugs such as foscarnet (FOS) act by direct inhibition of the polymerase. Others, such as the nucleoside analogs acyclovir (ACV), penciclovir (PCV), and ganciclovir (GCV), must first be phosphorylated to the monophosphate form by virus-encoded kinases and then further phosphorylated to the triphosphate form by cellular enzymes before they become active inhibitors (11, 36). The triphosphate forms of these nucleoside analogs inhibit polymerases by competing with the binding of natural deoxynucleoside triphosphates (dNTPs). One of the limitations of the currently available drugs is that they are active against only a few of the eight human herpesviruses (HHVs). ACV and PCV inhibit HSV and VZV replication but these drugs have poor activity against cytomegalovirus (CMV) because these compounds are poor substrates for the CMV UL97 kinase. Conversely, GCV inhibits CMV, HSV, and VZV but has proved to be too toxic to use against HSV and VZV.

The DNA polymerase encoded by the herpesviruses is a multifunctional enzyme (35). A 3'-5' exonuclease editing function and a deoxyribonucleotide polymerizing function are characteristics of this enzyme. The herpesvirus DNA polymerase amino acid sequences have several regions of sequence similarity with the catalytic subunits of {alpha}-like DNA polymerases, which include the eukaryotic polymerases {alpha}, {delta}, {varepsilon}, and {zeta} (2). There are seven regions, I through VII, that are shared by most members of the family, with region I being the most similar among the various polymerases and region VII being the least similar (37, 38). An eighth conserved region ({delta}-region C) is found in some members of this family (38). The crystalline structure of a herpesvirus polymerase has not been determined, but protein modeling comparisons of the amino acid sequence of the HSV-1 polymerase with the structure of Klenow polymerase have determined that regions I, II, and III are most likely involved in the essential catalytic function: substrate recognition (19). In support of this model, point mutations mapped within conserved regions I, II, III, V, VII, and delta region C have been shown to result in resistance to commercially available nucleoside analogs ACV, GCV, and cidofovir (CDV) (5, 7-10, 15, 17, 20, 21, 29, 30).

In order to identify novel antiviral compounds that would have the potential to inhibit replication of most of the HHVs, we developed a high-throughput in vitro assay to screen for inhibitors of herpesvirus DNA polymerase activity (3, 22, 26, 32). An automated screen of a proprietary chemical library for specific inhibitors of the HCMV DNA polymerase identified a naphthalene carboxamide, PNU-26370. A subsequent synthetic chemistry effort based upon this initial lead identified the 4-hydroxyquinoline-3-carboxamides as a novel class of nonnucleoside, broad-spectrum inhibitors of herpesvirus DNA polymerases. Additional chemical modification of this class yielded the 4-oxo-dihydroquinolines (4-oxo-DHQs), as represented by PNU-182171 and PNU-183792 (3, 22, 26). Kinetic studies with these compounds demonstrated that they inhibit the DNA polymerase function of the HSV, HCMV, and VZV DNA polymerases (26). These compounds do not inhibit the polymerase activity of cellular polymerase {alpha} or {delta}, nor do they inhibit the mitochondrial DNA polymerase {gamma}. In cell culture assays these compounds inhibit HSV-1, HSV-2, HCMV, and VZV. In the present study we also demonstrate that PNU-183792 inhibits the replication of HHV-8 but not HHV-6. In addition, we explored the mechanism of action of these compounds by analysis of drug-resistant mutant viruses and in vitro-mutated viral polymerases. The resistance phenotype was mapped to a single amino acid change of valine to alanine within conserved domain III of the HSV-1 and HSV-2 DNA polymerases. This valine (V823 in HSV-1) is conserved in the DNA polymerases of six (HSV-1, HSV-2, HCMV, VZV, EBV, and HHV-8) of the eight human herpesviruses and appears to play a critical role in the observed herpesvirus-specific antiviral activity of the 4-oxo-DHQs.


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MATERIALS AND METHODS
 
Cells and viruses. African green monkey kidney cells (Vero) and human foreskin fibroblasts (HFF) were grown in Dulbecco modified Eagle medium containing 10% fetal bovine serum and supplemented with antibiotics as described previously (3, 26). Cells were maintained at 37°C in a humidified atmosphere of 5% CO2. HSV-1 strains KOS, F, and DJL, HSV-2 strains MS, and 186 and, HCMV strain AD169 were used in these studies (3, 22, 26). Strain DJL is a clinical isolate of HSV-1 isolated in our lab from a primary oral lesion. HHV-6 strain GS and HHV-8 in BCBL-1 (body cavity-based lymphoma latently infected with HHV-8) cells were obtained from the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program (catalog numbers 350 and 3233; McKesson Bioservices, Rockville, Md.).

