Srn-Kaposi's sarcoma (KS) associated herpesvirus (KSHV or HHV8) has been strongly implicated in the development of Kaposi's sarcoma and primary effusion lymphomas1'2• This herpesvirus was ini-tially described in Kaposi's sarcoma associated with AIDS patients1, but has since been found in all epidemiological forms of Kaposi's sarcoma3-5. KSHV is a gammaherpesvirus, similar to other oncogenic herpesviruses such as the New World monkey virus, herpesvirus saimiri, and Epstein-Barr virus6, Genomic sequencing of KSHV has thus far failed to identify sequence similarity to genes of Epstein-Barr virus or herpesvirus saimiri which are known to have important oncogenic functions (P. SM, unpublished data). However, we report here that KSHV contains an open reading frame (ORF 72) with sequence similarity to cellular o/clins, in particular the D-type cyclins (a in the figure).(Herpesvirus saimiri also has an open reading frame with similarity to cyclins8• 9.) Various lines of evidence suggest that deregulated expression of cellular D-type cyclins is linked to the development of tumours in humans10• Cyclin D proteins are regulatory subunits which activate cellular kinases (CDKs) to phosphorylate checkpoint molecules such as the retinoblastoma-tumour suppressor protein (RB) 11• The similarity of KSHV ORF 72 to cellular D-type cyclins suggests that its encoded product, KSHV-cyclin, could also stimulate such kinases resulting in their unlicensed activation and, consequently, cause deregulation of cellular proliferation inherent to KSHV associated diseases. To determine whether KSHV-cyclin could activate kinases and perturb control of cell growth in a manner analogous to aberrant expression of D-type cyclins, we engineered an epitope-tagged (c-myc epitope) version of KSHV ORF 72 driven by the cytomegalovirus immediate early promoter. On transfection into COS-1 cells, this construct gives rise to expression of a polypeptide of relative molecular mass 28,000 that, like the D-type cyclins, is localized to the cell nucleus. The KSHV-cyclin protein is associated with kinase activity capable of phosphorylating RB in vitro at authentic sites, including those previously shown to be a hallmark of RB inactivation12 (b in the figure). This supports the hypothesis a, Amino-acid sequence alignment of the putative'cyclin box· of KSHV-cyclin (residues 49-82) with HVS cyclin (residues 50-83) and human a

•• av-crc:: H~ RK Lrr: lG T w MF sv~ ol!] r N~ E p N~ VA~ AL NL L DR L Bvs-c1c: D RTI l! Jltwhhllceafeldk 6 VFPL VSILDRY cyclh DJ Y MR RM VAT WM LEV CE~ Q KC EE EV f'PL AM NY L DR F cyclin D2 (residues 54-87). Identical residues to KSHV-cyclin are boxed. b, Twodimensional tryptic peptide pattern of full-length RB phosphorylated by KSHV-cyclin immunocomplex in vitro (v-cyc) and of natural RB immunopurified with anti-RB antiserum from 32P labelled human cells (in vivo) demonstrating that authentic sites are phosphorylated by the KSHV-cyclin associated kinase12. c, SAOS-2 senescence assay. Photomicrograph C of transfected cell monolayers after puromycin selection for 12 days are shown. Monolayers of senescent cells with enlarged cell bodies are seen when cells are transfected with wildtype RB (middle panel) but not in cells transfected with RB 76t mutant b v-cyc