Ganciclovir Phosphorylation Research Articles

Suicide gene therapy of graft-versus-host disease: immune reconstitution with transplanted mature T cells

After allogeneic hematopoietic stem cell transplantation (HSCT), mature transplanted T cells play a major role in restoration of the immune system. However, they can also induce a life-threatening complication: graft-versus-host disease (GVHD). Suicide gene therapy of GVHD aims to selectively eliminate alloreactive T cells mediating GVHD while sparing nonalloreactive T cells that should contribute to immune reconstitution. It was demonstrated previously that treatment with ganciclovir (GCV) can control GVHD in mice by killing donor T cells engineered to express the thymidine kinase (TK) suicide gene. TK allows phosphorylation of nontoxic GCV into triphosphate GCV, which is selectively toxic for dividing cells. Thus, in the TK-GCV system, the specificity of cell killing depends on the cycling status of TK T cells rather than allogeneic recognition. This is a potential drawback because in recipients of lymphopenic allogeneic HSCT, alloreactive and homeostatic signals drive the proliferation of donor T cells. It is shown here that the onset of alloreactive T-cell division occurs earlier than that of nonalloreactive T cells, thus establishing a time frame for GCV administration. A 7-day GCV treatment initiated at the time of HSCT allowed efficient prevention of GVHD, while sparing a pool of nondividing donor TK T cells. These cells later expanded and contributed to the replenishment of the recipient immune system with a diversified T-cell receptor repertoire. These results provide a rationale for designing the therapeutic scheme when using TK-GCV suicide gene therapy in allogeneic HSCT.

Inhibitors of human cytomegalovirus replication drastically reduce the activity of the viral protein kinase pUL97

The UL97-encoded protein kinase (pUL97) of human cytomegalovirus (HCMV) plays a critical role in the control of virus replication. Deletion of the UL97 gene results in a drastic reduction in the replication efficiency. Although the exact function of pUL97 remains unclear and its sensitivity to specific inhibitors is speculative, protein kinase inhibitors of the indolocarbazole class are effective inhibitors of cytomegalovirus. Based on the phosphorylation of ganciclovir (GCV), a novel quantification system for pUL97 kinase activity was established: the phosphorylated form of GCV exerts an easily quantifiable cytotoxic effect in transfected cells. Importantly, the addition of indolocarbazole compounds, Gö6976 and NGIC-I, which were highly effective at nanomolar concentrations while other protein kinase inhibitors were not, led to a significant reduction of pUL97 kinase activity. It was also demonstrated that a catalytically inactive mutant of pUL97, K355M, and a GCV-resistant mutant, M460I, were both negative for GCV phosphorylation, although protein phosphorylation remained detectable for the latter mutant. In vitro kinase assays were used to confirm the levels of pUL97-mediated phosphorylation recorded. To generate a tool for screening large numbers of putative inhibitors that preferentially interfere with GCV as well as protein phosphorylation, pUL97-expressing cell clones with stable pUL97 kinase activity were selected. This study demonstrates that certain indolocarbazole compounds are potent pUL97 inhibitors and, therefore, represent novel candidates for antiviral drugs that target viral protein kinase functions.

Coexpression of guanylate kinase with thymidine kinase enhances prodrug cell killing in vitro and suppresses vascular smooth muscle cell proliferation in vivo.

