Inhibitors of Nucleotidyltransferase Superfamily Enzymes Suppress Herpes Simplex Virus Replication

Updated information and services can be found at: http://aac.asm.org/content/58/12/7451 These include: SUPPLEMENTAL MATERIAL REFERENCES

CONTENT ALERTS

Supplemental material This article cites 74 articles, 25 of which can be accessed free at: http://aac.asm.org/content/58/12/7451#ref-list-1 Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more»

Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/

Downloaded from http://aac.asm.org/ on November 21, 2014 by NORTHERN ILLINOIS UNIV

John E. Tavis, Hong Wang, Ann E. Tollefson, Baoling Ying, Maria Korom, Xiaohong Cheng, Feng Cao, Katie L. Davis, William S. M. Wold and Lynda A. Morrison Antimicrob. Agents Chemother. 2014, 58(12):7451. DOI: 10.1128/AAC.03875-14. Published Ahead of Print 29 September 2014.

Inhibitors of Nucleotidyltransferase Superfamily Enzymes Suppress Herpes Simplex Virus Replication John E. Tavis, Hong Wang, Ann E. Tollefson, Baoling Ying, Maria Korom, Xiaohong Cheng, Feng Cao,* Katie L. Davis, William S. M. Wold, Lynda A. Morrison

Herpesviruses are large double-stranded DNA viruses that cause serious human diseases. Herpesvirus DNA replication depends on multiple processes typically catalyzed by nucleotidyltransferase superfamily (NTS) enzymes. Therefore, we investigated whether inhibitors of NTS enzymes would suppress replication of herpes simplex virus 1 (HSV-1) and HSV-2. Eight of 42 NTS inhibitors suppressed HSV-1 and/or HSV-2 replication by >10-fold at 5 ␮M, with suppression at 50 ␮M reaching ⬃1 millionfold. Five compounds in two chemical families inhibited HSV replication in Vero and human foreskin fibroblast cells as well as the approved drug acyclovir did. The compounds had 50% effective concentration values as low as 0.22 ␮M with negligible cytotoxicity in the assays employed. The inhibitors suppressed accumulation of viral genomes and infectious particles and blocked events in the viral replication cycle before and during viral DNA replication. Acyclovir-resistant mutants of HSV-1 and HSV-2 remained highly sensitive to the NTS inhibitors. Five of six NTS inhibitors of the HSVs also blocked replication of another herpesvirus pathogen, human cytomegalovirus. Therefore, NTS enzyme inhibitors are promising candidates for new herpesvirus treatments that may have broad efficacy against members of the herpesvirus family.

H

erpesviruses are enveloped viruses with large, doublestranded DNA genomes (1). Herpes simplex viruses (HSVs) replicate lytically in epithelial cells near their site of entry into the body (2). The virus then enters sensory nerves and establishes latent infection of the neurons, where it remains latent for the life of the host. Episodic reactivation from latency causes lytic replication at mucosal surfaces, triggering recurrent disease and providing the opportunity for transmission to uninfected individuals (3). The eight human herpesviruses cause an array of severe diseases associated with primary and recurrent infections. Herpes simplex virus 1 (HSV-1) and HSV-2 are closely related viruses with colinear genomes. HSV-1 infects more than half of all Americans (4) and causes gingivostomatitis, cold sores, encephalitis, herpetic stromal keratitis, and an increasing proportion of anogenital lesions (2, 5, 6). HSV-2 infects nearly one in five of Americans (4) and is the primary cause of ulcerative anogenital lesions (4). It also increases the risk of human immunodeficiency virus (HIV) acquisition (7, 8). HSV-1 and HSV-2 can be transmitted from a pregnant woman to her child during birth, often causing potentially fatal disseminated disease in the newborn (9). Treatment of herpesvirus infections primarily relies on nucleoside analog inhibitors of the viral DNA polymerase, including acyclovir (ACV), penciclovir, ganciclovir, valaciclovir, valganciclovir, brivudine, and famciclovir (10, 11). Several newer agents are undergoing clinical development (11, 12), but none of them can fully suppress herpesvirus infections (12). Viral strains resistant to the current drugs exist and are especially common in immunocompromised individuals (11, 13–16), but they are also significant in patients with ocular infections and in children (14, 17, 18). Cross-resistance to existing nucleoside analog drugs is common, because these drugs depend on the viral thymidine kinase (TK) and/or polymerase for their efficacy (18–20). Thus, new drugs with a different mechanism of action are needed. HSV genomic replication employs numerous viral enzymes. Replication (21, 22) begins when the viral linear double-stranded

December 2014 Volume 58 Number 12

DNA genome circularizes shortly after infection, possibly via recombination (23, 24). DNA replication initiates at one or more of three viral origins of DNA replication and is primed by the viral helicase-primase complex (HSV-1 proteins pUL5, pUL8, and pUL52). DNA replication requires the single-stranded DNAbinding protein pUL29 (ICP8), which is predicted to contain an RNase H-like fold (25). The viral DNA polymerase holoenzyme complex (pUL30 DNA polymerase plus pUL42) catalyzes DNA elongation by a presumed double-stranded rolling-circle mechanism. This complex possesses 5=-3= exonuclease, 3=-5= exonuclease, and RNase H activities (26). DNA replication initially produces head-to-tail linear concatemers, and branched concatemers accumulate later in the replication cycle through recombination and/or reinitiation mechanisms. Formation of the branched forms via recombination is stimulated by the pUL12 exonuclease (27). Last, the viral terminase complex (pUL15, pUL28, and pUL33) cleaves the viral DNA to unit length during packaging of the genome into viral capsids, and the crystal structure of pUL15 shows an RNase H-like fold (28). The nucleotidyltransferase superfamily (NTS) is a group of diverse enzymes whose members share a similar protein fold and enzymatic mechanisms (29). These enzymes function in nucleic

Received 14 July 2014 Returned for modification 9 September 2014 Accepted 26 September 2014 Published ahead of print 29 September 2014 Address correspondence to John Tavis, [email protected], or Lynda Morrison, [email protected]. * Present address: Feng Cao, John Cochran Division, Department of Veteran’s Affairs Medical Center, St. Louis, Missouri, USA. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.03875-14. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.03875-14

Antimicrobial Agents and Chemotherapy

p. 7451–7461

aac.asm.org

7451

Downloaded from http://aac.asm.org/ on November 21, 2014 by NORTHERN ILLINOIS UNIV

Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, Missouri, USA

Tavis et al.

MATERIALS AND METHODS Cells and viruses. Vero cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 3% newborn calf serum, 3% bovine growth serum, 2 mM L-glutamine, and 100 IU/ml penicillin– 0.1 mg/ml streptomycin (P/S). Human foreskin fibroblasts (HFFs) (ATCC CRL2429) were cultured in DMEM containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, and P/S. The HSV-1 and HSV-2 strains used for screening were deidentified clinical isolates from the Saint Louis University Hospital passaged once in culture. Wild-type HSV-1 and HSV-2 used in Fig. 6 were laboratory strains 17 and 333, respectively. ⌬TK is a thymidine kinase (TK)-deficient mutant of HSV-1 strain 17 (53). ⌬TK⫺ is a TK-deficient mutant of HSV-2 strain 333 (54). Virus stocks were grown, and the titers of the virus were determined on Vero cells (55, 56). Human cytomegalovirus (HCMV) strain AD169 was obtained from the American Type Culture Collection (ATCC) (VR-538). Compound acquisition. Compounds were purchased (company names are in Fig. S1 in the supplemental material) or obtained through the National Cancer Institute Developmental Therapeutics compound repository (compounds 46 to 56). Compounds were dissolved in dimethyl sulfoxide (DMSO) at 10 mM and stored at ⫺80°C. HSV-1 and HSV-2 replication inhibition assay. Compounds were diluted in phosphate-buffered saline (PBS) containing 2% newborn calf serum and 2 mM L-glutamine and then added to confluent cell monolayers in 24-well plates. Equivalent dilutions of DMSO were used as vehicle controls. HSV-1 and HSV-2 were diluted in the supplemented PBS medium and added so that the final compound concentrations were 50 ␮M or 5 ␮M and the HSV multiplicity of infection (MOI) was 0.1. The cells

