Role of the Human Herpesvirus 6 U69-Encoded Kinase in the Phosphorylation of Ganciclovir LEEN DE BOLLE, DETLEF MICHEL, THOMAS MERTENS, CHAYSAVANH MANICHANH, HENRI AGUT, ERIK DE CLERCQ, and LIEVE NAESENS Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium (L.D.B., E.D.C., L.N.); Abteilung Virologie, Institut fu ¨r Mikrobiologie, Universita ¨ t Ulm, Ulm, Germany (D.M., T.M.); and Laboratoire de Virologie, Groupe Hospitalier Pitie ´ -Salpe ˆ trie ` re, Paris, France (C.M., H.A.) Received February 8, 2002; accepted May 31, 2002 This article is available online at http://molpharm.aspetjournals.org ABSTRACT The human herpesvirus 6 (HHV-6) U69 gene product (pU69) is the presumed functional homolog of the human cytomegalovi- rus (HCMV) UL97-encoded kinase (pUL97), which converts ganciclovir to its monophosphate metabolite in HCMV-infected cells. It has been reported that insertion of U69 into baculovirus confers sensitivity to ganciclovir in insect cells (J Virol 73:3284 – 3291, 1999). Our metabolic studies in HHV-6 –infected human T-lymphoblast cells indicated that the efficiency of ganciclovir phosphorylation induced by HHV-6 was relatively poor. Re- combinant vaccinia viruses (rVVs), expressing high levels of pU69 from two HHV-6 strains (representing the A and B vari- ant), were constructed and used to compare the ganciclovir- phosphorylating capacity of pU69 and pUL97 in human cells. Metabolic studies with [8- 3 H]ganciclovir showed that ganciclo- vir was phosphorylated in human cells infected with pU69- expressing rVVs, although the levels of phosphorylated ganci- clovir metabolites were approximately 10-fold lower than those observed with pUL97. We also demonstrated that pU69, like pUL97, is expressed as a nuclear protein. Our results indicate that the limited phosphorylation of ganciclovir by pU69 may contribute to its modest antiviral activity against HHV-6 in cer- tain cell systems. HHV-6 is a lymphotropic and neurotropic -herpesvirus that is closely related to human cytomegalovirus (HCMV) and human herpesvirus 7. It was first isolated in 1986 from the peripheral blood of patients with lymphoproliferative disorders (Salahuddin et al., 1986). HHV-6 exists as two distinct variants (designated A and B), that differ in anti- genic properties (Ablashi et al., 1991) and in DNA sequence, with 90% nucleotide homology (Dominguez et al., 1999; Ise- gawa et al., 1999). Primary HHV-6 infection usually occurs before the age of 2 years and is associated with exanthema subitum (Yamanishi et al., 1988). Exanthema subitum is caused almost exclusively by the B variant; the pathogenic potential of HHV-6A in primary infection remains to be clar- ified. In adults, the overall seropositivity to HHV-6 is 90% (Dockrell and Paya, 2001). HHV-6 infection in the majority of cases results from the reactivation of latent virus during immunosuppression, as in transplant recipients and in per- sons infected with HIV. Apart from acting as a transactivator for other viruses in these patients, HHV-6 by itself has been implicated in a wide range of clinical manifestations such as fever, encephalitis, pneumonitis, hepatitis, graft failure, and bone marrow suppression (Singh and Carrigan, 1996; Emery, 2001; Mendez et al., 2001). Moreover, as a result of the complex interactions between -herpesviruses, HHV-6 may act as a cofactor in cytomegalovirus disease (Dockrell and Paya, 2001). HHV-6 has also been associated with certain lymphomas, chronic fatigue syndrome, and multiple sclero- sis, although a causal link with these syndromes is highly debated (Campadelli-Fiume et al., 1999). The antiherpetic drugs active against HHV-6 are phospho- noformic acid (foscarnet), cidofovir (CDV), and the nucleoside analogs ganciclovir (GCV) and acyclovir, the latter showing only weak activity. Foscarnet has the best in vitro activity against HHV-6 but its long-term use in patients is hampered by serious side effects, mainly nephrotoxicity (MacGregor et al., 1991). Ganciclovir shows good and consistent activity against several HHV-6 clinical isolates in peripheral blood This work was supported by a grant from the Wetenschappelijk Onderzoek Multiple Sclerose vzw. L.D.B. is a Research Assistant of the Fonds voor Wetenschappelijk Onderzoek Vlaanderen. ABBREVIATIONS: HHV-6, human herpesvirus 6; pU69, human herpesvirus 6 U69 gene product; HCMV, human cytomegalovirus; pUL97, human cytomegalovirus UL97-encoded kinase; rVV, recombinant vaccinia virus; CBLC, cord blood lymphocyte; EGFP, enhanced green fluorescent protein; CDV, cidofovir; GCV, ganciclovir; FCS, fetal calf serum; PCR, polymerase chain reaction; RT, reverse transcriptase; VV, vaccinia virus; CC 50 , compound concentration that causes 50% inhibition of cell growth as determined by cell counting; IC 50 , the compound concentration that produced 50% inhibition of viral DNA replication; PBS, phosphate-buffered saline; HPLC, high-performance liquid chromatography; aa, amino acid; MCMV, murine cytomegalovirus; wtVV, wild-type vaccinia virus. 0026-895X/02/6203-714 –721$7.00 MOLECULAR PHARMACOLOGY Vol. 62, No. 3 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1662/1005388 Mol Pharmacol 62:714–721, 2002 Printed in U.S.A. 714 at ASPET Journals on June 15, 2017 molpharm.aspetjournals.org Downloaded from