Antiviral assays. Using previously described plaque reduction assays the antiviral activity (i.e., the 50% inhibitory concentration [IC50]) of selected compounds against HSV-1, HSV-2, HCMV, and VZV was measured (3, 22, 27). The antiviral assays for HHV-6 and HHV-8 were done by an outside contract laboratory (Southern Research Institute, Frederick, Md.). A virus-induced cytopathogenic effect (CPE) inhibition assay was used to evaluate compounds for antiviral activity against HHV-6 in HSB-2 cells. HSB-2 cells in 96-well tissue culture plates were infected with virus in the presence or absence of drug, and CPE inhibition was determined by measuring dye (XTT) uptake (26). A microtiter plate assay utilizing real-time PCR as a method of detection was employed to evaluate compounds for antiviral activity against HHV-8. BCBL-1 cells were plated in 96-well tissue culture plates at a density of 5 x 103 cells per well (50 µl/well). Cells were induced to enter a lytic cycle of HHV-8 replication by treatment with tetradecanoyl phorbol acetate (final concentration per well, 10 ng/ml), and dilutions of drug were added (100 µl/well). On day 6, supernatant samples (100 µl/well) were collected into 96-well polypropylene PCR plates. Samples were treated with 0.75 mg of pronase/ml for 30 min at 37°C and 1 U of DNase for 60 min at 37°C to degrade any unencapsidated viral DNA. The DNase was then inactivated by heating the samples to 95°C for 30 min. Three microliters of clarified supernatant was subjected to real-time quantitative PCR. The HHV-8 copy number for each sample was determined by comparison to a standard curve of known DNA copy number, the data were plotted as a percentage of virus control, and the drug concentration (IC50) that reduced the DNA copy number was calculated.

Selection of drug-resistant HSV-1 and HSV-2. Vero cells were plated out at a density of 1.0 x 105 cells per well in a six-well tissue culture plate. Cells were infected with HSV at a multiplicity of infection of 10 PFU/cell, and at 1 h postinfection the cells were overlaid with 3 ml of medium containing 20 µM PNU-182171. Cultures were incubated for 20 h at 37°C and then freeze-thawed to release cell-associated virus, and 0.1 ml of culture was used to infect a new monolayer (one well of a six-well dish) of Vero cells. Serial passage was repeated a total of six times in the presence of 20 µM PNU-182171. Virus isolates were then plaque purified three times prior to the preparation of stocks.

Selection of drug-resistant HCMV. HFF cells were plated into T25 tissue culture flasks to achieve 80% confluency at the time of the transfection. Wild-type HCMV AD169 DNA and plasmid DNA containing the mutant HCMV polymerase gene were mixed (2 µg of viral DNA to 4 µg of plasmid DNA), and the DNAs were transfected by using Superfect transfection reagent according to methods recommended by the manufacturer (Qiagen, Valencia, Calif.). Cells were harvested 5 days posttransfection and freeze-thawed to release virus, and half of the sample was used to infect HFF cell monolayers. Cells were overlaid with media containing 20 µM PNU-183792. Serial passage was repeated seven times in the presence of 20 µM PNU-183792, and virus isolates were then plaque purified three times prior to preparation of viral stock.

Isolation of HSV and HCMV viral DNA. HSV DNA was purified from the cytoplasm of infected Vero cells. Vero cells (50% confluent) were infected at a multiplicity of 0.01 PFU/cell. At 3 to 5 days postinfection infected cells were harvested by centrifugation at 1,000 rpm in a Beckman GS-6R centrifuge. The pelleted cells were resuspended in Tris-EDTA (TE) buffer and placed on ice for 15 min. NP-40 was then added to a final concentration of 0.2%, followed by incubation on ice for another 15 min. The cells were centrifuged at 2,000 rpm for 10 min in a Beckman GS-6R centrifuge. The supernatant was removed, and EDTA was added to a final concentration of 20 mM, followed by the addition of sodium dodecyl sulfate to a final concentration of 0.3% and proteinase K to a concentration of 50 µg/ml, and then incubated at 45°C for 2 h. HCMV DNA was isolated by infecting HFF cells with HCMV at a multiplicity of 0.1 PFU/cell. Cells and media were harvested 5 to 7 days postinfection and subjected to low-speed centrifugation to remove intact cells and cell debris, followed by a high-speed spin to pellet the virus particles (2,500 rpm in a Beckman SW28 rotor for 1 h). After incubation of the HSV and HCMV samples, 1.5 volumes of saturated NaI was added to the digested extract, and the refractive index was adjusted to 1.434 to 1.435. Ethidium bromide was added to a final concentration of 50 µg/ml. The samples were loaded into a VTI 50 centrifuge tube and spun for 24 h at 45,000 rpm. The DNA band was harvested, extracted three times with n-butanol, and then dialyzed against TE buffer, followed by a dialysis against 95% ethanol and a final dialysis against TE buffer.

Marker transfer. Wild-type HSV-1 (strain KOS) DNA and plasmid containing the entire polymerase gene or a subcloned region of the polymerase gene were mixed (2 µg of viral DNA plus 4 µg of plasmid DNA). The DNAs were transfected by using Superfect transfection reagent onto Vero cells (80% confluent) in T25 tissue culture flasks. When the transfected Vero culture showed 100% CPE, the cells were harvested, and the virus titer with or without 20 µM PNU-183792 was determined. The percent rescue was determined by comparing the number of plaques in the drug-treated titration to the number of plaques in the nontreated titration.