Herpes simplex virus-thymidine kinase (HSV-TK) phosphorylates the prodrugs ganciclovir (GCV) and acyclovir (ACV), leading to disruption of DNA synthesis and inhibition of cell proliferation. HSV-TK vectors have been successfully employed in cardiovascular and cancer gene therapy. Activation of GCV and ACV, after an initial phosphorylation step by the viral thymidine kinase, is carried out by guanylate kinase. We reasoned that coexpression of guanylate kinase (GK) with HSV-TK would augment phosphorylation of GCV or ACV, leading to increased cell killing. To test this hypothesis, a vector expressing TK with GK (TKciteGK) was developed and tested on vascular smooth muscle cells (vsmcs) in vitro and in vivo. Compared to HSV-TK vectors, killing of vascular cells transduced with TKciteGK and exposed to GCV was significantly increased (P = 0.03). The TKciteGK construct was evaluated with three promoters: CMV, EF1alpha, and SM22alpha. TKciteGK expression driven by a CMV promoter induced cell killing more effectively than SM22alpha or EF1alpha promoters in primary vsmcs. Based upon these in vitro findings, TKciteGK vectors with a CMV promoter were tested in two animal models of cardiovascular disease: balloon angioplasty and stent deployment in pig arteries. Following vascular injury, expression of CMV-TKciteGK with GCV significantly reduced vsmc proliferation and intimal lesion formation compared to control vectors with GCV. In the angioplasty model, there was an 80% reduction in intima-to-media area ratio (P = 0.0002). These findings were paralleled in a stent model with 66% reduction in intimal lesions (P = 0.006). Coexpression of GK with TK increases cell killing and permits administration of GCV at lower doses. These modifications in TKciteGK vectors and GCV showed enhanced efficacy at lower prodrug doses, leading to improved safety for cardiovascular gene therapy.

Indolocarbazoles exhibit strong antiviral activity against human cytomegalovirus and are potent inhibitors of the pUL97 protein kinase

We have analyzed a panel of protein kinase inhibitors (PKIs) and found that some indolocarbazoles (Gö6976, K252a, K252c) proved to be highly effective inhibitors of GCV-sensitive and -resistant human cytomegalovirus (HCMV) strains, but did not show any effect against herpes simplex virus. Antiviral activity was determined by focus reduction assays (IC 50 ranging from 0.009 to 0.4 μM). Other inhibitors of serine/threonine kinases (Gö6850, H-7, roscovitine) were found to be ineffective. Virus yield at 5 days after infection was reduced by three orders of magnitude with nanomolar concentrations of the indolocarbazoles. These compounds were fully effective when added up to 24 h post infection and showed reduced activity up to 72 h post infection. Cytotoxicity assays in proliferating and non-proliferating cells demonstrated that the effective antiviral concentration of these compounds was significantly lower than either antiproliferative (IC 50/CC 50 ranging from 6.5 to 390) or cytotoxic (IC 50/CC 50 ranging from 72.5 to 1000) doses. The effects of PKIs on the virus-encoded protein kinase pUL97 were studied using recombinant vaccinia viruses. Indolocarbazoles strongly inhibited both pUL97 autophosphorylation (IC 50 ranging from 0.0012 to 0.013 μM) and pUL97-dependent ganciclovir phosphorylation (IC 50 ranging from 0.05 to 0.26 μM). Other inhibitors of serine/threonine kinases showed only weak (Gö6850) or no (H-7, roscovitine) effect on these pUL97 functions, while oxoflavone tyrosine kinase inhibitors had no effect at all.

15 Novel and potent anti-EBV compound, L-I-OddU dependents it's activity on EBV thymidine : Kinase Toshihiko Kira 1)2), Shiro Shigeta 1), Yung-Chi Cheng 2) 1)2)FuKushima Medical University, Dep. Microbiology, Hikarigaoka 1, Fukushima, Japan 960-1295 2)Yale University, Dep. Pharmacology, 333 Cedar St., New Haven, CT, 16520

