Vaccinia virus regulates expression of p 21 WAF 1 / Cip 1 in A 431 cells

Vaccinia virus (VACV) is one of the largest animal double-stranded DNA viruses and replicates in the cytoplasm of host cells. It is the prototypic member of the Poxviridae family and its genome has the capacity to encode more than 250 gene products (Moss 2007). About half of the VACV genes encode immediate-early or early genes associated with the establishment of infection, regulatory proteins involved in transcription or DNA replication and proteins implicated in immune evasion or the spread of infection (Seet et al. 2003, Moss 2007). During viral replication, a phenomenon of localized cellular proliferation (hyperplasia) is observed (Burnet 1936) and is associated with the release of a soluble growth factor by the infected cells (Twardzik et al. 1985, Buller et al. 1988). This effect is attributed to the VACV growth factor (VGF), which shares amino acid sequence homology and functional properties with the cellular growth factors (EGF) and transforming growth factor beta (TGF-β) (Brown et al. 1985, Twardzik et al. 1985). VGF is highly conserved among different members of the Poxviridae family and it is sometimes associated with their oncogenicity (Tzahar et al. 1998). The mitogenic activity of VGF has been considered beneficial for viral replication because the VACV-VGF-deficient virus (VACV-VGF-) replicates better in proliferating cells than in quiescent ones (Buller et al. 1988), which is in line with the requirement of mitogen-activated protein kinase (MAPK)/ extracellular-signal regulated kinase (ERK kinase) (MEK)/ERK for maximal viral replication (Andrade et al. 2004). Mitogenic signals triggered in A431 cells by VGF are mediated by the EGFR, which is tyrosinephosphorylated (Y1173) (King et al. 1986, Tzahar et al. 1998) and activates phospholipase C-γ1 (Kim et al. 1995). Upon tyrosine-phosphorylation of phospholipase C-γ1, it promotes phosphoinositide breakdown to generate diacylglycerol and inositol triphosphate. Exposure of A431 cells to EGF leads to phosphorylation of EGF receptor (EGFR) at Y1173 (Downward et al. 1984); coincidentally, the same tyrosine residue is activated upon VACV infection or exposure to VGF (King et al. 1986). Downstream targets of EGF signalling in A431 cells include protein kinase C (PKC), which is activated on threonine 654 (T654) as a consequence of phospholipid turnover (Hunter et al. 1984). VGF is also able to phosphorylate PKC on T654 (King et al. 1986). Thus, both EGF and VGF seem to share common signalling molecules to deliver intracellular signals. Although EGF acts as a mitogen for a number of normal or transformed cell lines, it appears to arrest cell growth under certain circumstances, as is the case for A431 cells (Gill & Lazar 1981, Barnes 1982). These cells express a high number of EGFRs, which, upon EGF stimulation, undergo receptor hyper-phosphorylation followed by cell growth arrest (Gill & Lazar 1981). The mechanism associated with cell growth arrest appears to require the cyclindependent kinase (CDK) inhibitor (CKI) p21�AF1/cyclin-dependent kinase (CDK)-interacting protein (Cip1), which is regulated by both EGF and interferon-У (Chin et al. 1996, Zhou et al. 2002) and controls the transition from the G0/G1 to the S-phase during the cell cycle (el-Deiry et al. 1993, Sherr & Roberts 1999). Vaccinia virus regulates expression of p21WAF1/Cip1 in A431 cells