7452

aac.asm.org

were incubated at 37°C for 1 h, virus-containing inoculum was removed, the wells were washed once in PBS, and compound (50 ␮M or 5 ␮M) in supplemented DMEM was added. Cells were incubated at 37°C an additional 23 h, and then the plates were inspected by phase-contrast microscopy for cytopathic effect (CPE) or toxicity. Wells in which the cells were substantially healthier than in the DMSO-treated control wells were harvested by scraping, along with additional wells with significant CPE for comparison. Samples were frozen at ⫺80°C, thawed, and sonicated, and then virus titers were determined by plaque assay on Vero cells (55). Each experiment was repeated at least once. Fifty percent effective concentrations (EC50s) were determined as described above except that serial dilutions of the compounds were employed. Values were calculated with GraphPad Prism using the three-parameter log (inhibitors) versus response algorithm with the bottom value set at zero. Toxicity assays. Quantitative cytotoxicity assays were done in triplicate in 96-well plates in which the compounds were added at 0.78 to 100 ␮M in a final concentration of 1% (vol/vol) DMSO. The cells were incubated for 24 h under conditions employed for the replication inhibition assays. Mitochondrial toxicity was measured by incubating the cells with 0.25 mg/ml thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich), the cultures were incubated for 60 min, metabolites were solubilized in acidic isopropanol, and absorbance was read at 570 nm. Cellular lysis was measured by detecting release of intracellular proteases with the CytoToxGlo assay (Promega) according to the manufacturer’s instructions. Percent viability was determined for each compound concentration, and 50% cytotoxic concentration (CC50) values were calculated using GraphPad Prism using the four-parameter variable response log (inhibitors) versus response algorithm with the bottom value set at zero. Quantitative PCR measurement of viral genome levels. Cells and virus associated with cell debris were pelleted by centrifugation for 30 min at 21,100 ⫻ g, and total DNA was isolated from the pellets using a QIAamp DNA minikit (Qiagen) according to the manufacturer’s instructions. The primers and probe for real-time PCR targeted the HSV-2 latency-associated transcript region. The primers and probe sequences were as follows: forward primer, 5=-GAGCTAACACTCGGCTTGCT-3=; reverse primer, 5=-TCTCCTCCCCGTCTTTCC-3=; and Universal Probe Library probe 10, 5=-FAM-GGAGGTG-dark quencher-3= (Roche) (FAM stands for 6-carboxyfluorescein). PCR mixtures contained FastStart Universal Probe master mix containing Rox (Roche), 900 nM forward and reverse primers, 250 nM probe, and 30 to 100 ng DNA template. Quantification was performed in triplicate using an ABI 7500 genetic analyzer (Applied Biosystems) with 1 cycle at 50°C for 2 min and 1 cycle at 95°C for 10 min, followed by 40 cycles of amplification, with each cycle consisting of 95°C for 15 s and 60°C for 1 min. A total of 103 to 108 copies of pcDNA3.1(⫹)5=Flag-ICP0 were used to generate a standard curve. Data were analyzed using ABI 7500 sequence detection system software (ABI) and expressed as HSV-2 genome equivalents per nanogram of total DNA. HCMV replication inhibition and toxicity assays. Compounds were tested against HCMV by the National Institute of Allergy and Infectious Disease’s Cooperative Antiviral Testing Group following their standardized protocols. Briefly, low-passage-number primary HFFs were infected with HCMV strain AD169 in the presence of serial dilutions of the compounds from 0.032 to 100 ␮M. Ten days later, the monolayers were stained with 1% neutral red solution in PBS. After the monolayers were rinsed, plaques were counted using a stereomicroscope, and the EC50 was interpolated from the data. Cytotoxicity was measured using a plate-based assay in which compounds were added to HFFs at 0.032 to 100 ␮M. At the end of the incubation period, plates were stained for 1 h with a neutral red solution in PBS. The stain was then removed, the plates were rinsed in PBS and dried, and dye internalized by viable cells was determined at an optical density at 550 nm (OD550). CC50 values were interpolated from the data.

RESULTS

Compound selection. Compounds to be screened against HSV-1 and HSV-2 were selected because they (i) inhibit the HIV RNase H

Antimicrobial Agents and Chemotherapy

Downloaded from http://aac.asm.org/ on November 21, 2014 by NORTHERN ILLINOIS UNIV

acid metabolic events, including RNA and DNA digestion, DNA recombination, DNA integration, replication fork repair, DNA repair, and microRNA (miRNA) maturation and function. NTS enzymes include Escherichia coli RNase H 1 and 2 (30, 31), human RNase H 1 and 2 (32, 33), the RuvC Holliday junction resolvase (34), the Argonaute RNase (35), the hepatitis B virus (HBV) RNase H (36–38), the HIV RNase H (39), and the HIV integrase (40). This class of enzymes is characterized by the spatial arrangement of three or four conserved carboxylates (the DDE or DEDD motifs) in their active sites. These carboxylates coordinate two divalent cations essential for the nucleic acid cleavage activities of the enzymes (29, 41, 42). The HSVs express numerous proteins involved in nucleic acid metabolism, and they upregulate expression of cellular proteins that assist viral DNA replication. Many of these enzymes, especially the nucleases, have catalytic properties consistent with NTS members. The HIV RNase H and integrase have attracted much attention as potential drug targets, with hundreds of compounds being reported to inhibit these enzymes (43, 44). Most of the inhibitors bind at the active site via interactions with the divalent cations (45–48). Three anti-HIV integrase drugs have been approved by the Food and Drug Administration (FDA) (raltegravir, elvitegravir, and dolutegravir), but no anti-RNase H drugs have yet been approved. As predicted from their common NTS membership (29, 39) and similar orientation of active site cations (49), some anti-HIV RNase H compounds inhibit the integrase, and some anti-integrase compounds inhibit the RNase H (46, 50–52). Furthermore, we recently demonstrated that the HBV RNase H is sensitive to many anti-HIV RNase H and integrase antagonists (36–38). This capacity of NTS enzyme inhibitors to cross-inhibit distant superfamily members led us to hypothesize that HSV-1 and HSV-2 would be sensitive to NTS inhibitors because enzymes with activities typical of this superfamily play multiple critical roles during HSV replication.

NTS Enzyme Inhibitors Suppress HSV Replication

December 2014 Volume 58 Number 12

that suppressed HSV replication by ⱖ2 log10 units at 5 ␮M, plus a number of other compounds for comparison. Cells were incubated for 24 h with 100 to 0.78 ␮M concentrations of the compound under the conditions employed for the replication inhibition assays. Mitochondrial toxicity was measured using an MTT assay, and cellular lysis was measured by assessing release of intracellular proteases into the culture medium using the CytoTox-Glo assay (Promega). All compounds selected for cytotoxicity assessment had 50% cytotoxic concentration (CC50) values of ⬎50 ␮M by both the MTT (Table 1) and cellular lysis assays (data not shown). NTS inhibitors suppress HSV-1 and HSV-2 replication in HFFs. Inhibition of viral replication was assessed in human foreskin fibroblast (HFF) cultures to determine whether NTS inhibitors could block HSV-1 and HSV-2 replication in a physiologically relevant, primary human cell type. HFFs were infected with HSV-1 or HSV-2 at an MOI of 0.1 and treated with ACV or compounds at 50 or 5 ␮M or with a DMSO vehicle control. Viral yields 24 h postinfection were measured by plaque assay. Eight inhibitors of HSV-1 and HSV-2 at 5 ␮M in Vero cells (compounds 30, 41, 46, 49, 50, 55, 56, and 59) were selected for analysis, and six compounds with ⬍1 log10 unit activity were selected as controls (compounds 47, 48, 52, 53, 61, and 62). As expected, ACV inhibited HSV-1 and HSV-2 with similar efficacies in Vero cells and HFFs (Table 1 and Fig. 2). The eight compounds that inhibited HSV-1 and HSV-2 in Vero cells also inhibited the viruses to similar or greater degrees in HFFs, with the exception of compound 30 which inhibited HSV-2 ⬃45-fold less effectively at 5 ␮M in HFFs. The six compounds that were ineffective in Vero cells remained inactive in HFFs. CC50 values in HFFs by the MTT assay were ⬎100 ␮M, with the exception of compounds 50 and 61 (Table 1). Therefore, NTS inhibitors can suppress HSV-1 and HSV-2 replication in a natural human host cell. EC50s. We determined EC50s in Vero cells for all compounds that inhibited HSV-1 or HSV-2 at 5 ␮M by ⱖ3 log10 units (Fig. 3 and Table 1). Compound 59, which had less activity, was included for comparison. EC50s against HSV-1 ranged from 4.20 ␮M for compound 30 down to 0.24 ␮M for compound 49. For HSV-2, the values ranged from 4.12 ␮M for compound 59 down to 0.22 ␮M for compound 49. ACV had EC50s of 0.16 and 1.44 ␮M versus HSV-1 and HSV-2, respectively. This led to therapeutic index (TI) values which ranged from ⬎52 for compound 46 to ⬎417 for compound 49 against HSV-1 and from 24 for compound 46 to ⬎455 for compound 49 against HSV-2. TI values in this assay for ACV were ⬎625 for HSV-1 and ⬎69 for HSV-2. EC50s were also measured in HFFs for compounds 30, 41, 46, 49, 55, 56, and 59 (Table 1). These values ranged from 2.81 to 0.13 ␮M for HSV-1 and from 1.67 to 0.38 ␮M for HSV-2. The TI values in HFFs ranged from ⬎36 for compound 55 to ⬎699 for compound 41 against HSV-1 and from ⬎60 for compound 55 to ⬎263 for compound 46 against HSV-2. For comparison, ACV had TI values of ⬎833 for both HSV-1 and HSV-2 in HFFs. NTS inhibitors affect multiple stages of herpesvirus replication. To begin defining the stage(s) of viral replication targeted by the compounds, we incrementally delayed the time postinfection that the inhibitors were added. ACV and compounds 41 and 46 as representative inhibitors from different chemical classes were added to Vero cells at 10 ␮M and concurrently infected with HSV-2 (0 h postinfection), or compound was added at 1 to 12 h postinfection. All cultures were incubated until 24 h postinfection,

aac.asm.org 7453

Downloaded from http://aac.asm.org/ on November 21, 2014 by NORTHERN ILLINOIS UNIV