The human cytomegalovirus (HCMV) gene UL97 product was shown to play an important role in phosphorylation of ganciclovir (GCV) in HCMV-infected cells. The deletion of the 4 amino-acid sequence AACR confers resistance to a laboratory mutant. The aim of this study was to develop a rapid and simple method to detect mutations within the 12 base pair (bp) fragment encoding AACR, from isolates and clinical specimens (urine, bronchoalveolar lavage, cerebral spinal fluid samples). A target region encompassing this 12 bp sequence was amplified by a single-step PCR assay from HCMV isolates and reference strains, and a two-step procedure from clinical specimens. Reaction products were submitted to restriction enzyme analysis and dot-blot hybridization assay. Two biotinylated probes were used: one probe (DL) overlapping the 12 bp region; and a control probe with similar length and GC content. Hybridization was performed under conditions allowing the detection of one bp deletion (HCMV strain susceptibility to GCV was determined by a rapid late antigen synthesis reduction assay.) The control probe hybridized to the UL97 sequence amplified from all 23 tested isolates and the reference strains. The DL probe gave a positive signal with GCV-susceptible strains; no signal was obtained for five out of seven resistant isolates, and for a laboratory mutant derived from the strain AD169. Restriction analysis of amplification products showed different patterns suggesting this region can be involved in various DNA changes. The results from assays directly performed on 10 clinical samples correlated with GCV strain susceptibility determination. This rapid and simple method for detecting mutations in this region of the gene UL97 could be a useful tool for identification of some GCV-resistant strains from isolates and directly from clinical specimens.

Differential Ganciclovir-mediated Cell Killing by Glutamine 125 Mutants of Herpes Simplex Virus Type 1 Thymidine Kinase

The therapeutic combination of the herpesvirus simplex virus type 1 (HSV-1) thymidine kinase (TK) gene and the prodrug, ganciclovir (GCV), has found great utility for the treatment of many types of cancer. After initial phosphorylation of GCV by HSV-1 TK, cellular kinases generate the toxic GCV-triphosphate metabolite that is incorporated into DNA and eventually leads to tumor cell death. The cellular and pharmacological mechanisms by which metabolites of GCV lead to cell death are still poorly defined. To begin to address these mechanisms, different mutated forms of HSV-1 TK at residue Gln-125 that have distinct substrate properties were expressed in mammalian cell lines. It was found that expression of the Asn-125 HSV-1 TK mutant in two cell lines, NIH3T3 and HCT-116, was equally effective as wild-type HSV-1 TK for metabolism and sensitivity to GCV, bystander effect killing and induction of apoptosis. The major difference between the two enzymes was the lack of deoxypyrimidine metabolism in the Asn-125 TK-expressing cells. In HCT-116 cells expressing the Glu-125 TK mutant, GCV metabolism was greatly attenuated, yet at higher GCV concentrations, cell sensitivity to the drug and bystander effect killing were diminished but still effective. Cell cycle analysis, 4', 6'-diamidine-2'-phenylindoledihydrochloride staining, and caspase 3 activation assays indicated different cell death responses in the Glu-125 TK-expressing cells as compared with the wild-type HSV-1 TK or Asn-125 TK-expressing cells. A mechanistic hypothesis to explain these results based on the differences in GCV-triphosphate metabolite levels is presented.

Amino acids of conserved kinase motifs of cytomegalovirus protein UL97 are essential for autophosphorylation.

Thirteen point mutations targeting predicted domains conserved in homologous protein kinases were introduced into the UL97 coding region of the human cytomegalovirus. All mutagenized proteins were expressed in cells infected with recombinant vaccinia viruses (rVV). Several mutations drastically reduced ganciclovir (GCV) phosphorylation. Mutations at amino acids G340, A442, L446, and F523 resulted in a complete loss of pUL97 phosphorylation, which was strictly associated with a loss of GCV phosphorylation. Our results confirm that in rVV-infected cells pUL97 phosphorylation is due to autophosphorylation and show that several amino acids conserved within domains of protein kinases are essential for this pUL97 phosphorylation. GCV phosphorylation is dependent on pUL97 phosphorylation.

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.

Human herpesvirus 8-encoded thymidine kinase and phosphotransferase homologues confer sensitivity to ganciclovir.

Human herpesvirus 8 (HHV-8) sensitivity to the nucleoside analog ganciclovir (GCV) suggests the presence of a virally encoded kinase that catalyzes the initial phosphorylation of GCV. Analysis of the HHV-8 genome identified two candidate kinases: proteins encoded by open reading frame (ORF) 21, with homology to the herpesvirus thymidine kinases (TK), and ORF 36, with homology to the herpesvirus phosphotransferases (PT). Experiments presented here show that both ORF 21 and ORF 36 encode GCV kinase activities as demonstrated by GCV phosphorylation and GCV-mediated cell death. In both regards the PT homologue ORF 36 was more active than the TK homologue ORF 21. ORF 21, but not ORF 36, weakly sensitized cells to killing by penciclovir. Neither ORF sensitized cells to killing by (E)-5-(2-bromovinyl)-2'-deoxyuridine.

