Characterization of the Nuclear Localization Signal of High Risk HPV16 E2 Protein

The papillomavirus E2 proteins have major functions during the viral life cycle in the regulation of viral DNA replication together with E1 and in transcriptional activation or repression. E2 can function either as an activator of viral transcription at low concentrations or as a repressor at high concentrations when it interferes with the binding of transcription factors such as TFIID and Sp1 (reviewed in Longworth and Laimins, 2004). In addition, E2 plays an essential role in viral genome segregation by tethering the viral DNA to the mitotic chromosomes and in the case of bovine papillomavirus (BPV) type 1 E2 protein this process is mediated by the bromodomain protein Brd4 (Baxter et al., 2005; McBride, McPhillips, and Oliveira, 2004; McPhillips, Ozato, and McBride, 2005; Oliveira, Colf, and McBride, 2006; You et al., 2004). The high risk types HPV16 and 18 E2 proteins have also proapoptotic activities (Demeret, Garcia-Carranca, and Thierry, 2003; Webster et al., 2000). For HPV18 E2 this proapoptotic activity depends on the induction of the extrinsic pathway via caspase 8 activation in the cytoplasm (Blachon et al., 2005; Demeret, Garcia-Carranca, and Thierry, 2003). Recently, the high risk HPV16/18 E2 proteins, but not the low risk types, have been shown to have the ability to induce genomic instability via a strong interaction with two activators of the Anaphase Promoting Complex (APC), Cdc20 and Cdh1 (Bellanger et al., 2005). The full length E2 protein contains two functional domains connected by a flexible hinge (H) region and forms dimers via the C terminal domain. The structures of both the N-terminal and C-terminal domains have been determined: the N domain consists of a bundle of α-helixes packed against a β-sheet framework (Harris and Botchan, 1999) whereas the C -DNA binding domain consists of a dimeric β-barrel structure with a pair of symmetrically disposed α-helices that bind and bend the DNA (Hegde and Androphy, 1998; Hegde et al., 1992; Kim et al., 2000). The N domain functions as a transactivation domain and can also recruit and bind the E1 helicase. The structure and functions of the N and C domains are relatively conserved among papillomaviruses, whereas the H region is variable both in sequence and length. Alternative promoter usage and mRNA splicing generate additional forms of the E2 polypeptides that contain intact H and C domains and either a truncated or substituted N domain (reviewed in Longworth and Laimins, 2004). Nucleocytoplasmic transport occurs through the nuclear pore complex, which is a large structure with an eight fold symmetry. Nuclear import of macromolecules is signal-mediated, energy-dependent and highly selective. The first identified nuclear localization signals (NLSs), referred to as classical NLSs, fall into two categories: monopartite NLSs consisting of a simple sequence of 3-5 basic amino acid residues, and bipartite NLSs consisting of a basic dipeptide upstream (10-12 amino acids) from a simple basic sequence. Other types of NLSs have been identified in hnRNP proteins, ribosomal proteins, U snRNPs, viral proteins while many others remain to be characterized. Most NLSs do not fit a well defined consensus, and for many, no consensus has been proposed (reviewed in Fried and Kutay, 2003; Moroianu, 1999). Two NLSs have been identified in the BPV-1 E2 protein: residues 107 to 115 (RKCFKKGAR) in the N domain and residues 339-352 (KCYRFRVKKNHRHR) in the C domain (Skiadopoulos and McBride, 1996). Partially homologous sequences are present in the E2 proteins of HPV types, although it is not clear if they actually function as NLSs. The E2 protein of low risk type HPV11 has been shown to contain a classic monopartite NLS in the hinge region, required for nuclear localization of HPV11 E2 and for its interaction with the nuclear matrix (Zou et al., 2000). This HPV11 E2’ NLS is not conserved in the high risk HPV16 and HPV18 E2 proteins. In this study we have identified and characterized the NLS of high risk HPV16 E2 protein using both in vivo and in vitro analysis of the intracellular localization of E2, E2 domains and different deletion and substitution variants.