Construction of plasmids for in vitro transcription-translation of the HSV-1, HCMV, HHV-6, and HHV-8 polymerases and the HHV-8 processivity factor. A 3,900-bp BclI-XbaI fragment containing the entire coding region of the HSV-1 KOS polymerase gene was gel isolated from pDP4 (obtained from D. Coen, Harvard Medical School [25]). The BclI restriction site is located 11 bp upstream from the start codon for the HSV-1 polymerase. The 3,900-bp fragment (with a BclI site filled in to generate a blunt end) was inserted into the EcoRI-XbaI site (an EcoRI site was filled in to generate a blunt end) of pGEM-3Zf(+). The resulting construct, pGEM-DP4, contains the HSV-1 polymerase gene under the control of the promoter for the T7 polymerase. In order to construct HSV-1 polymerase mutants with changes at amino acid 823, in vitro mutagenesis was used to insert a unique MunI site into pGEM-DP4 to generate pGEM-DP4-E/M. The MunI site was located 50 bp downstream of the codon for amino acid 823 of the HSV-1 polymerase and served as a convenient cloning site for generating mutations at the 823 codon. The MunI site was created in such a way that it did not change the amino acid sequence of the HSV-1 polymerase. The valine-to-alanine mutations at the codon for amino acid 823 were created by PCR mutagenesis with oligonucleotide 1 (5'-CGGCAGGAGTCCGTGCTGGCGTCCCGTGAACCCGTACACCG-3'; the underlined bases represent the V823A codon change) and oligonucleotide 2 (5'-CGCGGGGTGATCGGCCAGTACTGCATACAGG-3'), with pGEM-DP4-E/M as a template. A 673-bp fragment was generated and used as a template in a second PCR with oligonucleotide 2 and oligonucleotide 3 (5'-GCCAATTGTCGTCACCGTCGCGGAACGTGCAGGCACGGCAGGAGTCCGTGCTG-3'). A 723-bp fragment was generated with flanking SstI and MunI restriction enzyme sites. This fragment was digested with MunI and SstI and ligated back into pGem-DP4-E/M, which was digested with SstI and MunI. The entire DNA polymerase gene (3,900 bp) was sequenced to verify that there were no additional mutations.

The HCMV polymerase gene (strain Ad169) was cloned for expression in a coupled in vitro transcription-translation system. A MalE-HCMV polymerase fusion gene was partially digested with BamHI (34). The 3.8-kb fragment comprising the full-length HCMV polymerase gene was inserted into the BamHI site of pGEM-3ZF(+) expression vector with the ATG start codon downstream of the T7 promoter. A V823A mutant of HCMV polymerase in the pGEM vector was generated through site-directed mutagenesis by using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol with the primers 5'-GGTTTTACCGGCGCGGTCAACGGTATG-3' and 5'-CATACCGTTGACCGCGCCGGTAAAACC-3'. A V823A/V824L mutant of HCMV polymerase was generated with a second set of primers (5'-ATGGGCAGACACGGCATCATACCGTTCAGCGCGCCGGTAAAACCGTAGAAAGC-3' and 5'-TTTCTACGGTTTTACCGGCGCGCTGAACGGTATGATGCCGTGTCTGCCCATCG-3') with the pGEM-HCMV-V823A vector. The entire coding region of the polymerase gene was sequenced in order to verify the mutations.

HHV-6 (strain Z29 variant B) virion DNA was obtained from Advanced Biotechnologies (Columbia, Md.). The HHV-6 polymerase gene was cloned from virion DNA by using PCR. Primers designed for PCR contained an SstI site in the forward primer (5'-GTCGAGCTCAGCGTGATGGATTCGGT-3') and an XbaI site in the reverse primer (5'-GCCTCTAGACGTTCACATTACCTCTGC-3'). One microliter of template virion DNA (2 µg) and a 1 µM concentration of each of the forward and reverse primers were used in a PCR. The 3-kb fragment was inserted into the XbaI SstI site of pGem3-ZF(+) with the ATG start codon downstream from the T7 promoter. The polymerase gene was sequenced and compared with the published sequence af157706 (GenBank). An A678V mutant HHV-6 polymerase was generated through site-directed mutagenesis with the primers 5'-GGTGTACGGTGTCACGGGAGTGGCGCACGGGTTATTGC-3' and 5'-GCAATAACCCGTGCGCCACTCCCGTGACACCGTACACC-3'. The A678V/V675F mutant HHV-6 polymerase was generated by using a second set of primers (5'-CCGTGCGCCACTCCCGTGAAACCGTACACCGAGTTACATGTTG-3' and 5'-CAACATGTAACTCGGTGTACGGTTTCACGGGAGTGGCGCACGG-3'), with the pGEM-HHV6-A678V vector. Mutagenesis was confirmed by sequencing the entire coding region of the polymerase gene.

HHV-8-infected cell DNA from KS-1 cells was purchased from Advanced Biotechnologies (catalog number 08-735-000, lot number 4308-033198). The HHV-8 polymerase gene was PCR cloned with the primers 5'-CCGAATTCAGATCATGGATTTTTTCAATCCATTTATCG-3' and 5'-ATGGATCCTAGGGCGTGGGAAAAGTCACGGGAATG-3' that extend the full-length of the 3-kbp polymerase gene (bp 11363 to 14401 according to the sequence u75698). PCR fragments were ligated into the EcoRI/BamHI restriction sites of pGEM3-ZF(+), and the entire insert was sequenced. A nucleotide substitution was generated at nucleotide position 13440 in HHV-8 polymerase gene by site-directed mutagenesis with complementary oligonucleotide primers (5'-CGGCTTCACGGGCGCTGCCTCTGGCATACTG-3' and 5'-CAGTATGCCAGAGGCAGCGCCCGTGAAGCCG-3') that correspond to positions 13426 to 13456 of the HHV-8 sequence. The base change resulted in a V693A amino acid substitution in the HHV-8 DNA polymerase.