or integrase, (ii) inhibit the HBV RNase H as determined by our previous studies (36–38), or (iii) are close chemical relatives of inhibitors of the HIV RNase H, HIV integrase, or the HBV RNase H. Forty-two compounds were chosen to maximize the chemical diversity assessed and to evaluate multiple compounds within six chemical families: tropolones, polyoxygenated heterocycles, HIV integrase strand transfer inhibitors, cyanopyrans, aminocyanothiophenes, and hydroxyxanthenones (see Fig. S1 in the supplemental material). Primary screening for HSV-1 and HSV-2 replication inhibitors. The 42 compounds plus ACV as a representative nucleos(t)ide [nucleoside(nucleotide)] analog were tested for the ability to inhibit replication of primary HSV-1 and HSV-2 clinical isolates. Vero cells are the standard laboratory host cell for HSVs and were chosen for initial screening because they are primate cells that are highly permissive for HSV. Vero cell monolayers were treated with compound (50 or 5 ␮M) or a DMSO vehicle control and simultaneously infected at a multiplicity of infection (MOI) of 0.1. Monolayers were microscopically inspected 24 h postinfection, and cultures in which cells were healthier than the DMSOtreated controls were collected for determination of viral titer by plaque assay. Table 1 shows the results for all compounds that inhibited viral replication by ⱖ1 log10 unit, and Fig. 1 shows the data for all compounds at 5 ␮M; the complete data set is in Table S1 in the supplemental material. Inhibition by ⱖ3 log10 units at 50 ␮M was observed for 10 compounds against HSV-1 and 12 compounds against HSV-2. Importantly, four compounds (compounds 41, 46, 49, and 56) inhibited HSV-1 and HSV-2 by ⱖ4 log10 units at 5 ␮M. Two additional compounds inhibited HSV-1 or HSV-2 by ⱖ3 log10 units at 5 ␮M (compounds 55 and 30, respectively). Maximal suppression was achieved with compound 56 against HSV-2 (reduction by nearly 6 log10 units at 5 ␮M [Table 1 and Fig. 1]). These most effective inhibitors were in the tropolone, polyoxygenated heterocycle, and hydroxyxanthenone derivative families. The strand transfer inhibitors tested, including raltegravir, were ineffective under these conditions. Overall, inhibition by ⱖ1 log10 unit was observed at 5 ␮M for 19% of the compounds against HSV-1 and 17% of the compounds against HSV-2. The best antagonist at 5 ␮M was compound 56 (manicol), which inhibited HSV-1 by 5.14 log10 units (138,000-fold) and HSV-2 by 5.95 log10 units (891,000-fold). For comparison, the approved anti-HSV drug ACV inhibited HSV-1 replication at 5 ␮M by 3.61 log10 units (4,070-fold) and HSV-2 by 2.88 log10 units (758-fold) in this assay. The compounds had similar levels of activity against HSV-1 and HSV-2 in Vero cells (Table 1 and Fig. 1). The only prominent exception was compound 30, which was ⬎100-fold more active against HSV-2 than HSV-1 at 5 ␮M. Therefore, this screen of only 42 compounds identified five compounds (compounds 41, 46, 49, 55, and 56) with inhibitory activity against HSV-1 and/or HSV-2 comparable to or better than ACV. We next asked whether inhibition of viral replication could be overcome by infecting cells at an MOI of 5 instead of 0.1. Cells were treated with ACV, compound 46, or compound 55 at 50 or 5 ␮M, and viral yields at 24 h postinfection were measured by plaque assay. ACV, compound 46, and compound 55 at 5 ␮M all efficiently controlled virus replication after infection at a high MOI; 50-fold-more input virus yielded only 50-fold-higher titers at 24 h (data not shown). Cytotoxicity. Cytotoxicity was measured for all compounds

aac.asm.org

Antimicrobial Agents and Chemotherapy

Beta-thujaplicinol Beta-thujaplicin Gamma-thujaplicin Nootkatin 5-Nitrosotropolone NSC 79556 Tropolone NSC 282885 Manicol Chembridge 5945310 Chembridge 5946384

TRC 939800 Ciclopirox Sigma PH008969

Sigma S439274 Chembridge 7248520 Chembridge 5104346 Indofine D-009

Acyclovir

Tropolones 46 47 48 49 50 52 53 55 56 59 62

Polyoxygenated heterocycles 1 41 43

Hydroxyxanthenones 8 30 31 34

Nucleoside analog ACV

5.39

0.86 4.02 3.01 2.64

2.66 5.23 1.31

4.81 1.86 4.71 4.72 NQ 2.02 1.90 5.04 5.82 4.24 2.36

50 ␮M

3.61

⫺0.18 1.39 0.21 0.08

0.28 4.29 0.31

5.01 0.31 0.31 5.10 2.07 0.07 0.25 3.62 5.14 1.65 0.38

5 ␮M

0.16 ⫾ 0.17

4.20 ⫾ 2.45

0.27 ⫾ 0.00

0.42 ⫾ 0.19 0.35 ⫾ 0.35 1.09 ⫾ 0.47

0.24 ⫾ 0.06

1.94 ⫾ 0.14

EC50 (␮M)b

4.73

2.49 4.55 3.56 3.29

3.12 5.25 1.13

5.12 1.38 4.57 5.05 NQ 1.34 1.45 5.06 5.79 4.71 1.93

50 ␮M

2.88

0.18 3.69 0.22 -0.07

⫺0.11 5.50 0.31

5.32 0.22 0.20 4.91 1.98 0.10 0.45 2.93 5.95 1.22 0.03

5 ␮M

1.44 ⫾ 0.04

3.93 ⫾ 3.92

0.45 ⫾ 0.10

1.07 ⫾ 0.75 0.58 ⫾ 0.01 4.12 ⫾ 0.65

0.22 ⫾ 0.04

1.69 ⫾ 0.06

EC50 (␮M)b

⬎100

⬎100 ⬎100

⬎100 ⬎50

⬎100 ⬎100 ⬃100

⬎100

⬎100

6.69

5.23

5.57

6.39 5.06 5.99 6.27 NQ NQ 2.13 6.17 6.39 6.03 3.35

CC50 (␮M)c 50 ␮M

Log10 suppression

Log10 suppressiona

4.68

3.22

5.06

5.83 0.02 0.05 5.79 2.92 0.10 ⫺0.03 4.99 5.72 2.56 0.12

5 ␮M

0.12

0.32

0.14

2.81 0.24 1.63

0.74

0.97

EC50 (␮M)

HSV-2

6.27

6.68

4.97

5.83 5.16 5.76 5.98 NQ NQ 1.22 6.55 6.80 5.41 2.32

50 ␮M

3.84

2.04

5.21

5.17 0.08 0.20 5.47 2.88 ⫺0.01 0.29 3.94 5.93 1.16 0.02

5 ␮M

Log10 suppression

0.12

0.52 ⫾ 0.43

1.00

1.67 ⫾ 1.53 0.55 0.84

1.23 ⫾ 0.98

0.38

EC50 (␮M)d

⬎100

⬎100

⬎100

⬎100 ⬎100 ⬎100 ⬎100 47.0 ⫾ 2.3 ⬎100 ⬎100 ⬎100 ⬎100 ⬎100 ⬎100

CC50 (␮M)e

Difference between the titer of the DMSO-treated vehicle control and the titer of the compound-treated culture in log10 units. Data are the averages from two to four experiments, each done in duplicate. Standard deviations are shown in Fig. 1. NQ, not quantified due to obvious toxicity. b Average ⫾ 1 standard deviation from two or three experiments, each done in duplicate. c MTT assay confirmed by cell rupture assay; each assay was done in triplicate. Quantitative values not reported because the data did not fit the regression curve. d Average ⫾ 1 standard deviation from one or two experiments, each done in duplicate. e MTT assay done in triplicate. Quantitative values not reported except for compound 42 because the data did not fit the curve. Data for compound 42 are the average ⫾ 1 standard deviation.

a

Compound name

Class and compound no.

HSV-1

HSV-2

HSV-1 Log10 suppression

HFF cells

Downloaded from http://aac.asm.org/ on November 21, 2014 by NORTHERN ILLINOIS UNIV

7454

Vero cells

TABLE 1 Suppression of HSV-1 and HSV-2 replication by compounds related to known inhibitors of NTS enzymes

Tavis et al.

Polyoxygenated heterocycles

Tropolones

7.0

Hydroxyxanthenones

6.0

HSV-1

5.0

HSV-2

4.0 3.0 2.0

HIV strand transfer Aminocyanoinhibitors thiophenes

Cyanopyrans

*

* * *

Miscellaneous Compounds

* * *

*

*

2 3 4 7 9 22 38

* *

6 24 27 28

*

10 11 66 70

1 41 42 43 44 45

46 47 48 49 50 52 53 55 56 59 61 62

DMSO ACV

* *

8 30 31 34 35 39

*

0.0

5 19 21

1.0

Compound (5 μM)

FIG 1 Inhibition of HSV-1 and HSV-2 replication by NTS antagonists at 5 ␮M. Compounds were added to Vero cells simultaneously with HSV-1 or HSV-2 infection at an MOI of 0.1. Twenty-four hours later, the cultures were harvested, and infectious virus titers in the cultures were determined by plaque assay. Replication inhibition is expressed as the difference in recovered titers in the compound-treated cultures compared to values from the DMSO vehicle-treated control. Data are the averages plus 1 standard deviation (error bars) from two to four experiments per compound, each done in duplicate. Asterisks indicate that the titer was not determined because the CPE was visually equivalent to that of the DMSO control. ACV, acyclovir.

