The U69 Gene of Human Herpesvirus 6 Encodes a Protein Kinase Which Can Confer Ganciclovir Sensitivity to Baculoviruses

Abstract

The protein encoded by the U69 open reading frame (ORF) of human herpesvirus 6 (HHV-6) has been predicted to be a protein kinase. To investigate its functional properties, we have expressed the U69 ORFs from both HHV-6 variants, A and B, by using recombinant baculoviruses (BV6AU69 and BV6BU69). Nickel agarose and antibody affinity chromatography was used to purify the proteins to homogeneity and when incubated with [γ-32P]ATP, both U69 proteins became phosphorylated on predominantly serine residues. These data strongly suggest that U69 is a protein kinase which autophosphorylates. The phosphorylation reaction was optimal at physiological pH and low NaCl concentrations. It required the presence of Mg2+ or Mn2+, and Mg2+ was able to support phosphorylation over a wider range of concentrations than Mn2+. Both ATP and GTP could donate phosphate in the protein kinase assay and the former was more efficient. U69 was capable of phosphorylating histone and casein (serine/threonine kinase substrates) but not enolase (a tyrosine kinase substrate). For the autophosphorylation reaction, the Michaelis constants for ATP of baculovirus-expressed HHV-6A and HHV-6B U69 were calculated to be 44 and 11 μM, respectively. U69 is a homologue of the UL97 gene encoded by human cytomegalovirus which has been shown to phosphorylate the antiviral drug ganciclovir (GCV). We analyzed whether the U69 ORF alone was capable of conferring GCV sensitivity on baculoviruses BV6AU69 and BV6BU69. In plaque reduction experiments, these baculoviruses displayed a GCV-sensitive phenotype compared to a control baculovirus (BVLacZ). The 50% inhibitory concentrations (IC50) of BV6AU69 and BV6BU69 were calculated to be 0.35 and 0.26 mM, respectively, whereas the control baculovirus had an IC50 of >1.4 mM. This shows that the U69 gene product is the only one required to confer GCV sensitivity on baculovirus.

How to control an infectious bead string: nucleosome‐based regulation and targeting of herpesvirus chromatin

“…Previous results had already shown that the BGLF4 protein phosphorylates histones in in vitro kinase assays [206]. In fact, there are several examples of viral kinases capable of histone (usually H1 or H2B) phosphorylation in vitro, including HCMV UL97 [207], human herpesvirus 6 U69 [208], HSV-2 UL13 [209] as well as KSHV and MHV-68 viral cyclin/ cyclin-dependent kinase complexes [210][211][212][213]. However, the lack of in vivo data makes any conclusion about the actual relevance of these observations impossible.…”

Human herpesvirus 6 infection in hematopoietic stem cell transplant patients

“…Acyclovir and other thymidine kinase-dependent drugs, however, are only marginally effective (Agut et al, 1989;Burns & Sandford, 1990;Yoshida et al, 1998). The HHV-6 U69 gene product, the presumed functional homologue of HCMV UL97, phosphorylates ganciclovir (Ansari & Emery, 1999;De Bolle et al, 2002). A HHV-6 mutant exhibiting decreased sensitivity to ganciclovir in vitro was isolated after serial passages in the medium containing the drug; this mutant was later found to encode mutations in the U69 gene (Manichanh et al, 2001).…”

Antiviral agents active against human herpesviruses HHV‐6, HHV‐7 and HHV‐8

“…Since the identification of the CMV UL97-encoded protein kinase as the enzyme responsible for the activation of ganciclovir in CMV-infected cells [33], the homologous HHV-6 protein (pU69) has been thought to play a similar role in the activation of ganciclovir in HHV-6-infected cells. Ansari and Emery [34] demonstrated that the HHV-6 U69encoded serine/threonine kinase, when expressed in baculovirus, is able to confer ganciclovir sensitivity. In order to directly measure the HHV-6 pU69-catalysed phosphorylation of ganciclovir in human cells, we have recently cloned the U69 gene into vaccinia virus, according to a strategy that was originally developed for CMV UL97 [35].…”

“…Their homologous counterparts in EBV, HHV-6, HHV-7 and HHV-8 have either not been established (HHV-7) or have remained less well defined as far as the substrate specificity is concerned (HHV-6, HHV-8 and EBV). For HHV-6, we have already described the interaction of ganciclovir with the HHV-6 U69-encoded kinase [34], and for HHV-8 we addressed the ORF-21-and ORF-36-encoded kinases also with respect to ganciclovir as substrate [70,71]. The substrate specificity of the EBV BXLF-1-encoded TK is definitely narrower than that of its HSV-1, HSV-2 and VZV homologues: it would have good affinity for brivudin, but only poor activity for the acyclic nucleoside guanosine analogues acyclovir, ganciclovir and penciclovir [77][78][79].…”