The HHV-8 processivity factor was PCR cloned with primers (5'-GCCGGATCCATGCCTGTGGATTTTCACTATGGGG-3' and 5'-GCCCTGCAGAGATCTTCAAATCAGGGGGTTAAATG-3') from HHV-8 genomic DNA (bp 95549 to 96740), and the resulting PCR product was digested with BamHI and PstI and inserted into the BamHI PstI site of pGEM3-ZF(+). In order to increase expression by in vitro transcription-translation, a Kozak concensus sequence (A/GCCACC) was added downstream from the T7 promoter sequence by PCR.

In vitro transcription-translation. The cloned herpesvirus polymerases and the HHV-8 processivity factor were transcribed and translated from the T7 promoter in the pGEM vector by using a TnT T7-coupled reticulocyte lysate system (Promega) according to the manufacturer's protocol. Translation of full-length protein was confirmed by [35S]methionine labeling. Briefly, 1 µg of purified plasmid DNA was incubated in the presence of reticulocyte lysate at 30°C for 90 min, and the resulting proteins were separated on a 12 to 14% acrylamide gel by using a morpholinepropanesulfonic acid buffer system. After electrophoresis, gels were fixed for 20 min in 10% methanol-10% trichloroacetic acid (TCA) and then soaked in Enlightening (DuPont) for 20 min. The gels were dried 1 h at 65°C prior to autoradiography. Proteins that were transcribed or translated for use in the DNA polymerase activity assays were prepared in the absence of [35S]methionine.

DNA polymerase assays. HSV-1, HCMV, VZV, and human {alpha} and {delta} polymerases were expressed and isolated by using a baculovirus expression system as described previously (26). Polymerase activity for the HSV-1, HCMV, VZV, and human {alpha} polymerases was measured by using a scintillation proximity assay (26). Polymerase activity of human {delta} polymerase was measured by following the incorporation of 3H-labeled dTTP with a poly(dA-dT) primer template as described previously (26).

The activity of the polymerases made by in vitro transcription-translation were assayed in 100-µl volumes by using a buffer containing 70 mM (NH4)2SO4; 50 mM Tris-HCl (pH 8.0); 10 mM MgCl2; 0.1 mM EDTA; 0.1 mM dithiothreitol; 5% dimethyl sulfoxide; 5% glycerol; 25 mM NaCl; 10 µM concentrations each of dGTP, dCTP, and TTP; 0.075 U of activated calf thymus DNA (Amersham); and various amounts of dATP and [32P]dATP. A 1-µl portion of a 50-µl transcription-translation reaction was used per 100-µl polymerase reaction for all polymerases except HHV-8. Reactions were performed at 37°C for 30 min and terminated with the addition of 140 µl of 10% TCA. After incubation on ice, DNA was harvested on GF/C 96-well multiscreen filterplates (Millipore) by vacuum filtration (Millipore). Wells were washed four times with 200 µl of 5% TCA-20 mM sodium pyrophosphate and one time with 50 µl of 80% ethanol. Plates were air dried for 1 h, and 140 µl of Microscint PS (Packard) was added to each well. Plates were shaken 5 min and counted by using a Packard Topcount. To determine the polymerase inhibitory activity, compounds were initially diluted in 100% dimethyl sulfoxide before diluting them 1/20 in the reaction plate. The activity of the HHV-8 polymerase was difficult to detect in the absence of the HHV-8 processivity factor (6, 24). Therefore, the HHV-8 polymerase assay contained a ratio of 0.8 µl of polymerase to 1.2 µl of processivity factor. The HHV-8 polymerase activity was stimulated 10-fold by the addition of the HHV-8 processivity factor. This ratio was also used with the mutant HHV-8 polymerase, and a 20-fold stimulation in activity was observed.

DNA sequencing. HSV-1, HSV-2, or HCMV viral DNAs were sequenced directly using an ABI 377 fluorescence sequencer (Perkin-Elmer Applied Biosystems, Foster City, Calif.) and the ABI BigDye Prism dRhodamine terminator cycle sequencing ready reaction kit with AmpliTaq FSTM DNA polymerase (Perkin-Elmer Applied Biosystems). Each cycle sequencing reaction contained ca. 1.0 µg of purified viral DNA. Generally, sequence reads of 600 to 700 bp were obtained. Potential sequencing errors were minimized by obtaining sequence information from both DNA strands and by resequencing difficult areas with primers at different locations until all sequencing ambiguities were removed.


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RESULTS
 
During an automated screen of a proprietary chemical library for specific inhibitors of the HCMV DNA polymerase a compound, PNU-26370, with modest activity against HCMV was discovered (3, 22, 26, 32). A subsequent synthetic chemistry effort yielded a novel class of herpesvirus polymerase inhibitors, the 4-oxo-DHQs, represented by PNU-182171 and PNU-183792 (3, 22, 26, 32). The structures of PNU-182171 and PNU-183792 are shown in Fig. 1, and a summary of the biological activity for the two compounds is given in Table 1. The 4-oxo-DHQs are nonnucleoside inhibitors that specifically target herpesvirus DNA polymerases. The specificity for herpesvirus polymerases was demonstrated by the fact that these compounds did not inhibit human cell polymerases {alpha}, {delta}, and {gamma} (Table 1). In vitro polymerase assays demonstrated that PNU-182171 and PNU-183792 inhibited not only the HCMV polymerase but also the HSV-1 and VZV polymerases, and PNU-183792 was also shown to inhibit the HHV-8 polymerases (Table 1). The IC50s against these enzymes ranged from 0.37 to 1.20 µM. In contrast, PNU-183792 did not inhibit the HHV-6 polymerase (IC50 > 50 µM; Table 1). In antiviral cell culture assays, PNU-182171 and PNU-183792 were found to be good inhibitors of HCMV, HSV-1, HSV-2, and VZV, and PNU-183792 was also found to inhibit HHV-8 replication. However, in agreement with the in vitro polymerase assays, PNU-183792 did not inhibit the replication of HHV-6 in antiviral cell culture assays (Table 1).