when infectious viral titers were determined by plaque assay. Viral DNA in total DNA harvested from replicate cultures was measured by quantitative PCR and expressed as genome equivalents (GE) per nanogram of total DNA. ACV inhibited accumulation of both viral DNA and infectious virus (Fig. 4A). This effect was greater against viral progeny formation than DNA accumulation, especially when the compound was added early during infection. Delaying addition of ACV as much as 5 h postinfection did not alter inhibition of either DNA accumulation or viral titers, but delaying addition until past 5 h postinfection led to a gradual loss of inhibition. ACV became essentially ineffective when added at 12 h postinfection. Therefore, events targeted by ACV start approximately 5 h postinfection and are finished by ⬃12 h postinfection. This is consistent with ACV’s mechanism as a direct inhibitor of viral DNA elongation (57–59). Compounds 41 and 46 also inhibited both viral DNA replication and accumulation of infectious virus. Like ACV, the effect of both of these compounds was greater on infectious virus titers than on DNA accumulation (Fig. 4B and C), and the compounds had little efficacy when added 12 h postinfection. In sharp contrast to ACV, however, both compounds had progressively less effect on both viral DNA accumulation and virus titers if addition of the compounds was delayed by more than 1 h after infection. Thus,

compounds 41 and 46 inhibited a very early step in viral replication, and delay of compound addition until after this step restored some virus replication. Also in contrast to ACV, there was no subsequent, clearly defined point where efficacy of the NTS inhibitors began to fail. Rather, the effect of delaying addition of compounds 41 and 46 was gradual between 1 and 12 h postinfection. To investigate the possibility that a compound was directly toxic to virions, we preincubated compound 46 with concentrated virus stock and subsequently diluted the virus for infection of cells at an MOI of 0.1. Preincubation in compound had no effect on viral titer (Fig. 5). Together, these studies indicate that compounds 41 and 46 inhibit at least one event that occurs at a very early, postentry stage of viral replication, plus one or more events that occur during a later phase of replication. NTS inhibitors suppress replication of acyclovir-resistant HSV-1 and HSV-2 mutants. ACV is a nucleoside analog prodrug that must be phosphorylated by the viral thymidine kinase (TK) to become a substrate for the viral DNA polymerase (60). HSV TKdeficient mutants are therefore insensitive to ACV. Because viral resistance to ACV and other nucleoside analogs is a significant medical problem (13, 18, 61), especially in immunocompromised patients (13, 16, 62), we asked whether defined HSV-1 and HSV-2

Replicaon inhibion (Log10)

7.0 HSV-1 Vero

6.0

HSV-1 HFF 5.0

HSV-2 Vero

4.0

HSV-2 HFF

3.0 2.0 1.0 0.0 DMSO ACV

30

41

46

47

48

49

50

52

53

55

56

59

61

62

Compound (5 μM) FIG 2 Inhibition of HSV-1 and HSV-2 replication in human fibroblasts at 5 ␮M. Compounds were added to HFF cells simultaneously with HSV-1 or HSV-2 infection at an MOI of 0.1, and infectious virus titers in the cultures at 24 h postinfection were determined by plaque assay on Vero cells as described in the legend to Fig. 1. The HFF data are plotted next to the Vero cell data from Fig. 1 for comparison. Data are the averages ⫾ 1 standard deviation from two experiments per compound, each done in duplicate.

December 2014 Volume 58 Number 12

aac.asm.org 7455

Downloaded from http://aac.asm.org/ on November 21, 2014 by NORTHERN ILLINOIS UNIV

Replicaon inhibion (log10)

NTS Enzyme Inhibitors Suppress HSV Replication

Tavis et al.

TK-deficient mutants would be sensitive to NTS inhibitors. Vero cells were infected with laboratory strains of HSV-1 or HSV-2 and engineered TK-deficient mutants of the same strains. The cells were treated with 50 ␮M ACV or compound 30, 41, or 46, and viral yields 24 h postinfection were measured by plaque assay. ACV efficiently inhibited wild-type HSV-1 and HSV-2 replication, but it had little effect on the TK-deficient mutants (Fig. 6A). In marked contrast, compounds 30, 41, and 46 efficiently inhibited the wild-type and TK-deficient mutant strains of both HSV-1 and HSV-2. Similar patterns were seen when the compounds were used at 5 ␮M (Fig. 6B), although inhibition by compound 30 at 5 ␮M was too weak to definitively interpret. Therefore, NTS inhibitors from three different chemical families do not require phosphorylation by the viral TK enzyme to be active, confirming that these NTS inhibitors suppress HSV-1 and HSV-2 replication in a different manner than ACV does.

7456

aac.asm.org

NTS inhibitors have antiviral efficacy against HCMV. Last, we tested the ability of the most effective HSV inhibitors to inhibit replication of another major human herpesvirus pathogen, HCMV (63). This was based on the prediction that other members of the herpesvirus family would also be sensitive to NTS inhibitors due to their shared genomic replication mechanism. Compounds 41, 46, 49, 55, 56, and 59 and the FDA-approved anti-HCMV drug ganciclovir were evaluated with plaque reduction and toxicity assays in HFFs. HFFs were infected with HCMV in the presence of serial dilutions of the compounds. After 10 days, the cultures were stained with neutral red, and plaques were counted. Compound cytotoxicity for uninfected cells was determined in the same manner except that neutral red uptake by viable cells was measured by optical density. Compounds 41, 46, 49, 55, and 59 inhibited HCMV replication with EC50s of 0.33 to 1.94 ␮M (Table 2). In contrast to the 24-h HSV assays in HFFs, the NTS inhibitors had

Antimicrobial Agents and Chemotherapy

Downloaded from http://aac.asm.org/ on November 21, 2014 by NORTHERN ILLINOIS UNIV

FIG 3 EC50s for select inhibitors of HSV-1 and HSV-2. Serially diluted compounds were added to Vero cells simultaneously with HSV-1 or HSV-2 infection at an MOI of 0.1, and infectious virus titers in the cultures at 24 h postinfection were determined by plaque assay. The curves are for one representative compound in each of the hydroxyxanthenone (compound 30), polyoxygenated heterocycle (compound 41), and hydroxylated tropolone (compound 49) classes. The EC50s are the averages from two or three experiments, each done in duplicate.

GE Titer

DMSO

7 6 Compound #41

4

GE

3

Titer

2

DMSO

0 0 2 4 6 8 10 12 14 Time of compound addion (hours P.I.)

7 6

FIG 5 Effect of preincubating compound 46 and the viral inoculum on virus infectivity. A high-titer HSV-1 stock was preincubated in 50 ␮M compound 46 or DMSO control (mock preincubation). The inoculum was then diluted to reduce compound 46 to 0.05 ␮M and virus titer such that a standard replication inhibition assay at an MOI of 0.1 was performed. Compound 46 was added to 0.05 ␮M in the mock preincubation sample for consistency. A standard inhibition assay in which compound 46 was used at 50 ␮M throughout the assay was included as a control. Values are the averages ⫾ 1 standard deviation from two experiments, each done in duplicate.

Compound #46 GE

3

Titer

2 1 0 0 2 4 6 8 10 12 14 Time of compound addion (hours P.I.)

FIG 4 Effects of delaying inhibitor addition during the course of HSV-2 infection. Compounds were added to Vero cells at 10 ␮M either concurrently with infection at an MOI of 0.1 (time zero) or at the indicated times postinfection (hours postinfection [P.I.]), and cells were harvested 24 h postinfection. Viral titers were determined by plaque assay and expressed as the number of PFU/ml. Total DNA was isolated from replicate wells, viral DNAs were measured by quantitative PCR, and DNA content was expressed as genome equivalents (GE)/ng of total DNA. The experiment was repeated once. Values are the averages of two replicate samples per time point. DMSO indicates values from the parallel vehicle-treated control cultures at 24 h postinfection.

measurable toxicity in this 10-day assay, most notably compound 41 with a CC50 of 2.9 ␮M. The TI values for the five NTS inhibitors of HCMV ranged from 5 for compound 46 to 17 for compounds 55 and 59. Therefore, NTS inhibitors can suppress HCMV replication in addition to inhibiting the HSVs, although not as well as ganciclovir. DISCUSSION

The complex herpesvirus DNA replication mechanism uses enzymes with catalytic properties which imply that they would be

A Replicaon inhibion (log10)

4

December 2014 Volume 58 Number 12

Standard

sensitive to NTS enzyme inhibitors. Therefore, we screened 42 compounds from six chemical families that were known or predicted to inhibit HIV and/or HBV NTS enzymes for the ability to suppress HSV-1 and HSV-2 replication. Eighteen compounds (43%) inhibited HSV-1 replication by ⱖ10-fold in short-term cell culture experiments in Vero cells at ⱕ50 ␮M, and 19 inhibited HSV-2 (45%) (Table 1). Six of the compounds that inhibited HSV-1 or HSV-2 at 5 ␮M were tropolones (compounds 46, 49, 50, 55, 56, and 59), and one inhibitor was found in each of the

1

5

Pre-incubaon Compound #46

0 2 4 6 8 10 12 14 Time of compound addion (hours P.I.)

