The HPV E proteins are acidic oncoproteins
The HPV E7 proteins are acidic oncoproteins of approximately 98–103 Triflusal that contain 3 domains: the conserved region (CR) 1, CR2, and the carboxyl terminal (C-terminal) domain. The CR1 domain is necessary for cellular transformation and RB degradation in mucosal high risk HPVs. The CR2 domain contains a conserved pRB family binding site (LxCxE domain) and a consensus casein kinase II (CKII) phosphorylation site (McLaughlin-Drubin and Munger, 2009). The C-terminal domain contains a zinc-binding domain that is composed of two Cys-X-X-Cys motifs, separated by 29–30 amino acids, and is involved in dimerization of E7 proteins and association with host complexes (Jones and Munger, 1996, McLaughlin-Drubin and Munger, 2009, Zwerschke and Jansen-Durr, 2000).
We previously discovered that the E7 oncoproteins of mucosal high risk HPV16 and low risk HPV11 enter the nucleus via a Ran-dependent pathway, independent of karyopherins/importins, and this pathway is mediated by their zinc-binding domain via hydrophobic interactions with the FG nucleoporins Nup62 and Nup153 (Angeline et al., 2003, Knapp et al., 2009, McKee et al., 2013, Eberhard et al., 2013, Piccioli et al., 2010). We also determined that HPV16 E7 and HPV11 E7 contain a leucine-rich NES within their zinc-binding domain that mediates their nuclear export via a CRM1 pathway (Knapp et al., 2009, McKee et al., 2013). CRM1 (chromosome region maintenance 1), a major nuclear export receptor, binds to hydrophobic leucine-rich NES containing cargoes in a RanGTP dependent manner. The canonical NES is defined as a L-X2–3-L-X2–3-L-X-L motif, where the leucine residues can be isoleucine, valine, phenylalanine or methionine (Hutten and Kehlenbach, 2007). The RanGTP/CRM1/NES cargo complex translocates through the nuclear pore complex and the cargo is released into the cytoplasm by GTP hydrolysis of RanGTP mediated by RanGAP (Hutten and Kehlenbach, 2007).
Although the activities, interactions and nucleocytoplasmic traffic of mucosal alpha genus HPV E7 oncoproteins have been well documented (McLaughlin-Drubin and Munger, 2009), much less is known about the cutaneous beta genus HPV E7 proteins. The cutaneous HPV8 E7 oncoprotein has a weaker interaction with pRB than HPV16 E7 oncoprotein and can transform rodent cells only in collaboration with an activated H-ras gene (Yamashita et al., 1993). Expression of HPV8 E7 protein in primary human keratinocytes causes polyploidy that is associated with decreased levels of pRb and p21 (Akgul et al., 2007). It also causes invasion of human keratinocytes into the dermis of organotypic skin cultures that is accompanied by overexpression of extracellular metalloproteinases (Akgul et al., 2005, Akgul et al., 2006b).
Flag-HPV8 E7 expressed in transfected human keratinocytes and EGFP-HPV8 E7 expressed in transfected HeLa cells are both predominantly localized in the nucleus with some low levels in the cytoplasm (Sperling et al., 2012, Onder and Moroianu, 2014). We recently determined that cutaneous beta genus HPV8 E7 oncoprotein contains a cNLS within its zinc-binding domain that mediates its nuclear import and localization independent of karyopherins/importins. Furthermore, we discovered that a mostly hydrophobic patch 65LRLFV69 within the zinc-binding domain mediates nuclear import and localization of HPV8 E7 via direct hydrophobic interactions with the FG nucleoporins Nup62 and Nup153 (Onder and Moroianu, 2014).
In this study we investigated the nuclear export of cutaneous HPV8 E7 oncoprotein. We discovered and characterized a leucine-rich NES, 76IRTFQELLF84, within the zinc-binding domain that mediates nuclear export of HPV8 E7 via a CRM1 pathway. We determined that HPV8 E7 interacts with CRM1 and that the hydrophobic amino acid residues I76, F79 and L82 of the NES are essential for this interaction and consequently for nuclear export of HPV8 E7 oncoprotein.
Results and discussion