The ORF74 or vGCR gene encoded by Kaposi's sarcoma-associated herpesvirus (KSHV; also called human herpesvirus 8) has properties of a ligand-independent membrane receptor signaling protein with angiogenic properties that is predicted to play a key role in the biology of the virus. We have examined the expression of vGCR mRNA and protein in primary effusion lymphoma (

Lytic-cycle replication of Kaposi's sarcoma-associated herpesvirus (KSHV) in PEL cells causes G 1 cell cycle arrest mediated by the virus-encoded replication-associated protein (RAP) (or K8 protein), which induces high-level expression of the cellular C/EBP␣ and p21 proteins. Here we have examined the mechanism of this induction at both the transcriptional and posttranslational levels. RAP proved to bind very efficiently to both C/EBP␣ and p21 and stabilized them by up to 10-fold from proteasome-mediated degradation in vitro. Cross-linking revealed that RAP itself forms stable dimers and tetramers in solution and forms higher-order complexes but not heterodimers with C/EBP␣. Cotransfection of RAP with C/EBP␣ cooperatively stimulated both the C/EBP␣ and p21 promoters in luciferase reporter gene assays. Only the basic/leucine zipper region of RAP was needed for interaction with and stabilization of C/EBP␣, but both the N-terminal and C-terminal domains were required for transcriptional augmentation. In vitro-translated RAP interfered with DNA binding by C/EBP␣ in electrophonetic mobility shift assay (EMSA) experiments but did not itself bind to the target C/EBP␣ sites or form supershifted bands. However, in endogenous chromatin immunoprecipitation (ChIP) assays with tetradecanoyl phorbol acetate-induced PEL cells, RAP proved to specifically associate with the C/EBP␣ promoter in vivo, but only in a C/EBP␣-dependent manner, implying an in vivo piggyback interaction with DNA-bound C/EBP␣. Expression of exogenous RAP (Ad-RAP) caused G 1 /S cell cycle arrest in human dermal microvascular endothelial cells and also induced both the C/EBP␣ and p21 proteins, which formed punctate nuclear patterns that colocalized with RAP in PML nuclear bodies. In the presence of RAP, C/EBP␣ was also efficiently recruited into viral DNA replication compartments in both infected and cotransfected cells. In support of a direct role for this interaction in viral DNA replication, three C/EBP␣ binding sites were identified by in vitro EMSA experiments within a 220-bp core segment of the duplicated KSHV Ori-Lyt region, and although RAP did not bind to Ori-Lyt DNA directly in vitro, both endogenous RAP and C/EBP␣ were found to be associated with the Ori-Lyt region by ChIP assays in lytically induced PEL cells. Finally, we found that the KSHV lytic cycle could not be triggered by either synchronizing KSHV latently infected PEL cells in G 1 phase or inducing p21 in a C/EBP␣-independent process.

BackgroundArtesunate, an artemisinin-derived monomer, was reported to inhibit Cytomegalovirus (CMV) replication. We aimed to compare the in-vitro anti-CMV activity of several artemisinin-derived monomers and newly synthesized artemisinin dimers.MethodsFour artemisinin monomers and two novel artemisinin-derived dimers were tested for anti-CMV activity in human fibroblasts infected with luciferase-tagged highly–passaged laboratory adapted strain (Towne), and a clinical CMV isolate. Compounds were evaluated for CMV inhibition and cytotoxicity.ResultsArtemisinin dimers effectively inhibited CMV replication in human foreskin fibroblasts and human embryonic lung fibroblasts (EC50 for dimer sulfone carbamate and dimer primary alcohol 0.06±0.00 µM and 0.15±0.02 µM respectively, in human foreskin fibroblasts) with no cytotxicity at concentrations required for complete CMV inhibition. All four artemisinin monomers (artemisinin, artesunate, artemether and artefanilide) shared a similar degree of CMV inhibition amongst themselves (in µM concentrations) which was significantly less than the inhibition achieved with artemisinin dimers (P<0.0001). Similar to monomers, inhibition of CMV with artemisinin dimers appeared early in the virus life cycle as reflected by decreased expression of the immediate early (IE1) protein.ConclusionsArtemisinin dimers are potent and non-cytotoxic inhibitors of CMV replication. These compounds should be studied as potential therapeutic agents for the treatment of CMV infection in humans.

The protein array provided a sensitive platform for global screening; identified new, frequently recognized viral antigens; and revealed a broader humoral response to EBV compared with KSHV in the same patients.