5

7 6 5 4 3 2 1 0 Mock preincubaon

8 7 6 5 4 3 2 1 0

HSV-1 Wild-type HSV-1 TKHSV-2 Wild-type HSV-2 TK-

DMSO

ACV

#30

#41

#46

Compound (50 μM)

B Replicaiton inhibion (log10)

Viral DNA or virus accumulaon (log10 GE/ng DNA or PFU/ml)

C

ACV

DMSO

Viral DNA or virus accumulaon (log10 GE/ng DNA or PFU/ml)

B

7 6 5 4 3 2 1 0

8 7 6 5 4 3 2 1 0

HSV-1 Wild-type HSV-21 TKHSV-2 Wild-type HSV-2 TK-

DMSO

ACV

#30

#41

#46

Compound (5 μM)

FIG 6 Sensitivity of thymidine kinase-deficient HSVs to NTS inhibitors. (A and B) Vero cell monolayers were infected with wild-type or mutant HSV-1 or HSV-2 strains at an MOI of 0.1 in the presence of ACV or compound 30, 41, or 46 at 50 ␮M (A) or 5 ␮M (B). Cultures were collected 24 h postinfection, and infectious virus titers were determined by plaque assay. Values are the averages plus 1 standard deviation from two experiments, each done in duplicate.

aac.asm.org 7457

Downloaded from http://aac.asm.org/ on November 21, 2014 by NORTHERN ILLINOIS UNIV

Viral DNA or virus accumulaon (log10 GE/ng DNA or PFU/ml)

A

Replicaon inhibion (log10)

NTS Enzyme Inhibitors Suppress HSV Replication

Tavis et al.

TABLE 2 Efficacy of select HSV inhibitors against HCMV EC50 (␮M)

CC50 (␮M)

TI

41 46 49 55 56 59 Ganciclovir

0.33 1.30 0.45 0.47 2.66 1.94 3.20

2.90 6.70 6.70 8.00 6.70 33.30 ⬎100

9 5 15 17 2 17 ⬎31

polyoxygenated heterocycle (compound 41) and hydroxyxanthenone (compound 30) classes. Four of these compounds inhibited HSV-1 and HSV-2 replication in Vero cells and HFFs by at least 100,000-fold at 5 ␮M. No hits were found among the other chemical families (see Table S1 in the supplemental material and Fig. 1). All compounds that inhibited HSV-1 or HSV-2 by ⱖ1 log10 unit in Vero cells were also active in HFFs (Fig. 2 and Table 1), demonstrating efficacy against HSV replication in a relevant human cell type. This high hit rate for a small primary screen provides support for the hypothesis that NTS enzymes are promising targets for antiherpesvirus drug development. These NTS enzyme inhibitors function against HIV by binding to the RNase H or integrase enzymes in part through binding to the essential divalent cations within the active site (45–48). Therefore, their presumed mechanism of action against the HSVs is to inhibit one or more NTS enzymes essential for viral genomic replication by binding to their active site(s) and masking the cations. The proposed mechanism, however, cannot be tested until the target enzyme(s) is identified. Sufficient data exist to establish constraints on the structureactivity relationship for the tropolones against the HSVs. Comparing compound 46 with compounds 47, 48, 50, and 53 (see Fig. S1 in the supplemental material) indicates that efficient inhibition in the absence of extended R groups at the ␣, ␤, and ␥ positions of the tropolone ring requires three adjacent cation-binding moieties (the contiguous hydroxyl and carbonyl groups). Of the six compounds with larger R groups (compounds 49, 52, 55, 56, 59, and 62), four suppressed viral replication by ⱖ1 log10 unit at 5 ␮M (compounds 49, 55, 56, and 59). Three of these inhibitory compounds (compounds 49, 55, and 59) had only two metal-binding moieties on the tropolone ring, but compound 56 had three binding moieties and was the best inhibitor identified. These data imply that a carbonyl group and a modified hydroxyl group are insufficient to support robust inhibition and that three metalbinding moieties are superior to two. Last, four of the most effective inhibitors (compounds 49, 55, 56, and 59) had a variety of R groups opposite the metal-binding motif. This indicates that structural diversity is permitted in these elements, and the wide range of EC50s for the four most effective inhibitors (0.24 to 1.09 ␮M for HSV-1 and 0.22 to 4.12 ␮M for HSV-2) implies that the chemical elements in these positions can significantly impact compound efficacy. Little to no cytotoxicity was observed for the HSV inhibitors in short-term experiments by either the MTT assay that measures mitochondrial function or by assessing cell rupture (Table 1; Table S1 in the supplemental material; data not shown). Compounds 50 and 61 caused some toxicity in HFFs, but all other CC50 values were higher than the 50 ␮M maximal concentration used for

7458

aac.asm.org

screening. This indicates that cytotoxicity is not a confounding factor in our screening, and it is promising with regard to the drug potential of these compound classes. However, we do not wish to imply that toxicity will not be a concern during subsequent drug development. Substantial toxicity was detected for compounds 41, 56, and 59 in HFFs in the longer-term HCMV assays (Table 2). Furthermore, tropolones can induce mitochondrial toxicity in rats (64), and compound 46 has CC50 values of 2 to 16 ␮M in CEM-SS, Huh7, and HepG2 cells (37, 65), indicating that optimization to limit cytotoxicity in various cell types will be warranted. Our data imply that the NTS inhibitors block more than one event in the viral replication cycle. The time-of-addition experiment (Fig. 4) revealed that compounds 41 and 46 interfere with events occurring between 1 and 12 h postinfection. The partial loss of activity when addition of compounds 41 and 46 was delayed until 3 or 5 h postinfection is in sharp contrast to the lack of effect that delaying ACV addition had during this time. The NTS inhibitors therefore suppress an activity important for viral replication that occurs prior to the onset of viral DNA replication, perhaps genome circularization. This is also in sharp contrast to a recently identified HIV integrase inhibitor that also suppressed HSV replication, whose earliest detectable effect is at the stage of viral DNA replication (66). The continued gradual loss of activity if addition of compounds 41 and 46 was delayed more than 1 h postinfection suggests that these NTS inhibitors additionally inhibit events that occur concurrently with viral DNA replication, possibly including DNA replication and primer removal, production of branched genomic concatemers, and/or genome monomerization. Alternatively, because viral infection and replication are somewhat asynchronous, the compounds may inhibit a very early event such as genome circularization, but the inhibitory effect appears to gradually diminish over time because a progressively larger proportion of the viral population has already initiated infection prior to compound addition at later times. It is unknown whether the events suppressed by these NTS inhibitors represent a single target whose activity is needed at multiple stages of the viral replication cycle or multiple targets that are each needed for viral replication. However, the high hit rate in this small targeted screen, the high efficacy of the inhibitors, and the gradual loss of activity in the time-of-addition experiments lead us to favor the possibility that the NTS inhibitors act against multiple enzymes that function at different stages of the replication cycle. In this context, the gradual loss of activity with increasing time of addition would be due to exposure to compound after one or more of the targets had performed their functions. Alternatively, the events controlled by these enzymatic function(s) may occur less abruptly than onset of viral DNA replication. If antiviral efficacy is due to activity against multiple targets, it would suggest a higher barrier to resistance evolution than is often observed for drugs with a single target. The target(s) of the NTS inhibitors could be viral and/or cellular. Candidate HSV gene products with NTS enzyme activities include the predicted RNase H activity of the ICP8 single-stranded DNA-binding protein (25), the RNase H activity of the pUL30 DNA polymerase (67), the 3=-5= exonuclease activity of pUL30 (68), and the 5=-3= exonuclease activity of the pUL12 polymerase accessory protein (27) that are directly involved in virus DNA replication (21). The pUL15 terminase that cleaves the viral DNA into monomers is known to be an NTS enzyme and is also a possible target (28). Cellular candidates include human RNase H 1

Antimicrobial Agents and Chemotherapy

Downloaded from http://aac.asm.org/ on November 21, 2014 by NORTHERN ILLINOIS UNIV

Compound no. or name

NTS Enzyme Inhibitors Suppress HSV Replication

December 2014 Volume 58 Number 12

tion and deafness in neonates and retinitis in immunocompromised patients (63); human herpesvirus 6 (HHV-6), which causes roseola infantum and febrile seizures (74); Epstein-Barr virus (EBV), which causes infectious mononucleosis and is associated with cancers, including Burkitt’s lymphoma (75); HHV-7, which may stimulate HCMV reactivation and tissue transplant rejection (76); and HHV-8, which causes Kaposi’s sarcoma (77). More than 90% of adults have been infected with and retain a latent form of one or more of these viruses. Animal herpesviruses of significant economic importance include pseudorabies virus (78, 79), Marek’s disease virus (80), bovine herpesviruses (BHV) (81), and herpes simian B virus (82). Importantly, these viruses all share the same basic genomic replication mechanisms, so they should be sensitive to NTS inhibitors if the presumed mechanism by which the NTS enzymes inhibit HSV-1 and HSV-2 is correct. This prediction is at least partially confirmed by our detection of antiHCMV activity for five out of the six best NTS HSV inhibitors identified here and the recent identification of an HIV integrase inhibitor, XZ45, as an inhibitor of HCMV replication (66). ACKNOWLEDGMENTS This work was funded by seed grants from the Saint Louis University Department of Molecular Microbiology and Immunology, the Friends of the Saint Louis University Liver Center, and the Saint Louis University School of Medicine. We thank Mark Buller and Duane Grandgenett for helpful discussions; Allie Taniuchi, Aisha Uraizee, Ankit Gupta, and Matthew Coates for technical assistance; and David Leib and Jim Smiley for providing mutant viruses. Compounds 46 to 56 were obtained from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute. Saint Louis University School of Medicine has utilized the nonclinical and preclinical services program offered by the National Institute of Allergy and Infectious Diseases. L. A. Morrison and J. E. Tavis are inventors on U.S. provisional patent application 61/90825 that covers the inhibitors reported here.