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  		FIG. 1. Structures of PNU-182171 and PNU-183792.


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  		TABLE 1. Summary of biochemical and antiviral activity of PNU-182171 and PNU-183792

Isolation and characterization of HSV-1 and HSV-2 mutants that are resistant to the 4-oxo-DHQs. To investigate the mode of action of the 4-oxo-DHQs, we selected resistant viruses. A panel of viruses consisting of three strains of HSV-1 (KOS, DJL, and F) and two strains of HSV-2 (MS and 186) were used in these studies. Each parent virus strain was plaque purified, and the polymerase gene from each of the plaque-purified viruses was sequenced (data not shown). The amino acid sequences of the three HSV-1 strains were essentially identical, with only a few minor changes noted between the different strains. The same was found when the two HSV-2 strains were compared. Although the HSV-1 and HSV-2 polymerases share a high degree of amino acid sequence homology, there were notable regions of sequence divergence and several regions where there were gaps or amino acid insertions. The parent HSV-1 and HSV-2 viruses were tested in a plaque reduction assay against PNU-182171 and PNU-183792 and against acyclovir (ACV). PNU-182171 and PNU-183792 inhibited replication of the five viruses, with IC50 values ranging from 2 to 4 µM (Table 2). With the exception of the HSV-2 186, replication of these viruses was also inhibited by ACV. The fact that the amino acid sequence of the HSV-2 MS and HSV-2 186 polymerase were found to be nearly identical and that there were no amino acid changes in the polymerase previously reported to cause ACV resistance would suggest that the ACV resistance phenotype of HSV-2 186 is probably the result of a mutation in the viral thymidine kinase gene. HSV-2 186 has previously been shown to be susceptible to ACV (23). Since we plaque purified the HSV viruses prior to isolation of resistant mutants, the most likely explanation is that we isolated an HSV-2 186 virus with a thymidine kinase mutation that confers resistance to ACV.


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  		TABLE 2. Antiviral activity of PNU-182171, PNU-183792, and ACV against wild-type and mutant HSV-1 and HSV-2 selected for resistance to PNU-182171

In order to select for 4-oxo-DHQ-resistant mutants, the three HSV-1 and two HSV-2 strains were serially passaged in the presence of 20 µM PNU-182171. After the seventh passage, 4-oxo-DHQ-resistant virus from each strain was plaque purified three times in the presence of 20 µM PNU-182171, and high-titer stocks were prepared. The 3.7-kb coding region of the polymerase genes from the six resistant viruses (KOS-M1, KOS-M2, DJL-M1, F-M1, 186-M1, and MS-M1) were sequenced, and point mutations were identified in the resistant virus DNA. Five mutants—KOS-M1, DJL-M1, F-M1, 186-M1, and MS-M1—carried a single point mutation resulting in a V823A (or the equivalent amino acid in HSV-2) amino acid change (Fig. 2). The altered amino acid is located within conserved domain III of the HSV-1 and HSV-2 polymerases. DNA sequence analysis revealed that the KOS-M2 mutant contained three point mutations that resulted in three amino acid changes within the HSV-1 polymerase: V823M, T612I, and S742N. The mutants selected with PNU-182171 exhibited an ~10-fold increase in the IC50 when tested in a plaque reduction assay against PNU-182171 and PNU-183792 (Table 2). It should be noted that PNU-182171 was an early active compound that emerged from our chemistry efforts on these compounds and was used to isolate viral mutants because it was the most active compounds at that time. PNU-183792 later emerged as our prime candidate based on its biological activity and pharmacokinetic properties, so the remaining studies focused on this compound.



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  		FIG. 2. Amino acid sequence of conserved domain III of herpesvirus and human DNA polymerases. (Top) Schematic representation of the 1,235-amino-acid HSV-1 DNA polymerase, with the location of the conserved domains among the {alpha}-like DNA polymerases. (Bottom) Amino acid sequences of region III from the eight HHV DNA polymerases and human {alpha} and {delta} polymerases. The amino acid change, V823A, in the HSV-1 polymerase that confers resistance to the 4-oxo-DHQs is indicated. The amino acid numbers for each of the polymerases within conserved region III are listed on the right.

The resistant mutants grew to high titers in Vero cells, suggesting that the mutations in the resistant isolates did not significantly impair their growth. Intracellular replication of KOS, KOS-M1, and KOS-M2 was examined by establishing single-step growth curves in Vero cells (31). Samples from each infected culture were harvested at various times postinfection and assayed for infectious virus by determining the virus titers on Vero cells (Fig. 3). The two HSV-1 mutants showed growth similar to that of KOS, clearly demonstrating that the polymerase mutations did not alter replication of the two mutants.



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  		FIG. 3. Single-step growth curve of KOS, KOS-M1, and KOS-M2. Cultures of Vero cells were infected KOS ({square}), KOS-M1 ({triangleup}), and KOS-M2 (x) at a multiplicity of infection of 1. The cultures were harvested at the indicated times postinfection and freeze-thawed three times, and the yield of virus (in PFU) at each time point was determined by plaque assay on Vero cells.

