Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Thu, 06 Dec 2018 23:41:44 Journal of General Virology (2000), 81, 689–694. Printed in Great Britain .......................................................................................................................................................................................................... SHORT COMMUNICATION Binding of bovine papillomavirus type 4 E8 to ductin (16K proteolipid), down-regulation of gap junction intercellular communication and full cell transformation are independent events G. Hossein Ashrafi, 1 John D. Pitts, 2 AnnaMaria Faccini, 2 Pauline McLean, 2 Vincent O’Brien, 1 Malcolm E. Finbow 2 † and M. Saveria Campo 1 Papillomavirus Group 1 and Cell Communication Group 2 , The Beatson Institute for Cancer Research, Glasgow, UK The E8 open reading frame of bovine papilloma- virus type 4 encodes a small hydrophobic poly- peptide that contributes to primary cell trans- formation by conferring to cells the ability to form foci and to grow in low serum and in suspension. Wild-type E8 binds in vitro to ductin, a component of gap junctions, and this binding is accompanied by a loss of gap junction intercellular communication in transformed bovine fibroblasts. However, through the analysis of a panel of E8 mutants, we show here that binding of E8 to ductin is not sufficient for down-regulation of gap junction communication and that there is no absolute correlation between down-regulation of gap junction communication and the transformed phenotype. The E8 protein of bovine papillomavirus type 4 (BPV-4) is a member of the E5 family of viral transforming proteins. These are short hydrophobic peptides with a C terminus hydrophilic domain. E8 is 42 amino acids long ; its N-terminal 30 amino acids are hydrophobic and likely to form a transmembrane domain, while the 12 C-terminal amino acids make up the hydrophilic ‘ tail ’. E8 is expressed in the bottom layers of the mucous epithelium of early stage papillomas (Anderson et al., 1997) and is localized in the cell membranes (Pennie et al., 1993 ; Faccini et al., 1996). Author for correspondence : Saveria Campo. Present address : Department of Veterinary Pathology, Glasgow University, Garscube Estate, Glasgow G61 1QH, UK. Fax 44 141 330 5602. e-mail s.campoudcf.gla.ac.uk † Present address : Department of Biological Sciences, Glasgow Caledonian University, Cowcaddens, Glasgow G4 0BA, UK. E8 contributes to the transformed phenotype of primary cells by inducing focus formation, anchorage-independent growth and growth in low serum (Pennie et al., 1993 ; O’Brien et al., 1999). The last two phenotypic changes are accompanied by an E8-mediated increase in the amount of cyclin A and activation of the cyclin A–cdk2 complex (O’Brien & Campo, 1998 ; O’Brien et al., 1999). The presence of E8 also leads to a marked loss of cell–cell communication through gap junctions (GJIC ; Faccini et al., 1996). The human papillomavirus (HPV) type 16 and BPV-1 E5 proteins also down-regulate GJIC (Oelze et al., 1995; see below). Gap junctions are cell-surface structures that allow cell–cell communication via diffusion of small molecules (Simon & Goodenough, 1998). They contribute to the maintenance of homeostasis between cells in a tissue or in a proliferative unit (Kam & Hodgins, 1992). Gap junctions are often not functional in transformed cells and the isolation of transformed cells from their normal neighbours is thought to contribute to the neoplastic process (Budunova et al., 1995). One of the components of gap junctions is ductin (Finbow & Pitts, 1993). BPV-4 E8 and the E5 proteins of BPV-1, HPV-6 and HPV-16 bind to ductin (Goldstein et al., 1991; Conrad et al., 1993; Faccini et al., 1996). This interaction is deemed responsible for the observed down-regulation of gap junctions (Oelze et al., 1995 ; Faccini et al., 1996). Ductin is also a component of vacuolar H + -ATPase (Finbow et al., 1995), a universal eukaryotic proton pump responsible for the acidification of cytoplasmic organelles (Finbow & Harrison, 1997). The binding between ductin and these viral oncoproteins might also be responsible for the impairment of acidification of endosomes (Straight et al., 1995) and sustained activation of growth factor receptors (Banks & Matlashewski, 1996). We have reported that mutant forms of E8 can still induce cell growth in suspension but not in low serum (O’Brien et al., 1999), showing that these two parameters of the transformed phenotype can be dissociated. We have now examined the role of GJIC in the transformation process by E8 and find that 0001-6818  2000 SGM GIJ