Oncogenic activation of a human cyclin A2 targeted to the endoplasmic reticulum upon Hepatitis B virus genome insertion Carmen Berasain 1 , Deva Patil 1 , Eve Perara 2 , Shih-Min Huang 1 , He´le`ne Mouly 1 and Christian Bre´ chot 1 1 INSERM U370 and Liver Unit, Necker Institute and Hospital, 75015 Paris, France; 2 Department of Biology, San Francisco State University, San Francisco, California, USA Cyclins are major cell cycle regulators which role in malignant transformation remains controversial. In this report we describe a new mechanism of cyclin oncogenic activation. We demonstrate that an altered form of cyclin A2 (S2A) which N-terminal part is replaced by the hepatitis B virus envelope protein transforms normal rat kidney cells and cooperates with ras to transform rat embryo fibroblasts. In contrast, neither the viral moiety, nor a full length or N-terminally deleted cyclin A2 show these oncogenic properties. S2A oncogenicity arises from its binding to cyclin dependent kinases, since mutation in the MRAIL sequence abolishes transformation and correlates with an abnormal cellular localization in the endoplasmic reticulum membrane. Together, these results implicate modification in the cellular distribution of a cell cycle regulator as a mechanism of virally-induced transformation. Keywords: cyclin A; hepatitis B virus; hepatocellular carcinoma; oncogenic transformation Introduction Normal progression through the mammalian cell cycle occurs by the transient activation of protein kinase complexes, each consisting of a cyclin regulatory subunit and a cyclin dependent kinase (CDK) catalytic component (Pines, 1993; Sherr, 1994; Wuaring and Nurse, 1996; Nasmyth, 1996). The loss of cell cycle control is associated with oncogenesis and it has been suggested that cell cycle regulatory proteins, such as cyclins, CDKs, and their inhibitors (CKIs), may play a direct role in transforma- tion and act as oncogenes (Hunter and Pines, 1994; Sherr, 1996). Support for this hypothesis comes from the observed up-regulation of cyclins and CDKs gene expression in tumors and tumor cell lines and from in vivo and in vitro data which suggest that cyclins involved in G1 and G1/S transitions (cyclins D and E respectively) do, under certain circumstances, have an oncogenic potential (Wang et al., 1994; Hinds et al., 1994; Lovec et al., 1994; Mumberg et al., 1996; Bortner and Rosenberg, 1997). This is in contrast with the data obtained to date for cyclin A2, which acts downstream of cyclins D and E in two distinct cell cycle transitions, namely the initiation and progression of S phase and the G2/M transition, in association with CDK2 and CDK1, respectively (Zindy et al., 1992; Pagano et al., 1992). The ectopic expression of cyclin A2 in vitro shortens G1 (Rosenberg et al., 1995; Resnitzky et al., 1994) and abrogates adhesion dependency for S phase entry (Guadagno et al., 1993). In vivo, overexpression of cyclin A2 in the mammary gland of transgenic mice results in the induction of nuclear abnormalities and increased apoptosis (Bortner and Rosenberg, 1995). In addition, cyclin A2 is a target of viral oncogenes such as E1A, E7, or SV40 T antigen, that activate the cyclin A2 promoter (Buchou et al., 1993; Zerfass et al., 1996) and interact with cyclin A2 in multiproteic complexes also containing p107 and E2F (Nevins, 1994). However, despite these reports, there is no direct evidence for the transforming capacity of cyclin A2 and in general, although there is a strong correlation between an increased activity of cyclin/CDK complexes and neoplastic transformation, the evidence that this activity plays a causative role in oncogenesis is still circumstantial. We have previously reported the existence of an altered form of cyclin A2 in an hepatitis B virus (HBV)-associated hepatocarcinoma (Wang et al., 1990, 1992). HBV is an important etiological factor of liver cancer and there are several observations which point to a direct eect of the viral genome in transformation. The integration of the HBV genome is a frequent event in human hepatocarcinoma and induces chromosomal instability, translocations and deletions at the site of insertion (Rogler et al., 1992; Marchio et al., 1997). This integration also allows persistence of rearranged and truncated viral genomes that code for transactivators (the X protein of HBV (HBx) and truncated forms of the middle surface antigen) (Henkler and Koshi, 1996; Caselmann, 1996; Hildt et al., 1996). In isolated tumors, insertion occurs in genes coding for proteins with an essential role in cell proliferation or dierentiation, namely the retinoid acid receptor (Dejean et al., 1986; de The´ et al., 1987), cyclin A2 (Wang et al., 1990, 1992) and mevalonate kinase (Grae¨f et al., 1994, 1995) or in genes coding for the epidermal growth factor receptor (Zhang et al., 1992) and a carboxypeptidase (Pineau et al., 1996) the impact of which remains to be clarified. This contrasts strikingly with the Woodchuck animal model, where insertion of the Woodchuck hepatitis virus (WHV) into the N-myc2 and c-myc oncogenes in liver cancers is a frequent event of which the transforming eect has been well established (Buendia, 1992). Insertion of the HBV DNA into the cyclin A2 gene generates a chimaeric RNA initiated at the viral major Correspondence: C Bre´chot, INSERM U370, 156, rue de Vaugirard, 75015 Paris, France Received 18 November 1997; revised 2 February 1998; accepted 5 February 1998 Oncogene (1998) 16, 1277 – 1288  1998 Stockton Press All rights reserved 0950 – 9232/98 $12.00