Genetic mapping of resistance to 4-oxo-DHQs. To determine whether the polymerase genes carrying the 4-oxo-DHQ-resistance mutations could transfer this resistance phenotype to wild-type HSV-1, we conducted a series of marker transfer experiments. Vero cells were cotransfected with intact DNA from HSV-1 KOS and plasmids containing the entire HSV-1 DNA polymerase gene (3,700-bp fragment) isolated from wild-type KOS or from the 4-oxo-DHQ-resistant mutants, KOS-M1 or KOS-M2. The progeny virus from the transfection were titrated on Vero cells in the presence or absence of 20 µM PNU-183792. There was no rescue of PNU-183792 resistance when the transfections were done in the absence of plasmid DNA or when the plasmid contained the wild-type HSV-1 polymerase gene (Table 3). In contrast, cotransfection with a plasmid containing the entire coding region of the polymerase gene from KOS-M1 (V823A) or KOS-M2 (V823M, T612I, and S742N) resulted in the rescue of 4-oxo-DHQ resistance (Tables 3 and 4). These results demonstrate that the valine-to-alanine change at amino acid 823 by itself confers drug resistance. Along with the valine to methionine change at amino acid 823, mutant KOS-M2 also contained two mutations which altered amino acids 612 and 724 from threonine to isoleucine and serine to asparagine, respectively. A set of marker transfer experiments were done by using cloned fragments isolated from the polymerase gene of the KOS-M2 mutant (Table 4). Interestingly, only the full-length polymerase gene from this mutant was able to rescue the 4-oxo-DHQ-resistant phenotype. A cloned fragment containing the V823M region separated from the T612I and S724N region did not rescue. In addition, clones containing the T612I and S742N region of the polymerase gene or T612I region alone did not rescue. It appears that V823M is not sufficient to confer 4-oxo-DHQ resistance alone but requires at least one additional amino acid change within the polymerase. Finally, since we only tested polymerase clones in the marker transfer experiments, we cannot rule out the possibility that drug-resistant mutations may map to viral genes other than the polymerase gene. Marker transfer studies with cloned fragments from the rest of the viral genome need to be tested in order to determine whether only polymerase mutations are capable of conferring drug resistance.


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  		TABLE 3. Marker transfer of PNU-183792 resistance as determined by using an HSV-1 polymerase gene containing a V823A mutation


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  		TABLE 4. Marker transfer of 4-oxo-DHQ resistance determined by using the HSV-1 polymerase gene from KOS-M2

Isolation and characterization of a HCMV mutant that is resistant to the 4-oxo-DHQs. In order to select for a HCMV 4-oxo-DHQ-resistant mutant, virus (strain AD169) was serially passaged in the presence of 20 µM PNU-183792. Although we could readily select for HSV mutants by using this procedure, we failed to isolate an HCMV mutant, even when the virus was passaged at lower drug concentrations (5 µM). Comparison of the amino acid sequences of the HSV polymerase (Y-G-F-T-G-V-Q-H-G) and the HCMV polymerase (Y-G-F-T-G-V-V-N-G) in the region of amino acid 823 (the underlined amino acid) showed that the next amino acid (amino acid 824) in the HCMV polymerase is also a valine. In order to determine whether both V823 and V824 need to be changed in order to confer resistance to the 4-oxo-DHQs, in vitro polymerase assays were done with mutant HCMV polymerases containing a V823A change alone or a mutant HCMV polymerase in which V823 and V824 were changed to valine and leucine, respectively. The mutation V823A alone resulted in an ~10-fold increase in the PNU-183792 IC50 over that of the wild type (IC50s, 19 and 2 µM, respectively), whereas the polymerase with the double amino acid change had a nearly 25-fold increase in the IC50 (43 µM). In order to isolate an HCMV-resistant mutant, marker transfer experiments were done. Plasmids containing the mutant polymerase genes were transfected into HFF cells along with wild-type HCMV AD169 DNA. The resulting virus was then passaged in the presence of 20 µM PNU-183792. A 4-oxo-DHQ-resistant virus was isolated from the studies done with the HCMV polymerase gene containing mutations that result in the V823A, V824L amino acid changes, but not with the gene containing the V823A change alone. The mutant selected with PNU-183792 (HCMV AD169-M1) exhibited ~7-fold increase in IC50 when tested in a plaque reduction assay. (A plaque reduction of 4-oxo-DHQ-resistant HCMV yielded IC50 values of 0.7 and 4.7 µM, respectively, for AD169 and AD169-M1 [i.e., HCMV polymerase with mutations V823A and V824L].) The entire coding region of the HCMV polymerase genes from both the parent strain and the resistant virus were sequenced. Comparison of the DNA sequence of the two polymerase genes demonstrated that the resistant mutant contained two point mutations that resulted in the predicted V823A and V824L amino acid changes. As with the HSV resistant viruses, these results suggest that the region encompassing amino acid 823 is important for inhibition of herpesvirus polymerase activity by these compounds.

