Antiviral therapy of primary and recurrent infections with human cytomegalovirus is reserved for severe manifestations and faces several limitations. Presently candidates for novel drugs with lower adverse side effects and a minimized frequency of resistance formation are under investigation. Here we demonstrate that artesunate, an antimalaria drug with highly valuable pharmacological properties, possesses antiviral activity. A concentration-dependent inhibition of the replication of human cytomegaloviruses with wild-type phenotype was demonstrated in several cell lines. Inhibition was quantified using recombinant green fluorescent protein expressing virus variants. The IC50 values were in the same range for ganciclovir-sensitive and ganciclovir-resistant human cytomegalovirus, as calculated with 5.8+/-0.4 microM and 6.9+/-0.2 microM, respectively. This indicated a strong antiviral potential and a lack of cross-resistance. The optimal antiviral concentrations of artesunate were separable from those inducing cytotoxicity. In addition, the replication of viruses from three genera was seen to be artesunate-sensitive to varying degrees. This suggests a mechanism linked to cellular activation pathways. Both the protein levels and the DNA binding activity of the two virus-induced cellular transcription factors Sp1 and NF-kappaB were found to be markedly reduced in the presence of artesunate. We also analyzed the cellular signaling kinase phosphoinositide 3-kinase, required for the activation of factors such as Sp1 and NF-kappaB in infected fibroblasts. The phosphorylation of two downstream effectors of phosphoinositide 3-kinase, Akt and p70S6K, was markedly inhibited in the presence of artesunate. Thus, artesunate possesses attractive antiviral characteristics which are suggestively based on the interference with essential steps in the host cell kinase cascades.

Human cytomegalovirus (HCMV) infection of transplant recipients is frequently associated with allograft vasculopathy and rejection. One potential mechanism is vascular injury from HCMVtriggered, immunologically mediated processes. HCMV infection has been shown to increase the expression of intercellular adhesion molecule-1 (ICAM-1). The objective of this study was to determine the molecular basis of HCMV-enhanced ICAM-1 gene expression. Transient transfection experiments identified the IE2p86 protein as a potent activator of the ICAM-1 promoter. The tegument protein pp71 showed a strong synergistic effect on IE2p86-mediated ICAM-1 promoter activation. Mutagenesis experiments defined a DNA element from 2110 to 242 relative to the transcription start site as responsive for IE2p86. Further point mutations within this DNA element identified an Sp1-binding site that was essential for strong synergistic activation by IE2p86 and pp71. To confirm the activation of ICAM-1 gene expression, human fibroblasts (HFF) as well as endothelial cells (HUVEC) were infected with recombinant IE2p86-and pp71-expressing baculoviruses, respectively. In FACS analysis, a synergistic induction of ICAM-1 was detectable when cells were co-infected with the two recombinant baculoviruses. These findings clearly demonstrate that IE2p86 and pp71 are crucial regulatory factors for HCMV-induced ICAM-1 upregulation. INTRODUCTIONHuman cytomegalovirus (HCMV), a b-herpesvirus, is a ubiquitous human pathogen with a prevalence of infection in the adult population of 50-90 %. Although HCMV rarely causes severe disease on primary infection in immunocompetent people, it represents a highly pathogenic agent for immunosuppressed patients such as transplant recipients, as well as newborns, and establishes lifelong persistence in the infected host (Alford & Britt, 1990). So far, the exact site of latency is still uncertain; monocytes, haematopoietic precursor cells and endothelial cells have been discussed as potential reservoirs for latent HCMV (Fish et al., 1995;Sindre et al., 1996).Recent evidence suggests that HCMV not only causes acute infection in transplanted patients but may also be associated with extensive transplant vasculopathy, ultimately leading to chronic allograft rejection (Grattan et al., 1989;Skowronski et al., 1993;Koskinen et al., 1993;Lautenschlager et al., 1997a;Schnitzler et al., 1997;Borchers et al., 1999). One of the molecular mechanisms underlying the acceleration of vascular disease processes by HCMV infection may be enhancement of transplant immunogenicity. Several studies indicate that this involves the HCMV-induced upregulation of cellular adhesion molecules, which augments the adherance and infiltration of inflammatory cells that are capable of promoting vascular disease (Einsele et al., 1994;Lemstrom et al., 1995;Steinhoff et al., 1995;Koskinen et al., 1996;Yilmaz et al., 1996; Martelius et al., 1998;Waldman et al., 1998;Lautenschlager et al., 1999;The et al., 2001). For instance, cell culture experiments have demonstrated that...

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