Functional regions of the human cytomegalovirus protein pUL97 involved in nuclear localization and phosphorylation of ganciclovir and pUL97 itself.

In order to identify functional regions of the human cytomegalovirus protein pUL97 (i) different 5' fragments of the UL97 open reading frame (ORF) were fused to the coding region of the green fluorescent protein and (ii) recombinant vaccinia viruses (rVV) were generated carrying two full-length and 11 mutated UL97 ORFs. The results indicated the presence of an N-terminal region within pUL97 which changed the intracellular distribution of the fusion proteins. pUL97 was localized in the nucleus, but not in the nucleoli, and was detected in the nuclear matrix fraction. Expression of all pUL97 mutants could be confirmed by Western blot analysis. pUL97-associated ganciclovir (GCV) phosphorylation in rVV-infected cells, determined quantitatively by HPLC analysis, was abolished completely using individual UL97 deletion mutants. Phosphorylation of full-length and some of the mutated pUL97 was detected in cells infected with the rVVs. The UL97 constructs carrying point mutations from GCV-resistant HCMV isolates at positions 460M, 520H, 594V, and the 4 aa deletion 590AACR593, also resulted in decreased but not abolished phosphorylation of GCV in the rVV system, whereas the phosphorylation of pUL97 itself was not influenced. The rVV system is a suitable method for quantitatively testing the functional relevance of pUL97 mutations.

Synergistic anticancer effects of ganciclovir/thymidine kinase and 5-fluorocytosine/cytosine deaminase gene therapies.

A bacterial enzyme, Escherichia coli cytosine deaminase, which converts the prodrug 5-fluorocytosine into the toxic drug 5-fluorouracil, and a viral enzyme, herpes simplex virus thymidine kinase, which converts ganciclovir from an inactive prodrug to a cytotoxic agent by phosphorylation, are being actively investigated for use in gene therapy for cancer. The purpose of this study was to determine whether combining these prodrug-activating gene therapies might result in enhanced anticancer effects. Rat 9L gliosarcoma cells were transfected with plasmids containing the E. coli cytosine deaminase gene (9L/CD cells), with plasmids containing the herpes simplex virus thymidine kinase gene (9L/TK cells), or with both expression plasmids (9L/CD-TK cells). The drug sensitivities of the cell lines were evaluated; in addition, the sensitivities of 9L and 9L/CD-TK cells mixed in varied proportions were measured. The effects of prodrug treatment on 9L/CD-TK tumor growth (i.e., size and volume) in nude mice were monitored. The isobologram method of Loewe and the multiple drug-effect analysis method of Chou-Talalay were used to measure the interaction between the two prodrug-activating gene therapies. To elucidate the mechanism of interaction, the phosphorylation of ganciclovir in 9L/CD-TK cells after varying prodrug treatments was studied. The presence of transfected cytosine deaminase and thymidine kinase genes in 9L gliosarcoma cells reduced cell survival, both in vitro and in vivo, following treatment with the relevant prodrugs; the effects of the two components appeared to be synergistic and related mechanistically to the enhancement of ganciclovir phosphorylation by thymidine kinase following 5-fluorouracil treatment.

The Cys607-->Tyr change in the UL97 phosphotransferase confers ganciclovir resistance to two human cytomegalovirus strains recovered from two immunocompromised patients.

Two ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) strains recovered from an AIDS patient (strain VR4990) and a heart transplant recipient (strain VR5474) showed a Cys607-->Tyr change in the UL97-encoded phosphotransferase. No amino acid substitutions were observed in the viral DNA polymerase. Marker transfer experiments showed marked reduction in GCV phosphorylation and drug susceptibility of the recombinant HCMV strain VR4990rec2-1-1. These results further extend the region of the carboxy-terminal domain of the UL97 phosphotransferase involved in GCV substrate recognition.