REFERENCES 1. Pellet PE, Roizman B. 2013. Herpesviridae, p 1802–1822. In Knipe DM, Howley PM (ed), Fields virology, 6th ed. Lippincott Williams & Wilkins, Philadelphia, PA. 2. Roizman B, Knipe DM, Whitley RJ. 2013. Herpes simplex viruses, p 1823–1897. In Knipe DM, Howley PM (ed), Fields virology, 6th ed. Lippincott Williams & Wilkins, Philadelphia, PA. 3. Mark KE, Wald A, Magaret AS, Selke S, Olin L, Huang ML, Corey L. 2008. Rapidly cleared episodes of herpes simplex virus reactivation in immunocompetent adults. J. Infect. Dis. 198:1141–1149. http://dx.doi .org/10.1086/591913. 4. Xu F, Sternberg MR, Kottiri BJ, McQuillan GM, Lee FK, Nahmias AJ, Berman SM, Markowitz LE. 2006. Trends in herpes simplex virus type 1 and type 2 seroprevalence in the United States. JAMA 296:964 –973. http: //dx.doi.org/10.1001/jama.296.8.964. 5. Gilbert M, Li X, Petric M, Krajden M, Isaac-Renton JL, Ogilvie G, Rekart ML. 2011. Using centralized laboratory data to monitor trends in herpes simplex virus type 1 and 2 infection in British Columbia and the changing etiology of genital herpes. Can. J. Public Health 102:225–229. 6. Horowitz R, Aierstuck S, Williams EA, Melby B. 2010. Herpes simplex virus infection in a university health population: clinical manifestations, epidemiology, and implications. J. Am. Coll. Health 59:69 –74. http://dx .doi.org/10.1080/07448481.2010.483711. 7. Abu-Raddad LJ, Magaret AS, Celum C, Wald A, Longini IM, Jr, Self SG, Corey L. 2008. Genital herpes has played a more important role than any other sexually transmitted infection in driving HIV prevalence in Africa. PLoS One 3:e2230. http://dx.doi.org/10.1371/journal.pone.0002230. 8. Freeman EE, Weiss HA, Glynn JR, Cross PL, Whitworth JA, Hayes RJ. 2006. Herpes simplex virus 2 infection increases HIV acquisition in men

aac.asm.org 7459

Downloaded from http://aac.asm.org/ on November 21, 2014 by NORTHERN ILLINOIS UNIV

and the Fen1 endonuclease that may remove RNA primers during DNA synthesis (69), and the double-stranded break repair enzymes Mre11, Rad50, NBS1, Rad51 (70), and Rad52 (27). The base excision repair enzymes SSH2 and MLH1 (71) that form complexes that are recruited to viral replication sites and contribute to HSV genomic replication (71) are also possible targets. Our data indicate that the NTS inhibitors tested work by a different mechanism than the approved nucleos(t)ide analog antiherpesvirus drugs that terminate DNA elongation. We demonstrated that HSV-2 strains resistant to ACV are sensitive to NTS inhibitors in this targeted screen (Fig. 5) and that none of the NTS antagonists employed are nucleos(t)ide analogs (see Fig. S1 in the supplemental material). Furthermore, unlike most nucleos(t)ide analogs, compounds 30, 41, and 46 do not require phosphorylation by the viral thymidine kinase to be active (Fig. 6). This implies that NTS inhibitors could be used as salvage therapies for nucleoside analog-resistant infections, and it raises the possibility of additive or synergistic activity of NTS and nucleoside analog drugs that could significantly improve therapeutic efficacy. It also suggests that combination therapy employing nucleoside analogs and drugs that inhibit NTS enzymes could reduce the rate at which resistance would evolve to either drug class. ACV and ganciclovir are standard treatments and prophylactic agents for herpesvirus infections. Recently, approval of other drugs (i.e., penciclovir, brivudine, valaciclovir, famciclovir, cidofovir, fomivirsen, and foscarnet) for one or more herpesviruses has increased therapeutic options. In addition, an inhibitor of the HSV-2 helicase-primase complex, pritelivir, is in clinical development (72). Despite these expanded options, new antiherpesvirus agents are still needed to further improve efficacy and to address other clinical issues, such as safety in children and pregnant women, increased use of antiherpesvirus prophylaxis including in transplant recipients, and resistance to nucleos(t)ide analogs. Recently, Yan et al. observed that select HIV integrase inhibitors suppress replication of herpesvirus family members (66). Specifically, two HIV integrase inhibitors reduced HSV replication in HEp-2 cell cultures by up to ⬃3.9 log10 units (8,000-fold) at 10 ␮M (66). In contrast, the inhibitors we identified suppress HSV replication by up to 5.95 log10 units (890,000-fold) at 5 ␮M and were originally identified as HIV RNase H inhibitors. Although cross-inhibition of the HIV integrase and RNase H by certain compounds occurs (46, 50–52), we do not know whether HIV integrase inhibitors act against the same or different targets as the HIV RNase H inhibitors during suppression of HSV replication. A significant difference between the assays used in the two studies is measurement of inhibition under one-step versus multistep growth conditions. Comparison of the two protocols revealed that our single-step assay likely underestimates the efficacy of the RNase H inhibitors because virus in the DMSO control cultures has not yet reached maximal titer. Thus, the high efficacy of the primary screening hits that we identified among three different chemical families of RNase H inhibitors, coupled with their different mechanism(s) of action relative to the nucleoside analog inhibitors, makes the members of the RNase H antagonist subclass of NTS inhibitors attractive candidates for novel antiherpesvirus therapies to complement the existing drugs. The herpesvirus family contains many viruses beyond HSV-1 and HSV-2 of major medical or veterinary importance. Human pathogens include varicella-zoster virus (VZV) (73), which causes chicken pox and shingles; HCMV, which causes mental retarda-

Tavis et al.

9. 10. 11.

13. 14.

15. 16. 17.

18. 19.

20.

21. 22. 23. 24. 25.

26. 27.

28.

7460

aac.asm.org

29. 30.

31. 32. 33. 34.

35. 36.

37.

38.

39. 40.

41.

42.

43. 44. 45.

46.

47.

otic and prokaryotic viruses. J. Virol. 87:7140 –7148. http://dx.doi.org/10 .1128/JVI.00311-13. Yang W, Steitz TA. 1995. Recombining the structures of HIV integrase, RuvC and RNase H. Structure 3:131–134. http://dx.doi.org/10.1016 /S0969-2126(01)00142-3. Katayanagi K, Miyagawa M, Matsushima M, Ishikawa M, Kanaya S, Ikehara M, Matsuzaki T, Morikawa K. 1990. Three-dimensional structure of ribonuclease H from E. coli. Nature 347:306 –309. http://dx.doi.org /10.1038/347306a0. Lai L, Yokota H, Hung LW, Kim R, Kim SH. 2000. Crystal structure of archaeal RNase HII: a homologue of human major RNase H. Structure 8:897–904. http://dx.doi.org/10.1016/S0969-2126(00)00179-9. Lima WF, Wu H, Crooke ST. 2001. Human RNases H. Methods Enzymol. 341:430 – 440. http://dx.doi.org/10.1016/S0076-6879(01)41168-2. Frank P, Braunshofer-Reiter C, Poltl A, Holzmann K. 1998. Cloning, subcellular localization and functional expression of human RNase HII. Biol. Chem. 379:1407–1412. Ariyoshi M, Vassylyev DG, Iwasaki H, Nakamura H, Shinagawa H, Morikawa K. 1994. Atomic structure of the RuvC resolvase: a Holliday junction-specific endonuclease from E. coli. Cell 78:1063–1072. http://dx .doi.org/10.1016/0092-8674(94)90280-1. Song JJ, Smith SK, Hannon GJ, Joshua-Tor L. 2004. Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305:1434 – 1437. http://dx.doi.org/10.1126/science.1102514. Tavis JE, Cheng X, Hu Y, Totten M, Cao F, Michailidis E, Aurora R, Meyers MJ, Jacobsen EJ, Parniak MA, Sarafianos SG. 2013. The hepatitis B virus ribonuclease H is sensitive to inhibitors of the human immunodeficiency virus ribonuclease H and integrase enzymes. PLoS Pathog. 9:e1003125. http://dx.doi.org/10.1371/journal.ppat.1003125. Hu Y, Cheng X, Cao F, Huang A, Tavis JE. 2013. ␤-Thujaplicinol inhibits hepatitis B virus replication by blocking the viral ribonuclease H activity. Antiviral Res. 99:221–229. http://dx.doi.org/10.1016/j.antiviral .2013.06.007. Cai CW, Lomonosova E, Moran EA, Cheng X, Patel KB, Bailly F, Cotelle P, Meyers MJ, Tavis JE. 2014. Hepatitis B virus replication is blocked by a 2-hydroxyisoquinoline-1,3(2H,4H)-dione (HID) inhibitor of the viral ribonuclease H activity. Antiviral Res. 108:48 –55. http://dx.doi .org/10.1016/j.antiviral.2014.05.007. Nowotny M. 2009. Retroviral integrase superfamily: the structural perspective. EMBO Rep. 10:144 –151. http://dx.doi.org/10.1038/embor.2008 .256. Dyda F, Hickman AB, Jenkins TM, Engelman A, Craigie R, Davies DR. 1994. Crystal structure of the catalytic domain of HIV-1 integrase: similarity to other polynucleotidyl transferases. Science 266:1981–1986. http: //dx.doi.org/10.1126/science.7801124. Nowotny M, Gaidamakov SA, Crouch RJ, Yang W. 2005. Crystal structures of RNase H bound to an RNA/DNA hybrid: substrate specificity and metal-dependent catalysis. Cell 121:1005–1016. http://dx.doi.org/10.1016 /j.cell.2005.04.024. Klumpp K, Hang JQ, Rajendran S, Yang Y, Derosier A, Wong KI, Overton H, Parkes KE, Cammack N, Martin JA. 2003. Two-metal ion mechanism of RNA cleavage by HIV RNase H and mechanism-based design of selective HIV RNase H inhibitors. Nucleic Acids Res. 31:6852– 6859. http://dx.doi.org/10.1093/nar/gkg881. Quashie PK, Sloan RD, Wainberg MA. 2012. Novel therapeutic strategies targeting HIV integrase. BMC Med. 10:34. http://dx.doi.org/10.1186 /1741-7015-10-34. Ilina T, Labarge K, Sarafianos SG, Ishima R, Parniak MA. 2012. Inhibitors of HIV-1 reverse transcriptase-associated ribonuclease H activity. Biology 1:521–541. http://dx.doi.org/10.3390/biology1030521. Himmel DM, Maegley KA, Pauly TA, Bauman JD, Das K, Dharia C, Clark AD, Jr, Ryan K, Hickey MJ, Love RA, Hughes SH, Bergqvist S, Arnold E. 2009. Structure of HIV-1 reverse transcriptase with the inhibitor beta-thujaplicinol bound at the RNase H active site. Structure 17: 1625–1635. http://dx.doi.org/10.1016/j.str.2009.09.016. Billamboz M, Bailly F, Lion C, Touati N, Vezin H, Calmels C, Andreola ML, Christ F, Debyser Z, Cotelle P. 2011. Magnesium chelating 2-hydroxyisoquinoline-1,3(2H,4H)-diones, as inhibitors of HIV-1 integrase and/or the HIV-1 reverse transcriptase ribonuclease H domain: discovery of a novel selective inhibitor of the ribonuclease H function. J. Med. Chem. 54:1812–1824. http://dx.doi.org/10.1021/jm1014692. Kirschberg TA, Balakrishnan M, Squires NH, Barnes T, Brendza KM, Chen X, Eisenberg EJ, Jin W, Kutty N, Leavitt S, Liclican A, Liu Q, Liu

