Acyclovir is phosphorylated by the human cytomegalovirus UL97 protein.

Acyclovir (ACV) has shown efficacy in the prophylactic suppression of human cytomegalovirus (HCMV) reactivation in immunocompromised renal transplant patients without the toxicity associated with ganciclovir (GCV). The HCMV UL97 gene product, a protein kinase, is responsible for the phosphorylation of GCV in HCMV-infected cells. This report provides evidence for the phosphorylation of ACV by UL97. Anabolism studies with the HCMV wild-type strain AD169 and with recombinant mutants derived from marker transfer experiments performed by using mutant UL97 DNA from both clinical isolates and a laboratory-derived strain resistant to GCV showed that mutations in the UL97 gene cripple the ability of recombinant virus-infected cells to anabolize both GCV and ACV. These mutant UL97 recombinant viruses were less susceptible to both GCV and ACV than was the wild-type strain. A recombinant herpes simplex virus type 1 strain, in which the thymidine kinase gene is deleted and the UL13 gene is replaced with the HCMV UL97 gene, was able to induce the phosphorylation of ACV in infected cells. Finally, purified UL97 phosphorylated both GCV and ACV to their monophosphates. Our results indicate that UL97 promotes the selective activity of ACV against HCMV.

Inhibition of human cytomegalovirus DNA maturation by a benzimidazole ribonucleoside is mediated through the UL89 gene product.

2-Bromo-5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (BDCRB) is a member of a new class of benzimidazole ribonucleosides which inhibit human cytomegalovirus (HCMV) late in the replication cycle without inhibiting viral DNA synthesis. We show here that polygenomic concatemeric HCMV DNA does not mature to unit genome length in the presence of BDCRB. To discover the locus of action, virus resistant to BDCRB was selected by serial passage in the presence of the compound. Genetic mapping experiments with BDCRB-resistant virus demonstrated that the resistance phenotype mapped to one amino acid (Asp344Glu; low resistance) or two amino acids (Asp344Glu and Ala355Thr; high resistance) within the product of exon 2 of the HCMV U(L)89 open reading frame. The HCMV U(L)89 open reading frame and its homologs are among the most conserved open reading frames in the herpesviruses, and their products have sequence similarities to a known ATP-dependent endonuclease from the double-stranded DNA bacteriophage T4. These findings strongly suggest that BDCRB prevents viral DNA maturation by interacting with a U(L)89 gene product and that the U(L)89 open reading frame may encode an endonucleolytic subunit of the putative HCMV terminase. Further, since mammalian cell DNA replication does not involve a DNA maturation step, compounds which inhibit viral DNA maturation should be selective and safe.

Single amino acid changes in the DNA polymerase confer foscarnet resistance and slow-growth phenotype, while mutations in the UL97-encoded phosphotransferase confer ganciclovir resistance in three double-resistant human cytomegalovirus strains recovered from patients with AIDS.

Three human cytomegalovirus (HCMV) strains (VR4760, VR4955, and VR5120) showing double resistance to ganciclovir (GCV) and foscarnet (PFA) were isolated from three patients with AIDS who underwent multiple sequential courses of therapy with GCV and PFA (A. Sarasini, F. Baldanti, M. Furione, E. Percivalle, R. Brerra, M. Barbi, and G. Gerna, J. Med. Virol., 47:237-244, 1995). We previously demonstrated that the three strains were genetically unrelated and that each of them was present as a single viral population in vivo. Thus, in each of the three cases, a single viral strain was resistant to both GCV and PFA. In the present paper, we report the characterization of the molecular bases of the double resistance and demonstrate that the PFA resistance is associated with a slower replication of HCMV strains in cell cultures. Sequencing of the UL97 and UL54 genes, GCV anabolism assays, and marker transfer experiments showed that GCV resistance was due to single amino acid changes in the UL97 gene product (VR4760, Met-460 --> Ile; VR4955, Ala-594 --> Val; VR5120, Leu595 --> Ser), while single amino acid changes in domain II of the DNA polymerase (VR4760 and VR5120, Val-715 --> Met; VR4955, Thr-700 --> Ala) were responsible for both the PFA resistance and the slow-growth phenotype. Thus, in these three cases, double resistance to GCV and PFA was not due to a single mutation conferring cross-resistance or to the presence of a mixture of strains with different drug susceptibilities. The HCMV DNA polymerase recombinant strains carrying the mutations conferring PFA resistance were sensitive to GCV and (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC). In addition, the same UL54 mutations were responsible for the slow growth of the clinical isolates, since the recombinant strains showed a marked delay in immediate-early antigen plaque formation and a reduction of infectious virus yield compared with AD169, from which they were derived. These results may have some important implications for the successful isolation, propagation, and characterization of PFA-resistant strains from clinical samples containing mixed viral populations.

