Incorporation of 1-/3-D-Arabinofuranosylcytosine into DMA from Herpes Simplex Virus Resistant to 9-0-D-Arabinofuranosyladenine1

We have demonstrated previously that 1-0-D-arabinofuranosylcytosine (ara-C) incorporates specifically in cellular DMA and that the formation of (ara-C)DNA correlates significantly with inhibition of DMA synthesis and loss of clonogenic survival. Similar results have been obtained with 9-0-D-arabinofuranosyladenine (ara-A). These findings have been extended by studying the incorporation of ara-C in DNA of a wild-type herpes simplex virus (HSV) and a mutant virus resistant to ara-C and ara-A. The results demonstrate that HSV resistance to ara-A is associated with formation of less (ara-C)DNA and less inhibition of DNA synthesis when compared to wild-type virus. This effect on formation of (ara-C)DNA is reversed upon exposure to higher (>10~6 M) ara-C concentrations, and this pattern of resistance corresponds to drug effect on virus plaque formation. The results also demonstrate a highly significant relationship between incor poration of ara-C in HSV DNA and inhibition of DNA synthesis for both viruses. Further, higher concentrations of ara-C that result in greater inhibition of DNA synthesis are associated with an increasing number of ara-C residues at the 3'-terminus of the DNA strand, thus suggesting that ara-C functions as a poor primer terminus for viral chain elongation. These results also suggest that HSV cross-resistance to ara-A and ara-C may be related to an altered viral DNA polymerase and that incorporation of ara-C in HSV DNA is at least one mechanism responsible for slowing viral synthesis and inducing lethal events. INTRODUCTION ara-C3 and ara-A are inhibitors of DNA synthesis (4, 8) with antiviral (2,18) and antitumor activity (1, 9). These agents incor porate in DNA, and the inhibition of cellular DNA synthesis con-elates with the extent of (arabinosyl)DNA formation (13-16). The arabinosyl residue provides a poor primer terminus for further chain elongation and thereby slows DNA synthesis (16). Further, studies with ara-C and ara-A have demonstrated a highly significant relationship between their incorporation in DNA and loss of clonogenic survival (13-16), thus suggesting that incor poration of these drugs in DNA results in lethal cellular events. 1This work was supported by Grant CH-199 from the American Cancer Society, Contract AI-52530 from the National Institute of Allergy and Infectious Diseases, and USPHS Grant P01-AG0059. 2 Recipient of an American Cancer Society faculty research award. To whom requests for reprints should be addressed. 3The abbreviations used are: ara-C, 1-/3-o-arabinofuranosylcytosine; ara-A, 9/3-D-arabinofuranosyladenine; HSV, herpes simplex virus; PAA, phosphonoacetic acid; ACV, acydoguanosine 9-(2-hydroxyethoxymethyl)guanine; (ara-A)DNA, araA incorporated into DNA; (ara-C)DNA, ara-C incorporated into DNA; PCS, fetal calf serum; PFU, plaque-forming unit(s); ID.»,the concentration inhibiting plaque for mation by 50%; PBS, phosphate-buffered saline; HPLC, high-pressure liquid chromatography; ara-CTP, 1-/3-o-arabinofuranosylcytosine 5'-triphosphate; ara-CMP, 1-,(-D-arabinofuranosy Icytosine 5'-monophosphate; ara-ATP, 9-0-D-arabinofuranosyladenine 5'-triphosphate; ara-AMP, 9-i8-D-arabinofuranosyladenine 5'-monophosphate. Received April 4,1983; accepted September 27,1983. Recent observations with purified mammalian DNA polymer ase & have demonstrated that incorporated ara-A residues pro vide poor primer termini for further chain elongation and that excision of the ara-A residue is necessary for further DNA synthesis (17, 20). Similar observations have been made with purified HSV DNA polymerase (7). Recent studies with PAAresistant HSVs (10) that are cross-resistant to ara-A and ACV have demonstrated that less formation of (ara-A)DNA is associ ated with less inhibition of DNA synthesis.4 We have now ex tended these findings by monitoring the incorporation of ara-A into DNA of a mutant herpes virus (HSV-1 ara-A^ that is resistant to ara-A. The results of the present study demonstrate that resistance to ara-A is associated with cross-resistance to ara-C and for mation of less (ara-C)DNA. The resistance is reversed at higher drug concentrations, and inhibition of DNA synthesis is directly related to the extent of (ara-C)DNA formation. The ara-C residues are detectable in internucleotide linkage, although exposure to higher drug concentrations results in a greater number being detectable at the chain terminus. Further, inhibition of HSV replication is related to the extent of ara-C incorporation in DNA. MATERIALS AND METHODS Cells and Viruses. The Vero line of African green monkey kidney cells was obtained from Microbiological Associates (Walkersville, Md.) and maintained Mycoplasma free in Medium 199 (Grand Island Biological Co.) containing 2% PCS, penicillin (250 units/ml), and streptomycin (250 ^g/ml). The drug-resistant mutant HSV-1 (ara-A1) was derived by serial passage of HSV-1 strain Patton in ara-A and was a generous gift of Dr. Ronald Duff. This mutant and the wild-type HSV-1 (KOS) were grown in Vero cells by low-multiplicity infection (0.01 PFU/cell) and incubation at 37°. Pools were assayed by plaque formation in Vero cells, and all possessed a titer of approximately 2 x 107 to 10" PFU/ml (19). Drugs. ara-A and ara-C (Sigma Chemical Co., St. Louis, Mo.) stock solutions (10 mw) were prepared in distilled water and stored at -20°. Aphidicolin (NSC 234714) was obtained from Dr. H. Douros, Develop mental Therapeutics, National Cancer Institute, Bethesda, Md. The [53H]ara-C (specific activity, 10.2 to 15.5 Ci/mmol) was obtained from the Amersham/Searte Corp., Arlington Heights, III. Determination of Sensitivity and Resistance to Antiviral Com pounds. Resistance of HSV to antiviral compounds was determined by methods described previously (19). Vero cell monolayers were prepared in plastic tissue culture plates (Linbro; Flow Laboratories, Inc., McLean, Va.). Confluent monolayers were infected with virus and adsorbed for 1 hr at 37°. The inoculum was removed prior to adding 3 ml of 1% methylcelluk>se-2% FCS and appropriate concentrations of drug. After incubation at 37°in 5% CO2 for 3 to 5 days, the monolayers were fixed and stained for counting plaques. The ID.»was based on the number of plaques present in control wells without drug and was calculated from a semilogarithmic plot of surviving plaques in the presence of increasing 4 L Schnipper, C. Crumpacker, H. Allaudeen, D. Herrick, and D. W. Kufe. Mechanisms of resistance to ara-A in a mutant of herpes simplex virus type 1, manuscript in preparation.