Antimicrobial Agents and Chemotherapy

Downloaded from http://aac.asm.org/ on November 21, 2014 by NORTHERN ILLINOIS UNIV

12.

and women: systematic review and meta-analysis of longitudinal studies. AIDS 20:73– 83. http://dx.doi.org/10.1097/01.aids.0000198081.09337.a7. Kimberlin DW. 2007. Herpes simplex virus infections of the newborn. Semin. Perinatol. 31:19 –25. http://dx.doi.org/10.1053/j.semperi.2007.01 .003. King DH. 1988. History, pharmacokinetics, and pharmacology of acyclovir. J. Am. Acad. Dermatol. 18:176 –179. http://dx.doi.org/10.1016/S0190 -9622(88)70022-5. Field HJ, Vere Hodge RA. 2013. Recent developments in antiherpesvirus drugs. Br. Med. Bull. 106:213–249. http://dx.doi.org/10 .1093/bmb/ldt011. Vere Hodge RA, Field HJ. 2013. Antiviral agents for herpes simplex virus. Adv. Pharmacol. 67:1–38. http://dx.doi.org/10.1016/B978-0-12-405880-4 .00001-9. Morfin F, Thouvenot D. 2003. Herpes simplex virus resistance to antiviral drugs. J. Clin. Virol. 26:29 –37. http://dx.doi.org/10.1016/S1386 -6532(02)00263-9. Wang Y, Wang Q, Zhu Q, Zhou R, Liu J, Peng T. 2011. Identification and characterization of acyclovir-resistant clinical HSV-1 isolates from children. J. Clin. Virol. 52:107–112. http://dx.doi.org/10.1016/j.jcv.2011 .06.009. Levin MJ, Bacon TH, Leary JJ. 2004. Resistance of herpes simplex virus infections to nucleoside analogues in HIV-infected patients. Clin. Infect. Dis. 39(Suppl 5):S248 –S257. http://dx.doi.org/10.1086/422364. Gilbert C, Bestman-Smith J, Boivin G. 2002. Resistance of herpesviruses to antiviral drugs: clinical impacts and molecular mechanisms. Drug Resist. Updat. 5:88 –114. http://dx.doi.org/10.1016/S1368-7646(02)00021-3. van Velzen M, van de Vijver DA, van Loenen FB, Osterhaus AD, Remeijer L, Verjans GM. 2013. Acyclovir prophylaxis predisposes to antiviral-resistant recurrent herpetic keratitis. J. Infect. Dis. 208:1359 – 1365. http://dx.doi.org/10.1093/infdis/jit350. Duan R, de Vries RD, Osterhaus AD, Remeijer L, Verjans GM. 2008. Acyclovir-resistant corneal HSV-1 isolates from patients with herpetic keratitis. J. Infect. Dis. 198:659 – 663. http://dx.doi.org/10.1086/590668. Suzutani T, Ishioka K, De Clercq E, Ishibashi K, Kaneko H, Kira T, Hashimoto K, Ogasawara M, Ohtani K, Wakamiya N, Saijo M. 2003. Differential mutation patterns in thymidine kinase and DNA polymerase genes of herpes simplex virus type 1 clones passaged in the presence of acyclovir or penciclovir. Antimicrob. Agents Chemother. 47:1707–1713. http://dx.doi.org/10.1128/AAC.47.5.1707-1713.2003. Wang LX, Takayama-Ito M, Kinoshita-Yamaguchi H, Kakiuchi S, Suzutani T, Nakamichi K, Lim CK, Kurane I, Saijo M. 2013. Characterization of DNA polymerase-associated acyclovir-resistant herpes simplex virus type 1: mutations, sensitivity to antiviral compounds, neurovirulence, and in-vivo sensitivity to treatment. Jpn. J. Infect. Dis. 66:404 – 410. http://dx.doi.org/10.7883/yoken.66.404. Weller SK, Coen DM. 2012. Herpes simplex viruses: mechanisms of DNA replication. Cold Spring Harb. Perspect. Biol. 4:a013011. http://dx.doi.org /10.1101/cshperspect.a013011. Lehman IR, Boehmer PE. 1999. Replication of herpes simplex virus DNA. J. Biol. Chem. 274:28059 –28062. http://dx.doi.org/10.1074/jbc.274.40 .28059. Strang BL, Stow ND. 2005. Circularization of the herpes simplex virus type 1 genome upon lytic infection. J. Virol. 79:12487–12494. http://dx .doi.org/10.1128/JVI.79.19.12487-12494.2005. Wilkinson DE, Weller SK. 2003. The role of DNA recombination in herpes simplex virus DNA replication. IUBMB Life 55:451– 458. http://dx .doi.org/10.1080/15216540310001612237. Bryant KF, Yan Z, Dreyfus DH, Knipe DM. 2012. Identification of a divalent metal cation binding site in herpes simplex virus 1 (HSV-1) ICP8 required for HSV replication. J. Virol. 86:6825– 6834. http://dx.doi.org/10 .1128/JVI.00374-12. Boehmer PE, Lehman IR. 1997. Herpes simplex virus DNA replication. Annu. Rev. Biochem. 66:347–384. http://dx.doi.org/10.1146/annurev .biochem.66.1.347. Schumacher AJ, Mohni KN, Kan Y, Hendrickson EA, Stark JM, Weller SK. 2012. The HSV-1 exonuclease, UL12, stimulates recombination by a single strand annealing mechanism. PLoS Pathog. 8:e1002862. http://dx .doi.org/10.1371/journal.ppat.1002862. Selvarajan Sigamani S, Zhao H, Kamau YN, Baines JD, Tang L. 2013. The structure of the herpes simplex virus DNA-packaging terminase pUL15 nuclease domain suggests an evolutionary lineage among eukary-

NTS Enzyme Inhibitors Suppress HSV Replication

48.

49.

51.

52.

53.

54.

55. 56. 57. 58. 59. 60.

61.

62.