Analysis of the UL97 phosphotransferase coding sequence in clinical cytomegalovirus isolates and identification of mutations conferring ganciclovir resistance.

The UL97 phosphotransferase coding sequences of clinical cytomegalovirus (CMV) isolates, 10 resistant and 11 sensitive to ganciclovir, were compared to define mutations associated with drug resistance. In each ganciclovir-resistant isolate, a mutation was found that resulted in an amino acid substitution at codon 460 (4 isolates), codon 594 (2 isolates), or codon 595 (4 isolates). No sensitive isolate carried any of these mutations. Marker transfer studies showed that each mutation was capable of conferring ganciclovir resistance to the laboratory CMV strain AD169. Rapid diagnostic tests based on DNA amplification and restriction enzyme analysis were developed for these mutations. Specific mutant DNAs were detected when they constituted at least 10% of the population in the specimen. Several mutations in UL97 appear to be common markers for ganciclovir resistance, and their detection may be a rapid alternative to conventional cell culture susceptibility testing.

A three-nucleotide deletion in the UL97 open reading frame is responsible for the ganciclovir resistance of a human cytomegalovirus clinical isolate.

Multiple human cytomegalovirus (HCMV) strains frequently coexist in patients with AIDS, and chronic ganciclovir treatment may favor the emergence of ganciclovir-resistant viral mutants. We report the molecular and biochemical characterization of a HCMV ganciclovir-resistant strain (VR3480) previously recovered from a patient with AIDS who was undergoing multiple courses of ganciclovir treatment (G. Gerna, F. Baldanti, M. Zavattoni, A. Sarasini, E. Percivalle, and M. G. Revello, Antiviral Res. 19:333-345, 1992). Ganciclovir resistance of strain VR3480 was related to impaired ability to monophosphorylate the drug, as indicated by the finding that ganciclovir phosphorylation values for VR3480 were 30% of those shown by the HCMV reference strain AD169 in an in vitro activity assay. Sequencing of the UL97 gene of VR3480, which encodes the viral kinase responsible for ganciclovir phosphorylation, showed an in-frame deletion of three nucleotides resulting in the loss of a leucine at position 595 in the polypeptide. Mutant VR3480 UL97 DNA was able to transfer resistance to the AD169 strain in marker rescue experiments. Analysis of virus isolates and blood polymorphonuclear leukocyte samples spanning the 2-year follow-up period of the patient showed that ganciclovir-resistant strain VR3480 arose ex novo during prolonged antiviral treatment and accounted for the majority of virus load circulating in blood during the period of clinical resistance to ganciclovir treatment.

A point mutation in the human cytomegalovirus DNA polymerase gene confers resistance to ganciclovir and phosphonylmethoxyalkyl derivatives.

Ganciclovir-resistant mutant 759rD100 derived from human cytomegalovirus strain AD169 contains two resistance mutations, one of which is in the UL97 gene and results in decreased ganciclovir phosphorylation in infected cells [V. Sullivan, C. L. Talarico, S. C. Stanat, M. Davis, D. M. Coen, and K. K. Biron, Nature (London) 358:162-164, 1992]. In the present study, we mapped the second mutation to a 4.1-kb DNA fragment containing the DNA polymerase gene and showed that it confers ganciclovir resistance without impairing phosphorylation. Sequence analysis of the 4.1-kb region revealed a single nucleotide change that resulted in a glycine-to-alanine substitution at position 987 within conserved region V of the DNA polymerase. Recombinant viruses constructed to contain the DNA polymerase mutation but not the phosphorylation defect displayed intermediate resistance (4- to 6-fold) to ganciclovir relative to the original mutant 759rD100 (22-fold); the recombinant viruses also displayed resistance to ganciclovir cyclic phosphate (7-fold), 1-(dihydroxy-2-propoxymethyl)-cytosine (12-fold), and the phosphonylmethoxyalkyl derivatives (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)adenine and (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (8- to 10-fold). However, the recombinant viruses remained susceptible to certain related compounds. These results imply that the human cytomegalovirus DNA polymerase is a selective target for the antiviral activities of ganciclovir, certain of its derivatives and phosphonomethoxyalkyl derivatives; support a role for region V in substrate recognition; and suggest the possibility of clinical resistance of human cytomegalovirus to these compounds because of polymerase mutations.