Lobucavir is phosphorylated in human cytomegalovirus-infected and -uninfected cells and inhibits the viral DNA polymerase.

Lobucavir (LBV) is a deoxyguanine nucleoside analog with broad-spectrum antiviral activity. LBV was previously shown to inhibit herpes simplex virus (HSV) DNA polymerase after phosphorylation by the HSV thymidine kinase. Here we determined the mechanism of action of LBV against human cytomegalovirus (HCMV). LBV inhibited HCMV DNA synthesis to a degree comparable to that of ganciclovir (GCV), a drug known to target the viral DNA polymerase. The expression of late proteins and RNA, dependent on viral DNA synthesis, was also inhibited by LBV. Immediate-early and early HCMV gene expression was unaffected, suggesting that LBV acts temporally coincident with HCMV DNA synthesis and not through cytotoxicity. In vitro, the triphosphate of LBV was a potent inhibitor of HCMV DNA polymerase with a Ki of 5 nM. LBV was phosphorylated to its triphosphate form intracellularly in both infected and uninfected cells, with phosphorylated metabolite levels two- to threefold higher in infected cells. GCV-resistant HCMV isolates, with deficient GCV phosphorylation due to mutations in the UL97 protein kinase, remained sensitive to LBV. Overall, these results suggest that LBV-triphosphate halts HCMV DNA replication by inhibiting the viral DNA polymerase and that LBV phosphorylation can occur in the absence of viral factors including the UL97 protein kinase. Furthermore, LBV may be effective in the treatment of GCV-resistant HCMV.

O-10-146 - Primary spinal cord tumors in children

Herpes simplex virus-thymidine kinase (HSV-TK) phosphorylates the prodrugs ganciclovir (GCV) and acyclovir (ACV), leading to disruption of DNA synthesis and inhibition of cell proliferation. HSV-TK vectors have been successfully employed in cardiovascular and cancer gene therapy. Activation of GCV and ACV, after an initial phosphorylation step by the viral thymidine kinase, is carried out by guanylate kinase. We reasoned that coexpression of guanylate kinase (GK) with HSV-TK would augment phosphorylation of GCV or ACV, leading to increased cell killing. To test this hypothesis, a vector expressing TK with GK (TKciteGK) was developed and tested on vascular smooth muscle cells (vsmcs) in vitro and in vivo. Compared to HSV-TK vectors, killing of vascular cells transduced with TKciteGK and exposed to GCV was significantly increased (P = 0.03). The TKciteGK construct was evaluated with three promoters: CMV, EF1α, and SM22α. TKciteGK expression driven by a CMV promoter induced cell killing more effectively than SM22α or EF1α promoters in primary vsmcs. Based upon these in vitro findings, TKciteGK vectors with a CMV promoter were tested in two animal models of cardiovascular disease: balloon angioplasty and stent deployment in pig arteries. Following vascular injury, expression of CMV-TKciteGK with GCV significantly reduced vsmc proliferation and intimal lesion formation compared to control vectors with GCV. In the angioplasty model, there was an 80% reduction in intima-to-media area ratio (P = 0.0002). These findings were paralleled in a stent model with 66% reduction in intimal lesions (P = 0.006). Coexpression of GK with TK increases cell killing and permits administration of GCV at lower doses. These modifications in TKciteGK vectors and GCV showed enhanced efficacy at lower prodrug doses, leading to improved safety for cardiovascular gene therapy.