December 2014 Volume 58 Number 12

63. Ho M. 2008. The history of cytomegalovirus and its diseases. Med. Microbiol. Immunol. 197:65–73. http://dx.doi.org/10.1007/s00430-007-0066-x. 64. Nakagawa Y, Tayama K. 1998. Mechanism of mitochondrial dysfunction and cytotoxicity induced by tropolones in isolated rat hepatocytes. Chem. Biol. Interact. 116:45– 60. http://dx.doi.org/10.1016/S0009-2797(98)00078-7. 65. Chung S, Himmel DM, Jiang JK, Wojtak K, Bauman JD, Rausch JW, Wilson JA, Beutler JA, Thomas CJ, Arnold E, Le Grice SF. 2011. Synthesis, activity, and structural analysis of novel alphahydroxytropolone inhibitors of human immunodeficiency virus reverse transcriptase-associated ribonuclease H. J. Med. Chem. 54:4462– 4473. http://dx.doi.org/10.1021/jm2000757. 66. Yan Z, Bryant KF, Gregory SM, Angelova M, Dreyfus DH, Zhao XZ, Coen DM, Burke TR, Jr, Knipe DM. 2014. HIV integrase inhibitors block replication of alpha-, beta-, and gammaherpesviruses. mBio 5(4):e01318 – 14. http://dx.doi.org/10.1128/mBio.01318-14. 67. Liu S, Knafels JD, Chang JS, Waszak GA, Baldwin ET, Deibel MR, Jr, Thomsen DR, Homa FL, Wells PA, Tory MC, Poorman RA, Gao H, Qiu X, Seddon AP. 2006. Crystal structure of the herpes simplex virus 1 DNA polymerase. J. Biol. Chem. 281:18193–18200. http://dx.doi.org/10 .1074/jbc.M602414200. 68. Coen DM. 1996. Viral DNA polymerases, p 495–523. In DePamphilis ML (ed), DNA replication in eukaryotic cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 69. Zhu Y, Wu Z, Cardoso MC, Parris DS. 2010. Processing of laggingstrand intermediates in vitro by herpes simplex virus type 1 DNA polymerase. J. Virol. 84:7459 –7472. http://dx.doi.org/10.1128/JVI.01875-09. 70. Weizman M, Weller S. 2011. Interactions between HSV-1 and the DNA damage response, p 257–268. In Weller SK (ed), Alphaherpesviruses: molecular virology. Caister Academic, Norfolk, United Kingdom. 71. Mohni KN, Mastrocola AS, Bai P, Weller SK, Heinen CD. 2011. DNA mismatch repair proteins are required for efficient herpes simplex virus 1 replication. J. Virol. 85:12241–12253. http://dx.doi.org/10.1128 /JVI.05487-11. 72. Wald A, Corey L, Timmler B, Magaret A, Warren T, Tyring S, Johnston C, Kriesel J, Fife K, Galitz L, Stoelben S, Huang ML, Selke S, Stobernack HP, Ruebsamen-Schaeff H, Birkmann A. 2014. Helicase-primase inhibitor pritelivir for HSV-2 infection. N. Engl. J. Med. 370:201–210. http://dx .doi.org/10.1056/NEJMoa1301150. 73. Arvin AM, Gilden D. 2013. Varicella-zoster virus, p 2015–2057. In Knipe DM, Howley PM (ed), Fields virology, 6th ed. Lippincott Williams & Wilkins, Philadelphia, PA. 74. Zerr DM. 2006. Human herpesvirus 6: a clinical update. Herpes 13:20 –24. 75. Longnecker RM, Kieff E, Cohen JI. 2013. Epstein-Barr virus, p 1898 – 1959. In Knipe DM, Howley PM (ed), Fields virology, 6th ed. Lippincott Williams & Wilkins, Philadelphia, PA. 76. Chapenko S, Trociukas I, Donina S, Chistyakov M, Sultanova A, Gravelsina S, Lejniece S, Murovska M. 2012. Relationship between betaherpesviruses reactivation and development of complications after autologous peripheral blood stem cell transplantation. J. Med. Virol. 84:1953– 1960. http://dx.doi.org/10.1002/jmv.23412. 77. Damania BA, Cesarman E. 2013. Kaposi’s sarcoma-associated herpesvirus, p 2080 –2128. In Knipe DM, Howley PM (ed), Fields virology, 6th ed. Lippincott Williams & Wilkins, Philadelphia, PA. 78. Smith KC. 1997. Herpesviral abortion in domestic animals. Vet. J. 153: 253–268. http://dx.doi.org/10.1016/S1090-0233(97)80061-5. 79. Salwa A. 2004. A natural outbreak of Aujeszky’s disease in farm animals. Pol. J. Vet. Sci. 7:261–266. 80. Hirari K (ed). 2001. Current topics in microbiology and immunology, vol 255. Marek’s disease. Springer, Berlin, Germany. 81. Ali H, Ali AA, Atta MS, Cepica A. 2012. Common, emerging, vectorborne and infrequent abortogenic virus infections of cattle. Transbound. Emerg. Dis. 59:11–25. http://dx.doi.org/10.1111/j.1865-1682 .2011.01240.x. 82. Estep RD, Messaoudi I, Wong SW. 2010. Simian herpesviruses and their risk to humans. Vaccine 28(Suppl 2):B78 –B84. http://dx.doi.org/10.1016 /j.vaccine.2009.11.026.

aac.asm.org 7461

Downloaded from http://aac.asm.org/ on November 21, 2014 by NORTHERN ILLINOIS UNIV

50.

X, Mak J, Perry JK, Wang M, Watkins WJ, Lansdon EB. 2009. RNase H active site inhibitors of human immunodeficiency virus type 1 reverse transcriptase: design, biochemical activity, and structural information. J. Med. Chem. 52:5781–5784. http://dx.doi.org/10.1021/jm900597q. Agrawal A, DeSoto J, Fullagar JL, Maddali K, Rostami S, Richman DD, Pommier Y, Cohen SM. 2012. Probing chelation motifs in HIV integrase inhibitors. Proc. Natl. Acad. Sci. U. S. A. 109:2251–2256. http://dx.doi.org /10.1073/pnas.1112389109. Hare S, Maertens GN, Cherepanov P. 2012. 3=-Processing and strand transfer catalysed by retroviral integrase in crystallo. EMBO J. 31:3020 – 3028. http://dx.doi.org/10.1038/emboj.2012.118. Klarmann GJ, Hawkins ME, Le Grice SF. 2002. Uncovering the complexities of retroviral ribonuclease H reveals its potential as a therapeutic target. AIDS Rev. 4:183–194. Williams PD, Staas DD, Venkatraman S, Loughran HM, Ruzek RD, Booth TM, Lyle TA, Wai JS, Vacca JP, Feuston BP, Ecto LT, Flynn JA, DiStefano DJ, Hazuda DJ, Bahnck CM, Himmelberger AL, Dornadula G, Hrin RC, Stillmock KA, Witmer MV, Miller MD, Grobler JA. 2010. Potent and selective HIV-1 ribonuclease H inhibitors based on a 1-hydroxy-1,8-naphthyridin-2(1H)-one scaffold. Bioorg. Med. Chem. Lett. 20:6754 – 6757. http://dx.doi.org/10.1016/j.bmcl.2010.08.135. Billamboz M, Bailly F, Barreca ML, De Luca L, Mouscadet JF, Calmels C, Andreola ML, Witvrouw M, Christ F, Debyser Z, Cotelle P. 2008. Design, synthesis, and biological evaluation of a series of 2-hydroxyisoquinoline-1,3(2H,4H)-diones as dual inhibitors of human immunodeficiency virus type 1 integrase and the reverse transcriptase RNase H domain. J. Med. Chem. 51:7717–7730. http://dx.doi .org/10.1021/jm8007085. Korom M, Wylie KM, Wang H, Davis KL, Sangabathula MS, Delassus GS, Morrison LA. 2013. A proautophagic antiviral role for the cellular prion protein identified by infection with a herpes simplex virus 1 ICP34.5 mutant. J. Virol. 87:5882–5894. http://dx.doi.org/10.1128/JVI.02559-12. McDermott MR, Smiley JR, Leslie P, Brais J, Rudzroga HE, Bienenstock J. 1984. Immunity in the female genital tract after intravaginal vaccination of mice with an attenuated strain of herpes simplex virus type 2. J. Virol. 51:747–753. Knipe DM, Spang AE. 1982. Definition of a series of stages in the association of two herpesviral proteins with the cell nucleus. J. Virol. 43:314 – 324. Morrison LA, Knipe DM. 1996. Mechanisms of immunization with a replication-defective mutant of herpes simplex virus 1. Virology 220:402– 413. http://dx.doi.org/10.1006/viro.1996.0328. Martin TE, Barghusen SC, Leser GP, Spear PG. 1987. Redistribution of nuclear ribonucleoprotein antigens during herpes simplex virus infection. J. Cell Biol. 105:2069 –2082. http://dx.doi.org/10.1083/jcb.105.5.2069. de Bruyn Kops A, Knipe DM. 1988. Formation of DNA replication structures in herpes virus-infected cells requires a viral DNA binding protein. Cell 55:857– 868. http://dx.doi.org/10.1016/0092-8674(88)90141-9. McGuirt PV, Furman PA. 1982. Acyclovir inhibition of viral DNA chain elongation in herpes simplex virus-infected cells. Am. J. Med. 73:67–71. http://dx.doi.org/10.1016/0002-9343(82)90066-3. Elion GB, Furman PA, Fyfe JA, de Miranda P, Beauchamp L, Schaeffer HJ. 1977. Selectivity of action of an antiherpetic agent, 9-(2hydroxyethoxymethyl) guanine. Proc. Natl. Acad. Sci. U. S. A. 74:5716 – 5720. http://dx.doi.org/10.1073/pnas.74.12.5716. Duan R, de Vries RD, van Dun JM, van Loenen FB, Osterhaus AD, Remeijer L, Verjans GM. 2009. Acyclovir susceptibility and genetic characteristics of sequential herpes simplex virus type 1 corneal isolates from patients with recurrent herpetic keratitis. J. Infect. Dis. 200:1402–1414. http://dx.doi.org/10.1086/606028. Reyes M, Shaik NS, Graber JM, Nisenbaum R, Wetherall NT, Fukuda K, Reeves WC, Task Force on Herpes Simplex Virus Resistance. 2003. Acyclovir-resistant genital herpes among persons attending sexually transmitted disease and human immunodeficiency virus clinics. Arch. Intern. Med. 163:76 – 80. http://dx.doi.org/10.1001/archinte.163.1.76.