Critical role of amino acid 823 of the HSV-1 polymerase or its equivalent amino acid in HCMV, HHV-6, and HHV-8 polymerases for inhibition of polymerase activity by PNU-183792. The valine at 823 is conserved in the DNA polymerases of six (HSV-1, HSV-2, HCMV, VZV, EBV, and HHV-8) of the eight HHVs (Fig. 2). The polymerase for HHV-6 and HHV-7 contain an alanine at this amino acid. The human {delta} polymerase shares a high degree of homology with the herpesvirus polymerases especially within domain III. Similar to the HHV-6 and HHV-7 polymerases, {delta} polymerase contains an alanine at the amino acid (A706) equivalent to HSV-1 823 (Fig. 2). In order to determine the importance of the conserved valine with regard to polymerase inhibition by PNU-183792, the valine in the HSV-1, HCMV, and HHV-8 polymerases was changed to alanine, while in the HHV-6 and human {delta} polymerases the alanine was changed to valine. Inhibition of the wild-type and mutant polymerases by PNU-183792 was evaluated in an in vitro polymerase assay (Table 5). PNU-183792 inhibited the wild-type HSV-1, HCMV, and HHV-8 polymerases. The valine-to-alanine change in the HSV-1, HCMV, and HHV-8 polymerases resulted in the loss of polymerase inhibition by PNU-183792 (the IC50 increased 4- to 10-fold). In contrast, changing alanine to valine in the HHV-6 polymerase resulted in a 4- to 5-fold decrease in the IC50 against this enzyme, whereas the same change had no effect on the activity of PNU-183792 against the human {delta} polymerase. Interestingly, a further change in another variant HHV-6 amino acid, V675F (amino acid F820 in HSV), that is conserved in most of the other HHV polymerases resulted in further sensitivity of the HHV-6 polymerase to inhibition by PNU-183792 (Table 5). These results demonstrate that the valine and surrounding amino acids located within conserved domain III are critical for PNU-183792 inhibition of herpesvirus polymerases. The lack of activity of PNU-183792 against human {delta} polymerase containing the A706V amino acid change suggest that, although they share amino acid sequence homology within conserved domain III, the overall structure of this region differs between the herpesvirus and human polymerases.


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  		TABLE 5. Activity of PNU-183792 against mutant herpesvirus polymerases

Inhibition of HSV and HCMV 4-oxo-DHQ-resistant mutants by nucleoside analogs. The activity of several previously described herpesvirus antivirals against the HSV-1, HSV-2, and HCMV 4-oxo-DHQ-resistant mutants were tested in plaque reduction assays (Table 6). The nucleoside and/or nucleotide analogs tested included ACV, GCV, and CDV are licensed compounds that are commonly used to treat HSV and HCMV infections. The activities of these compounds against the mutants were compared to their activities against the wild-type strains that were used to isolate the HSV and HCMV mutants. In the plaque reduction assays there was little if any difference in the IC50 values for FOS, CDV, and GCV against the HCMV AD169-M1. The same was found for the HSV-1 KOS-M1 and HSV-2 MS-M1 mutants when tested against iododeonyuridine, ACV, or arabinosyladenine. In contrast, the HSV-1 KOS-M1 mutant was found to be hypersensitive to both bromovinyldeoxyuridine and arabinosylthymine, and the HSV-2 MS-M1 mutant was hypersensitive to arabinosylthymine. These results demonstrate that the V823A change in the HSV polymerase and the V823A/V824L changes in the HCMV polymerase resulted in resistance to the 4-oxo-DHQs, but they did not alter the antiviral activity of these viruses to other classes of polymerase inhibitors and, in fact, the mutations resulted in the mutants being more sensitive to some of these drugs.


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  		TABLE 6. Antiviral activity of nucleoside and nonnucleoside polymerase inhibitors against HSV-1, HSV-2, and HCMV isolates selected for 4-oxo-DHQ resistance


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DISCUSSION
 
In this study we describe a novel class of nonnucleoside antivirals the 4-oxo-DHQs, represented by PNU-182171 and PNU-183792, that are capable of specifically and effectively inhibiting the replication of six of the eight HHVs. These compounds inhibit HCMV, HSV-1, HSV-2, EBV, VZV, and HHV-8 replication. In order to elucidate the mechanism of action of the 4-oxo-DHQs, it was necessary to determine its target in the context of a viral infection. Drug-resistant mutants were generated by passaging HSV-1 and HSV-2 in the presence of PNU-182171. Using this strategy we demonstrated that a single point mutation resulting in a valine-to-alanine amino acid change within conserved domain III of the HSV-1 or HSV-2 DNA polymerase conferred resistance to PNU-182171 and PNU-183792. In addition, an HCMV-resistant mutant was constructed that contained two amino acid changes in the HCMV polymerase gene: V823A and V824L. The change of both valines was necessary to confer HCMV resistance to the 4-oxo-DHQs. Biochemical assays demonstrated that the HSV-1, HCMV, and HHV-8 polymerases were inhibited by PNU-183792 and that the valine-to-alanine change at amino acid 823 in the HSV-1 and HCMV polymerases or the equivalent amino acid in the HHV-8 polymerase resulted in a loss of inhibition by PNU-183792 (the IC50 increased >10-fold; Table 5). In contrast, the wild-type HHV-6 polymerase was not inhibited by PNU-183792, and the lack of activity correlated with the presence of alanine instead of valine at the conserved doman III amino acid of the HHV-6 polymerase. Finally, we demonstrated that replication of the 4-oxo-DHQ-resistant HSV and HCMV mutants were inhibited by nucleoside and nucleotide analogs and that the mutants were hypersensitive to some of these drugs. These results demonstrate (i) that the 4-oxo-DHQs inhibit by interacting with a region of the herpesvirus polymerases that either overlaps with or resides close to the binding site for nucleoside or nucleotide analogs and (ii) that this binding site is unique to the herpesvirus DNA polymerases since these compounds do not inhibit the cellular polymerases {alpha}, {delta}, and {gamma}.