A protein kinase homologue controls phosphorylation of ganciclovir in human cytomegalovirus-infected cells.

Human cytomegalovirus (HCMV) is a major pathogen in immunosuppressed individuals, including patients with acquired immune deficiency syndrome. The nucleoside analogue ganciclovir (9-(1,3-dihydroxy-2-propoxymethyl)-guanine) is one of the few drugs available to treat HCMV infections, but resistant virus is a growing problem in the clinic and there is a critical need for new drugs. The study of ganciclovir-resistant mutants has indicated that the selective action of ganciclovir depends largely on virus-controlled phosphorylation in HCMV-infected cells. The enzyme(s) responsible have not been identified. Here we report that the HCMV gene UL97, whose predicted product shares regions of homology with protein kinases, guanylyl cyclase and bacterial phosphotransferases, controls phosphorylation of ganciclovir in HCMV-infected cells. A four-amino-acid deletion of UL97 in a conserved region, which in cyclic AMP-dependent protein kinase participates in substrate recognition, causes impaired ganciclovir phosphorylation. The implications of these results for antiviral drug development and drug resistance are discussed.

Ganciclovir-resistant cytomegalovirus clinical isolates: mode of resistance to ganciclovir.

Cytomegalovirus strains with reduced in vitro susceptibilities to ganciclovir have been recovered from patients who failed long-term ganciclovir therapy. The ganciclovir-resistant clinical isolates in this study were unable to induce ganciclovir phosphorylation in virus-infected cells. The viral DNA polymerase function appeared unaltered in one genetically pure ganciclovir-resistant strain, compared with that of its wild-type ganciclovir-sensitive counterpart. All nine of the ganciclovir-resistant strains were susceptible to foscarnet. Moreover, these strains were sensitive to inhibition both by vidarabine and 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC), antiviral agents that are activated by cellular enzymes, and by (S)-1(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC), which is a monophosphate nucleoside analog. The in vitro resistance to ganciclovir of the ganciclovir-resistant clinical isolates studied was attributed to the inability of the cells infected with these isolates to phosphorylate ganciclovir; the virally encoded DNA polymerase did not appear to play a role in this ganciclovir resistance.

Rapid antiviral DNA-DNA hybridization assay for human cytomegalovirus.

A rapid DNA-DNA hybridization technique that can be accomplished in 4 to 5 days was compared with plaque reduction assay to determine its reliability in performing antiviral assays for human cytomegalovirus (HCMV). The assay involves lysing infected cells, direct wicking of denatured DNA onto membranes and hybridization using a 125I-labeled HCMV DNA probe. Using ten ganciclovir sensitive clinical HCMV strains for comparison, the DNA hybridization technique correlated well with the plaque assay. Clinical HCMV strains previously identified as resistant to ganciclovir were also readily identified. The DNA-DNA hybridization assay is less tedious and more rapid than plaque reduction assays, and thus, provides an excellent alternative for evaluation of the antiviral activity of drugs against HCMV.

Acyclovir-resistant varicella zoster virus infection after chronic oral acyclovir therapy in patients with the acquired immunodeficiency syndrome (AIDS).

Four patients with human immunodeficiency virus (HIV) infection who received chronic oral acyclovir therapy for suppression of recurrent varicella zoster or herpes simplex virus infection developed persistent disseminated hyperkeratotic papules that failed to heal with intravenous or high-dose oral acyclovir therapy. Varicella zoster virus, resistant to acyclovir in vitro, was isolated from skin lesions of all four patients. Three patients were adults in whom the acquired immunodeficiency syndrome (AIDS) had been diagnosed 12 to 20 months before isolation of acyclovir-resistant varicella zoster virus. The fourth patient was a perinatally HIV-infected child who developed primary varicella infection at age 7 years when profoundly immunosuppressed (absolute CD4+ lymphocyte count less than 50 cells/microL). Mean antiviral susceptibilities (ED50 values) of the four clinical isolates compared with the ED50 values of the reference strain Oka were 85 compared with 3.3 mumol/L for acyclovir, 1.4 compared with 0.8 mumol/L for vidarabine, and 123 compared with 117 mumol/L for foscarnet. When assayed by [125I]-dC plaque autoradiography, 90% to 100% of the viral isolate populations had altered or no measurable thymidine kinase function. Acyclovir-resistant varicella zoster virus infection may complicate long-term oral acyclovir administration in patients with AIDS and may be associated with the appearance of atypical hyperkeratotic papules.