Metabolism of ganciclovir and cidofovir in cells infected with drug-resistant and wild-type strains of murine cytomegalovirus

Murine cytomegalovirus (MCMV) has been used extensively as an animal model for human cytomegalovirus (HCMV). Understanding drug resistance and its treatment in MCMV may lead to more effective treatments of HCMV disease. Most ganciclovir-resistant HCMV clinical isolates exhibit a decreased capacity to induce ganciclovir phosphorylation (to its biologically active form) in infected cells. Using an MCMV strain resistant to both ganciclovir and cidofovir, the intracellular metabolism of these drugs was studied to determine if MCMV resistance correlates with decreases in drug phosphorylation. The wild-type (WT) MCMV used for comparison was inhibited in plaque reduction assays, by ganciclovir and cidofovir by 50% at 5.1 and 0.24 μM, respectively; the resistant strain was inhibited at 72 and 2.7 μM, respectively. In uninfected, WT, or resistant virus-infected cells, the extent of metabolism of 10 μM ganciclovir or 1 μM cidofovir to intracellular triphosphorylated species was similar. Phosphorylation and catabolism (following drug removal) rates over time were also similar. Intracellular levels of ganciclovir triphosphate and cidofovir diphosphate increased less than two-fold with increasing multiplicity of virus infection. Because few differences in drug phosphorylation between WT and resistant virus-infected cells were found, virus resistance to ganciclovir and cidofovir apparently is not linked to altered drug phosphorylation. Since the viral DNA polymerase is the antiviral target for these compounds, the resistant MCMV is most likely a DNA polymerase mutant.

The Human Cytomegalovirus UL97 Protein Is Phosphorylated and a Component of Virions

The expression of the human cytomegalovirus (HCMV) UL97 open reading frame in infected or transfected cells in the presence of the antiherpes compound ganciclovir (GCV) results in the intracellular phosphorylation of GCV. There are conventional kinase domains within the UL97-encoded protein (pUL97). However, the role of pUL97 in the HCMV replication cycle, and the mechanism by which it causes phosphorylation of GCV, are currently unknown. Herein, the biosynthesis and biogenesis of pUL97 was studied in HCMV-infected cells. pUL97 is expressed with early-late kinetics and is posttranslationally modified by phosphorylation. This phosphorylation occurs within 1 hr after synthesis, affects the electrophoretic mobility of pUL97, and is independent of the presence of other HCMV proteins. pUL97 was localized to the nucleus of infected cells and found in the HCMV virions. Thus, pUL97 is a virion phosphoprotein, and a likely tegument component.

Selective Activity of Various Nucleoside and Nucleotide Analogues against Human Herpesvirus 6 and 7

We have developed a new sensitive enzyme immunoassay (EIA) and MTT (tetrazolium salt) assay for screening compounds against two variants of human herpesvirus 6 (HHV-6A, HHV-6B) and human herpesvirus 7 (HHV-7) and evaluated the anti-HHV-6 and HHV-7 activity of a series of anti-herpesvirus compounds and acyclic nucleoside phosphonate analogues. The results indicate that the pattern of activity of these compounds against these betaherpesviruses is similar to that for human cytomegalovirus (HCMV). The highest potency and selectivity against the two variants of HHV-6 and HHV-7 was demonstrated by S2242 (N7-isomer of 6-deoxy-ganciclovir). Also, ganciclovir (GCV), foscarnet, (phosphonoformic acid; PFA) and the acyclic nucleoside phosphonate analogues such as cidofovir (HPMPC) exhibited selective inhibitory activity against these viruses. Thymidine kinase (TK)-dependent drugs (acyclovir, ACV; brivudin, BVDU; and sorivudine, BVaraU) showed little, if any, activity. These results suggest a structural homology of the DNA polymerase and a lack of TK gene among these three betaherpesviruses (HHV-6, HHV-7 and HCMV). The finding that HHV-7 was highly sensitive to GCV also suggests that HHV-7 may have an HCMV-UL97-homologue gene for the phosphorylation of GCV. The present EIA method is more rapid and sensitive than the previously reported procedures and could be useful for the large-scale screening of compounds against HHV-6 and HHV-7.

The human cytomegalovirus UL97 protein is a protein kinase that autophosphorylates on serines and threonines.

