SHORT REPORT The CXXC Zn binding motifs of the human papillomavirus type 16 E7 oncoprotein are not required for its in vitro transforming activity in rodent cells Joris Braspenning 1 , Antonio Marchini 1 , Valentina Albarani 1 , Laura Levy 2 , Francesca Ciccolini 1 , Caterina Cremonesi 1 , Robert Ralston 3 , Lutz Gissmann 1 and Massimo Tommasino 1 1 Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany; 2 Chiron Corporation, Virology Department, Emeryville, California, USA; 3 GeneMedicine Inc. The Woodlands, Texas, USA The conserved region 3 (CR3) of the E7 protein of human papillomaviruses contains two CXXC motifs involved in zinc binding and in the homodimerization of the molecule. Studies have suggested that the intact CXXC motifs in the CR3 of HPV16 and HPV18 E7 are required for the in vitro transforming activity of these proteins. CR3 also contains a low anity pRb binding site and is involved in the disruption of the E2F/Rb1 complex. E7 is structurally and functionally related to Adenovirus E1A protein, which also has two CXXC motifs in CR3. However, the Ad E1A transforming activity appears to be independent of the presence of such domains. In fact, this viral protein exists in vivo as two dierent forms of 289 and 243 amino acids. The shorter Ad E1A form (Ad E1A243), where both CXXC motifs are deleted by internal splicing, retains its in vitro transforming activity. We have investigated if the HPV16 E7 CR3 can be functionally replaced by the Ad E1A243 CR3, which lacks both CXXC motifs. A chimeric protein (E7/E1A243) containing the CR1 and CR2 of HPV16 E7 fused to the CR3 of Ad E1A 243 was constructed. The E7/E1A243 while not able to homo- dimerize in the S. cerevisiae two-hybrid system retains several of the properties of the parental proteins, HPV16 E7 and Ad E1A. It associates with the `pocket' proteins, induces growth in soft agar of NIH3T3 cells and immortalizes rat embryo ®broblasts. These data suggest that the CXXC motifs in CR3 of E7 do not play a direct role in the transforming properties of this viral protein but probably are important for maintaining the correct protein con®guration. Keywords: Human papillomavirus type 16 E7 protein; E7-E1A243 chimeric protein; CXXC motifs; cellular transformation; E7 homodimerization In the last two decades the association of certain types of human papillomavirus (HPV) with cervical cancer, e.g. HPV type 16 and 18 has been clearly demonstrated (zur Hausen, 1991). Several lines of evidence suggest that the products of two early genes, E6 and E7, play a key role in the induction of malignant transformation of the host cells. For instance, HPV DNA has been found to be integrated into the genome of the malignant cells in most of the cases (Choo et al., 1987; Cullen et al., 1991). This viral DNA integration results in the disruption of various viral genes with consistent preservation of only the early E6 and E7 genes, which are actively transcribed (Schwarz et al., 1985). The E7 proteins of the `high risk' HPV16 and 18 genotypes are able to induce focus formation and growth in soft agar in a variety of established rodent ®broblast lines, e.g. NIH3T3 and Y31 cells (for review see Mansur and Androphy, 1993). They also cooperate with the activated ras gene product to transform primary rodent cells and with E6 to immortalize primary human keratinocytes, the natural host of the virus (Mansur and Androphy, 1993). E7 is structurally and functionally related to other viral oncoproteins, such as the Adenovirus E1A (Ad E1A) and the SV40 large T antigen (Tag) Phelps et al., 1988; Vousden and Jat, 1989). These viral oncoproteins exert their transforming activity by interacting with and altering the function of a number of cellular proteins. The Ad E1A and HPV E7 associate with the so called `pocket' proteins, pRb, p107 and p130 (Whyte et al., 1988, 1989; Dyson et al., 1989; Davies et al., 1993; Hu et al., 1995), which are involved in controlling the activity of several transcription factors, including proteins of the E2F family (Kouzarides, 1995). The pRb/E7 interaction results in the inactivation of the regulatory function of the `pocket' protein with consequent constitutive activa- tion of the transcription factors (reviewed in Tomma- sino and Crawford, 1995). On the basis of its homology with Ad E1A, HPV16 E7 protein can be divided into three domains: conserved region 1 (CR1), conserved region 2 (CR2) and conserved region 3 (CR3). The disruption of the E2F/pRb by Ad E1A protein is mediated by two independent pRb binding domains, the low and high anity binding sites, which are located in the CR1 and CR2 of Ad E1A, respectively (Fattaey et al., 1993; Ikeda and Nevins, 1993). In HPV16 E7, while the high anity `pocket' protein binding site is located in CR2 as in Ad E1A, the low anity site is present in the CR3 (Patrick et al., 1994). In agreement with this, independent studies have shown that the HPV16 E7 CR3, together with CR1 and CR2, is required for the disruption of E2F/pRb complex (Huang et al., 1993; Wu et al., 1993). Another feature of the HPV16 E7 CR3 is the presence of two cys-x-x-cys motifs (CXXC) Correspondence: M Tommasino The ®rst two authors have equally contributed to this work Received 30 July 1997; revised 1 October 1997; accepted 3 October 1997 Oncogene (1998) 16, 1085 ± 1089  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00