A human cytomegalovirus mutant resistant to the nucleoside analog 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (BW B759U) induces reduced levels of BW B759U triphosphate.

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 approximately equal to 1/10th 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.

Murine cytomegalovirus infection of mouse testes.

With the aim of illustrating a mechanism of cytomegalovirus (CMV) venereal transmission, we induced murine CMV infection in the mouse testes of immunologically competent mice. Using in situ cytohybridization, we were able to show that murine CMV-specific DNA was associated with spermatocytes and mature sperm. Electron microscopy studies also supported sperm infection. The virus could be reisolated from infected epididymal sperm by cocultivation with mouse embryo fibroblasts. We found no difference in either the sexual performance or the fertilization efficiency of the sperm between infected and uninfected males.

Metabolic activation of the nucleoside analog 9-[( 2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine in human diploid fibroblasts infected with human cytomegalovirus.

9-[( 2-Hydroxy-1-(hydroxymethyl)ethoxy]-methyl)guanine (BW B759U) is a more potent inhibitor of human cytomegalovirus (HCMV) in vitro than is the related nucleoside analog acyclovir (ACV). BW B759U was selectively activated to the 5'-triphosphate (BW B759U-triphosphate) in cells infected with HCMV to levels at least 10-fold higher than those measured for ACV-triphosphate and up to as much as 100-fold higher than the levels found in uninfected cells. BW B759U-triphosphate accumulated in HCMV-infected cells with time; the rate of this increase was dependent upon the drug dose and virus multiplicity of infection. Enzyme activities that catalyzed the phosphorylation of thymidine and 2'-deoxycytidine increased 3- to 7-fold in extracts of cells early after HCMV infection but thereafter declined. No concomitant increase in the rate of BW B759U phosphorylation was detected under these assay conditions. Maximal rate of accumulation of both BW B759U-triphosphate and ACV-triphosphate after a short exposure to drug occurred in the late phase of the infective cycle, as the titer of extracellular virus reached a peak in untreated cultures, but after the decline of stimulated host deoxypyrimidine kinase activities. Once formed, the BW B759U-triphosphate pool decreased very slowly and thus it persisted for several days in both HCMV-infected and uninfected cells.

Murine cytomegalovirus-induced congenital defects and fetal maldevelopment.

The effects of congenital infection with cytomegalovirus on fetal development were studied by injection of murine cytomegalovirus (MCMV) into the endometrial lumina of mice in the early stages of pregnancy. The mice were later killed, and the sites of embryonic implantation were examined. Litter size was reduced and the incidence of abnormal fetuses was significantly increased among MCMV-infected animals. Morbid, moribund, severely growth-retarded, asynchronously developed, and malformed fetuses were found. Virus was reisolated from infected mothers, placentas, and fetal tissues but not from control animals that had received either heat-inactivated virus or culture medium. The recovered virus was identical to the original virus, as verified by restriction enzyme analysis. MCMV-specific DNA was detected in experimental tissue sections by in situ nucleic-acid hybridization. Seroconversion occurred in infected female mice. Thus, the presence of MCMV in the genital tract at the time of embryonic implantation causes not only embryonic infection but also fetal maldevelopment.

Cytomegalovirus infection of murine testicular interstitial Leydig cells.

We studied the susceptibility of mouse testicular interstitial Leydig cells to cytomegalovirus both in vivo and in vitro. The in vivo studies included intratesticular and intraperitoneal infection of 6-week-old mice with murine cytomegalovirus (MCMV); the in vitro studies involved an MCMV-Leydig cell interaction using a Leydig tumor cell line (I-10). MCMV-specific antigens were detected in interstitial Leydig cells in sections of MCMV-inoculated testes by an indirect immunofluorescence test. MCMV DNA was also localized in the same testes cells derived from mice, which received intratesticular and intraperitoneal MCMV inoculations, respectively, by in situ DNA-RNA hybridization. Cytopathic effects were seen in MCMV-infected I-10 cell cultures 2 or more days after exposure to MCMV. The infected cells showed intranuclear inclusions characteristic of cytomegalovirus when stained with May-Grunwald-Giemsa stain. The indirect immunofluorescence test was also positive with MCMV-infected I-10 cells. MCMV DNA was detected in these cells by in situ DNA-RNA cytohybridization, and the presence of viral particles in MCMV-infected I-10 cells was confirmed by electron microscopy. Thus, we conclude that the interstitial Leydig cell is susceptible to MCMV infection both in vivo and in vitro.