The product of the human cytomegalovirus (CMV) UL97 gene, which controls ganciclovir phosphorylation in virus-infected cells, is homologous to known protein kinases but diverges from them at a number of positions that are functionally important. To investigate UL97, we raised an antibody against it and overexpressed it in baculovirus-infected insect cells. Recombinant baculovirus expressing full-length UL97 directed the phosphorylation of ganciclovir in insect cells, which was abolished by a four-codon deletion that confers ganciclovir resistance to CMV. When incubated with [gamma-32P]ATP, full-length UL97 was phosphorylated on serine and threonine residues. Phosphorylation was severely impaired by a point mutation that alters lysine-355 in a motif that aligns with subdomain II of protein kinases. However, phosphorylation was impaired much less severely by the four-codon deletion. A UL97 fusion protein expressed from recombinant baculovirus was purified to near homogeneity. It too was phosphorylated upon incubation with [gamma-32P]ATP in vitro. This phosphorylation, which was abolished by the lysine 355 mutation, was optimal at high NaCl and high pH. The activity required either Mn2+ or Mg2+, with a preference for Mn2+, and utilized either ATP or GTP as a phosphate donor, with Kms of 2 and 4 microM, respectively. The phosphorylation rate was first order with protein concentration, consistent with autophosphorylation. These data strongly argue that UL97 is a serine/threonine protein kinase that autophosphorylates and suggest that the four-codon deletion affects its substrate specificity.

Molecular analysis of human cytomegalovirus strains from two lung transplant recipients with the same donor.

A total of four cytomegalovirus (CMV) isolates were obtained from two CMV seronegative patients, each of whom received a lung transplant from the same seropositive donor. CMV was isolated from Patient 1 from two bronchial alveolar lavage (BAL) specimens, one obtained during treatment with ganciclovir (GCV) and a second during later treatment with foscarnet. Both of these isolates are sensitive to GCV and foscarnet. CMV was isolated from Patient 2 from a blood and a BAL specimen obtained during treatment with GCV. Both of these isolates are resistant to GCV and show reduced GCV phosphorylation. Patient 1 is still alive 33 months posttransplant. Patient 2 died 6 1/2 months posttransplant. Although the four strains differ with respect to GCV susceptibility and phosphorylation, their DNA restriction fragment hybridization patterns and UL97 kinase gene sequences indicate that they are closely related. The restriction fragment hybridization patterns are identical among the strains, while these patterns differ markedly from those of unrelated strains. The DNA sequences of the UL97 genes of the strains from Patient 2 differ by only one nucleotide from those of Patient 1. The same comparison with unrelated strains shows a minimum of 12 nucleotide differences. The nucleotide change in the strains from Patient 2 produces an amino acid substitution of serine for leucine at residue 595, a substitution that was previously shown to transfer GCV resistance. Both patients, therefore, were apparently infected with the same donor strain, but during the course of GCV prophylaxis and treatment, a GCV-resistant mutant strain was selected in Patient 2.

Cidofovir, a New Agent with Potent Anti-Herpesvirus Activity

Cidofovir is a potent, broad spectrum antiviral agent with activity in vitro and in vivo against cytomegalovirus and other members of the herpesvirus family, as well as certain other DNA viruses. After uptake into cells it is converted enzymatically to cidofovir diphosphate, a structural analogue of deoxycytidine triphosphate, which selectively inhibits viral DNA polymerases relative to host cell polymerases. Cross-resistance to cidofovir is not usually seen with human cytomegalovirus isolates that are foscarnet-resistant, or isolates that are ganciclovir-resistant due to a deficiency in ganciclovir phosphorylation. Cross-resistance is seen, however, with isolates that are ganciclovir resistant due to polymerase mutations. A prolonged elimination phase seen in vivo, correlates with a long intracellular half-life seen in vitro and allows for efficacy in animal models of virus infection with infrequent dosing or prophylaxis. Clinical studies of intravenous cidofovir in cytomegalovirus retinitis in patients with AIDS are claimed to show delay of retinitis progression with maintenance doses given once every 2 weeks.