and its congener 9-{[2-hydroxy-1-(hydroxymethyl)ethoxy]-methyl}guanine (BW B759U) exhibit comparable antiviral activity against . However, BW B759U is a more potent inhibitor of both human CMV and Epstein-Barr virus (EBV) in vitro than is ACV (1-3, 5). As is the case with ACV, BW B759U is activated to the triphosphate form in HSV-and VZV-infected cells and the initial step of this phosphorylation is associated with the activity of the virus-specified thymidine kinase (TK) (refs. 4, 6-8; unpublished data Table 1). In the present study, we report that BW B759U, in contrast to ACV, is preferentially activated in human diploid fibroblasts infected with HCMV. MATERIALS AND METHODSCells and Virus. Human foreskin fibroblast (HFF) cells were derived in this laboratory from tissues obtained from Duke University Hospital Pediatrics Department (Durham, NC) and were used up to passage 11. Human diploid embryonic lung fibroblast (MRC-5) cells were obtained from American Type Culture Collection and were used between passage 22 and passage 26. Monolayer cultures were grown in Eagle's minimal essential medium (ME medium) containing 50 units of penicillin per ml and 50 ,g of streptomycin per ml supplemented with 10% fetal bovine serum (Sterile Systems, Logan, Utah) and 1% L-glutamine. HCMV strain AD169 was also obtained from American Type Culture Collection. Kerr strain of HCMV was obtained from E. S. Huang (Cancer Research Center, University of North Carolina, Chapel Hill). The clinical strain Wade was isolated. from a congenitally infected infant by J. Zeller (Duke University Hospital Infectious Diseases Laboratory).Virus stocks of AD169 were stored in liquid nitrogen and were amplified before use by low multiplicity of infection (moi) passage in MRC-5 cells [<0.05 plaque-forming units (pfu) per cell]. Supernatant virus was used at time of peak titer (days 8-11) and was back titrated at the time of use to determine approximate moi.Cells and virus stocks were routinely checked for mycoplasma contamination by electron microscopy and [125i]iododeoxycytidine autoradiography methods.Cell Cytotoxicity Assay. The cytotoxicity of the nucleoside analogs was determined by Naomi Cohn (The Wellcome Research Laboratories) by using a cell growth assay that allows Abbreviations: EBV, Epstein-Barr virus; HCMV, human cytomegalovirus; HSV, herpes simplex virus; VZV, varicella zoster virus; ACV, acyclovir or 9-[(2-hydroxyethoxy)methyl]guanine; FIaC, 2'-fluoro-5-iodoarabinosylcytosine; BW B759U, 9-{[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl}guanine, also abbreviated 2' NDG, BIOLF-62, and DHPG by other investigators; HFF, human foreskin fibroblast; TK, thymidine kinase; pfu, plaque-forming unit(s); moi, multiplicity of infection. 2473The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
We have isolated a human cytomegalovirus mutant that is resistant to the antiviral drug 9-{[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl}guanine (BW B759U), yet exhibits wild-type sensitivity to inhibitors of herpesvirus DNA polymerases such as phosphonoformic acid and aphidicolin. Cells infected with the mutant accumulate -1/1Oth the amount of drug triphosphate as do those infected with the wild-type parent. This reduction in drug triphosphate could not be attributed to altered drug uptake or to reduced stability of the triphosphate, once formed. The induction of normal nucleoside and deoxynucleoside triphosphates and certain cellular nucleoside kinases was comparable in mutant and wild-type virus infections. These results provide strong evidence for the importance of phosphorylation in the selectivity of this antiviral compound and raise the possibility that human cytomegalovirus encodes a nucleoside kinase. The mutant may identify the existence of a cytomegalovirus function whose properties could facilitate genetic analysis of this important pathogen.Human cytomegalovirus (HCMV) infections can produce serious consequences, including birth defects in newborns and life-threatening and sight-threatening disease in immunosuppressed individuals, such as transplant and cancer chemotherapy recipients as well as patients with acquired immunodeficiency syndrome (1).Currently, there are no licensed treatments for HCMV infections in the U.S. One promising anti-HCMV agent is the deoxyguanosine analog, 9-{[2-hydroxy-1-(hydroxymethyl)-ethoxy]methyllguanine (BW B759U; also known as 2'NDG, DHPG, and BIOLF-62) (2-4). BW B759U is a congener ofthe antiherpetic drug acyclovir (ACV), which is licensed for the treatment of herpes simplex virus (HSV). However, BW B759U is much more potent against HCMV than is ACV (2-6). Recent clinical studies suggest that BW B759U has consistent antiviral activity against HCMV in vivo and may be useful in treatment of HCMV disease (7-9).BW B759U is converted to its triphosphate form in HCMVinfected cells 10-100 times more efficiently than in uninfected cells, and much more efficiently than is ACV (5, 6). BW B759U triphosphate can then inhibit HCMV-induced DNA polymerase with Ki values below 1.5 x 10-6 M (4, 6, 10). As yet, an enzyme(s) specifically induced by HCMV infection that phosphorylates BW B759U has not been identified. Furthermore, it has not been possible to detect a HCMVencoded enzyme with properties similar to the HSV-encoded thymidine kinase (TK) (refs. 11 and 12; J.A.F. and K.K.B., unpublished data), an enzyme that is principally responsible for the monophosphorylation of BW B759U and ACV in HSV-infected cells (13-15). On the other hand, it has been shown that HCMV induces increases in host cell nucleoside kinases (5,6,11,12,16,17), but none of these has been identified as the enzyme responsible for BW B759U phosphorylation. Thus, it is still an open question whether BW B759U anabolism in the HCMV-infected cell results from the activity of an unidentified virus-encoded enzyme or ...
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