NATURE M EDICINE • VO LUM E 6 • N UM BER 1 0 • OCTOBER 2000 1121 ARTICLES Kaposi sarcoma-associated herpesvirus (KSHV, or human her- pesvirus 8) is associated with three tumor types: Kaposi sarcoma (KS), a plasmablastic variant of multicentric Castleman disease and primary effusion lymphoma 1–4 . In KS, KSHV is latently pre- sent in most tumor cells (the spindle cells) 5–8 , with only a few cells supporting lytic viral replication 9,10 . In multicentric Castleman disease, KSHV is latently present in plasmablastic B cells 4,8 . Primary effusion lymphoma is a B-cell neoplasm in which KSHV is latently present in all tumor cells 2,8,10 . Primary ef- fusion lymphoma cell lines that express latent KSHV proteins have been established 11,12 . These lymphoma cells are transformed and grow in soft agar and in athymic nude mice 12 . In vitro, KSHV can infect endothelial cells 13 , confer a growth advantage to pri- mary bone marrow endothelial cultures 14 and, in association with the E6 and E7 proteins of human papillomavirus type 16, induce a transformed phenotype in dermal microvascular en- dothelial cells 15 . These data indicate that KSHV encodes latent proteins that may function like the nuclear proteins of other DNA tumor viruses: Simian virus (SV) 40, adenovirus and human papilloma viruses encode latent nuclear proteins that target the retinoblas- toma protein (RB1)–E2F and p53 transcriptional regulatory path- ways 16–19 . Open reading frame 73 encodes the main immunogenic latent nuclear antigen LNA-1 (refs. 20–22). Experiments with monoclonal antibodies against LNA-1 have confirmed that it is expressed in the tumor cells of all three neo- plasia associated with KSHV infection 8,10,23 . LNA-1 binds histone H1, which tethers KSHV DNA to chromatin during mitosis, en- suring the delivery of viral progeny to daughter cells 24,25 . Several features of LNA-1 are characteristic of cellular and viral transcrip- tion factors, including an extended acidic domain separated into two regions by a glutamine-rich sequence. The first region con- sists almost exclusively of aspartic and glutamic acid residue re- peats, whereas the second glutamic acid-rich region has a putative leucine zipper motif 26 . LNA-1 transactivates E2F-regulated promoters Given the nuclear location of LNA-1 and its predicted amino- acid sequence, we first tested whether LNA-1 acts as a transcrip- tion co-factor. We investigated this by testing the ability of LNA-1 to regulate E2F-dependent transcription and tested the ef- fect of LNA-1 on an artificial promoter that contains three E2F DNA-binding sites (3 × E2F) derived from the adenovirus E2A gene promoter region 27 . We used cells from the DG75 line, de- rived from Burkitt lymphoma and lacking the Epstein-Barr virus, and plasmids containing a hemagglutinin (HA) tag and either full-length LNA-1 (pSG5-HA-LNA-1) or amino acids 1–735 of LNA-1 (pSG5-HA-LNA-1[aa1-735]). We transiently transfected the cells with a constant amount (3 μg) of the luciferase reporter plasmid pGL2-3 × E2F and increasing concentrations (1, 5 or 10 μg) of the LNA-1 effector plasmids. LNA-1 transactivated the luciferase reporter gene in a dose-dependent manner, whereas the truncated mutant did not (Fig. 1 a). In a parallel transient transfection experiment, the same amounts of the control effec- tor plasmid encoding adenovirus 2 early region 1 A (E1A; pCE- EIA), also transactivated the reporter plasmid pGL2-3xE2F (Fig. 1 a). Transfection of DG75 cells with the same amount of a mutant reporter plasmid, pGL2-3xE2F(mut), which lacks E2F binding, produced no transactivation with increasing concentra- tions (1, 5 and 10 μg) of pSG5-HA-LNA-1 (Fig. 1 b). LNA-1, LNA- The latent nuclear antigen of Kaposi sarcoma-associated herpesvirus targets the retinoblastoma–E2F pathway and with the oncogene Hras transforms primary rat cells STO YAN A. RADKOV, PAUL KELLAM & C HRIS BO SH O FF The CRC Viral Oncology Group, The W olfson Institute for Biomedical Research, Cruciform Building and The W ohl Virion Centre, Departments of Oncology and Molecular Pathology, University College London, London W C1E 6AE, UK Correspondence should be addressed to C.B.; email: c.boshoff@ucl.ac.uk Kaposi sarcoma-associated herpesvirus (KSHV) is involved in the etiopathogenesis of Kaposi sar- coma and certain lymphoproliferative disorders. Open reading frame (ORF) 73 encodes the main immunogenic latent nuclear antigen (LNA-1) of KSHV. LNA-1 maintains the KSHV episome and tethers the viral genome to chromatin during mitosis. In addition, LNA-1 interacts with p53 and represses its transcriptional activity. Here we show that LNA-1 also interacts with the retinoblas- toma protein. LNA-1 transactivated an artificial promoter carrying the cell cycle transcription factor E2F DNA-binding sequences and also upregulated the cyclin E ( CCN E1 ) promoter, but not the B-myb ( M YBL2 ) promoter. LNA-1 overcame the flat-cell phenotype induced by retinoblas- toma protein in Saos2 cells. In cooperation with the cellular oncogene Harvey rat sarcoma viral oncogene homolog ( Hras ), LNA-1 transformed primary rat embryo fibroblasts and rendered them tumorigenic. These findings indicate that LNA-1 acts as a transcription co-factor and may contribute to KSHV-induced oncogenesis by targeting the retinoblastoma protein–E2F transcrip- tional regulatory pathway. © 2000 Nature America Inc. • http://medicine.nature.com © 2000 Nature America Inc. • http://medicine.nature.com