Kinetic analysis with the HCMV polymerase demonstrated that this class of compounds are competitive inhibitors of nucleotide binding (26). Amino acid 823 is contained within conserved domain III, which has been implicated in nucleotide binding (2, 19). V823 is conserved within the DNA polymerases for six of the eight HHVs and also for a number of animal herpesviruses (2). The DNA sequences for 3 HSV-1 and 2 HSV-2 lab strains (sequenced in the present study) and for 40 HCMV clinical isolates (7) demonstrated that the valine at amino acid 823 was present in the DNA polymerases of all of these viruses. The correlation of antiviral activity of these compounds against wild-type HSV-1, HSV-2, and HCMV and the drop in antiviral activity against the V823A HSV-1 and HSV-2 mutants and the V823A/V824L HCMV mutant provides strong evidence that the mechanism of action of these compounds is inhibition of the viral DNA polymerase. In addition, in vitro DNA polymerase assays confirmed that a single amino acid change of V823A results in a loss of 4-oxo-DHQ inhibition with the HSV-1, HSV-2, HCMV, VZV, and HHV-8 polymerases. It is interesting that the V823M mutation found in the HSV-1 KOS-M2 mutant required at least one additional amino acid change in the HSV-1 polymerase in order to confer resistance to the 4-oxo-DHQs (Table 4). The S724N amino acid change found in the KOS-M2 polymerase is located in conserved domain II of the HSV polymerase (Fig. 2). Both conserved domains II and III are involved in substrate recognition (19). The S724N mutation by itself confers resistance to ACV, FOS, and phosphonoacetic acid (9, 17). The S724N change alone does not confer resistance to PNU-183792, as shown by the fact that a clone containing S724N and T612I amino acid changes did not rescue PNU-183792 resistance (Table 4). Kinetic analysis with the S724N HSV-1 polymerase demonstrated an increase in Km for both ACV-TP and dGTP, along with a >3-fold decrease in kcat for ACV-TP incorporation (19). A proposed model of the HSV-1 polymerase active site, based on the crystal structure of bacteriophage RB69 polymerase, puts amino acids S724 and V823 in close proximity and may explain why changing both amino acids results in PNU-183792 resistance.

The recently published structure of the DNA polymerase from bacteriophage RB69 complexed with primer template DNA and dTTP demonstrated the importance of region III for interaction with incoming nucleotide and primer template DNA (16). Several region III amino acids from the RB69 polymerase (including amino acid N572, which is equivalent to amino acid V823 of the HSV-1 polymerase) interact with the template strand of the primer-template DNA and with the incoming dNTP. These interactions appear to be important for holding the template in place during the addition of the incoming nucleotide to the primer strand. If the RB69 structure is applicable to the HSV-1 polymerase, then binding of a 4-oxo-DHQ to the 823 region of the polymerase could disrupt the interaction of the polymerase with the template strand of the DNA and interfere with the binding or incorporation of the incoming dNTP.

PNU-183792 has good oral bioavailability that ranged from 50 to 100% in mice, rats, and dogs (3). Pharmacokinetic studies in uninfected mice demonstrated that the blood levels (Cmax = 12 µM) after a 25-mg/kg oral dose of PNU-183792 were >3-fold above the cell culture IC50 for HCMV, murine CMV, and HSV (3). In a murine CMV lethal challenge model, PNU-183792 was found to protect mice whether the drug was initiated at 0, 24, or 48 h after infection, and PNU-183792 proved to be as effective as GCV in this model (3). PNU-183792 was well tolerated in rodent safety studies at doses 10 times greater (250 mg/kg/day) than the efficacious dose. The ability of the 4-oxo-DHQs to inhibit the growth of ACV- and GCV-resistant mutants highlights the potential of these compounds in the treatment of drug-resistant herpesvirus infections found in immunocompromised individuals. The broad-spectrum anti-herpesvirus activity of these compounds also makes these compounds interesting as potential agents for the prophylactic treatment of herpesviruses in immunocompromised patents. These compounds would represent the first class in which a single drug could be used to inhibit both EBV and CMV replication, especially in transplant patients, in whom these two viruses are a major cause of morbidity and mortality (12, 14). Several other classes of herpesvirus antivirals have been reported, but none of them show the broad-spectrum anti-herpesvirus activity of the 4-oxo-DHQs (1, 4, 13, 18). The 4-oxo-DHQs offer a unique opportunity in which a single drug could be used to treat many of the diseases caused by HHVs.


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ACKNOWLEDGMENTS
 
We thank Janet Wieber for help with the polymerase assays and Robert Ricketts for help with the single-step growth curve.


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FOOTNOTES
 
* Corresponding author. Mailing address: Infectious Disease Research, 7263-267-507, Pharmacia Corporation, 301 Henrietta St., Kalamazoo, MI 49007. Phone: (616) 833-9724. Fax: (616) 833-2599. E-mail: fred.l.homa{at}pharmacia.com. Back


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Journal of Virology, February 2003, p. 1868-1876, Vol. 77, No. 3
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.3.1868-1876.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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  • Tchesnokov, E. P., Gilbert, C., Boivin, G., Gotte, M. (2006). Role of Helix P of the Human Cytomegalovirus DNA Polymerase in Resistance and Hypersusceptibility to the Antiviral Drug Foscarnet. J. Virol. 80: 1440-1450 [Abstract] [Full Text]  
  • Gershburg, E., Pagano, J. S. (2005). Epstein-Barr virus infections: prospects for treatment. J Antimicrob Chemother 56: 277-281 [Abstract] [Full Text]  
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