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

Abstract

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.

FIG. 1

Alignments of the HHV-8 sequences to the proposed TK and PT catalytic domains of other herpesviruses by the CLUSTAL method (MEGALIGN Lasergene software) (31). (A) HHV-8 ORF 21 is compared to identified TKs in HSV (UL23), VZV (ORF 46), EBV (BXLF1), and HVS (ORF 21) within five conserved domains, which comprise the hypothetical catalytic site (3, 38). (B) HHV-8 ORF 36 is compared to the PTs characterized in HCMV (UL97), HSV (UL13), and VZV (ORF 47) and to putative PTs identified in EBV (BGLF4) and HVS (ORF 36).

FIG. 2

HPLC analysis of phosphorylated [³H]GCV products from TK- and PT-expressing cells. (A) 293T cells transfected with the vector control, ORF 21, or ORF 36-expressing plasmids were incubated with 8 μM [³H]GCV (specific activity, 1,000 dpm/pmol) for 30 h. Phosphorylated nucleosides were extracted and analyzed by HPLC (expressed in picomoles of GCV/10⁶ cells [determined for each sample]). Histograms show GCV MP, GCV DP, and GCV TP levels in the vector, TK, and PT transfectants relative to untransfected 293T control cells. Data shown are the averages of triplicate measurements in single experiments. Each experiment was repeated three times, and results were consistent. Error bars indicate standard deviations. (B) Western blot analysis was performed on protein extracts from TK and PT transfectants by using an anti-HA antibody specific for HA fusion tags at the N terminus of expressed TK and PT proteins. Efficiencies of transfection for each sample were corrected with an internal β-galactosidase control. The HA antibody shows cross-reactivity to a 90- to 100-kDa protein present in all 293T cells tested.

FIG. 3

Cytotoxic effects of GCV, PCV, and BVDU compounds in cells expressing HHV-8 ORFs 21 and 36 compared to those of other herpesvirus kinases. Following transfection, 293T cells were incubated in the absence or presence of GCV (25 μM) (A), PCV (25 μM) (B), or BVDU (25 μM) (C) for 4 days and assayed for cell viability. The data shown for each nucleoside analog are the means ± standard deviations of six separate experiments, and within each experiment, samples were performed in replicates of eight.

FIG. 4

Dose response for GCV sensitivity in cells expressing HHV-8 ORFs 21 and 36. Transfectants were incubated with various concentrations (5, 15, 25, 50, and 100 μM) of GCV. HCMV UL97-expressing cells, samples with no DNA, and vector transfectants were included as negative controls. The means ± standard deviations determined in three separate experiments are shown. In each experiment, cell killing was assessed in eight replicate wells.

FIG. 5

Expression of HHV-8-encoded TK and PT genes in infected cells in the presence or absence of butyrate and tetradecanoyl phorbol acetate (TPA) induction. JSC-1 cells were treated with sodium butyrate (1 mM) or TPA (20 ng/ml) for 24 h and harvested in parallel with control cells. Northern blot hybridization was performed on total RNA (10 μg) to determine the relative levels of PT and TK transcripts in induced and uninduced cells. Hybridizations to a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe demonstrate a comparable loading of RNA in each lane. Several overlapping transcripts were detected with the full-length ORF 36 probe. Hybridization with probes corresponding to ORFs 34, 35, 37, and 38 allowed the assignment of the 4.4-kb band to a transcript carrying ORFs 34 and 35, a 2.0-kb band to the transcript carrying ORF 37, and a 3.6-kb band to the transcript carrying ORF 36 (data not shown).