Papillomavirus E6 oncoproteins

Abstract

Papillomaviruses induce benign and malignant epithelial tumors, and the viral E6 oncoprotein is essential for full transformation. E6 contributes to transformation by associating with cellular proteins, docking on specific acidic LXXLL peptide motifs found on the associated cellular proteins. This review examines insights from recent studies of human and animal E6 proteins that determine the three-dimensional structure of E6 when bound to acidic LXXLL peptides. The structure of E6 is related to recent advances in the purification and identification of E6 associated protein complexes. These E6 protein-complexes, together with other proteins that bind to E6, alter a broad array of biological outcomes including modulation of cell survival, cellular transcription, host cell differentiation, growth factor dependence, DNA damage responses, and cell cycle progression.

Introduction to Papillomaviruses

Papillomaviruses are small encapsidated double-stranded DNA viruses that induce benign squamous epithelial neoplasms called papillomas in vertebrates, and replicate within the papilloma. Although virus-induced papillomas are initially benign, some may evolve over time to produce malignancies, an observation first made over 75 years ago (Rous and Beard, 1935). Medically, a subset of human papillomaviruses (HPV) is notable for inducing human upper respiratory and ano-genital carcinomas; that subset of viruses is referred to as “high risk” HPV types, and the related HPV viruses that cause benign but not malignant mucosal lesions are called “low risk”.

This review is part of the Papillomavirus Episteme PAVE online source for papillomavirus information (http://pave.niaid.nih.gov/#home) and will be periodically updated with corrections and new information, which can be emailed to the authors at E6.PAVE.review@gmail.com. E6 has been the subject of other excellent reviews recently (Fan and Chen, 2004; Klingelhutz and Roman, 2012; Li et al., 2005; Liu and Baleja, 2008; Narisawa-Saito and Kiyono, 2007; Tungteakkhun and Duerksen-Hughes, 2008; Vande Pol, 2012; Wise-Draper and Wells, 2008). This review focuses upon the recently solved structure of E6 and its relation to the proteomic identification of E6 associated protein complexes, and biological effects of E6.

The papillomavirus life cycle

Papilloma formation and the infectious life cycle begins with an injury to the cutaneous or mucosal squamous epithelium, exposing the basement membrane and basal cell layer to virus (Fig. 1). The viral DNA initially replicates as a plasmid to low copy numbers in proliferative basal epithelial cells. When an infected basal cell divides, the progeny cells may move laterally on the basement membrane or up into the spinous cell layer where a subset of these infected spinous cells aberrantly re-enter the cell cycle to amplify viral DNAs from low to high copy number (Figure 1 and reviewed in (Chow et al., 2010)). As cells with amplified viral DNA move to higher layers within the stratified epithelium they express late gene capsid proteins to encapsidate viral DNA. Infectious virus is released from the surface of the papilloma within desquamated cells. This is unlike the uninfected adjacent squamous epithelium where cells divide in the basal layer, but commit to a terminal differentiation pathway upon moving into the spinous cell layer.

Figure 1.

The papillomaviruses life cycle. Papillomaviruses infect keratinocytes in the basal layer of the epithelium. Upon infection, the viral genome replicates to low-copy episomes in the basal layer; upon cell division, a daughter cell will move up from the basal layer and undergo Notch dependent differentiation. Differentiation induces the productive phase of the viral life cycle. The expression of viral oncoproteins induces re-entry of cells into S phase in the spinous layer, amplifying viral DNA copy number. A subset of cells that amplified the viral genome will then express the late-phase L1 and L2 capsid proteins in the last layers of the epithelium, encapsidate viral genomes, and are shed as cells containing virus.

Three papillomavirus early gene products, termed E5, E6, and E7, are proteins that stimulate cell proliferation, cell survival, and modulate keratinocyte differentiation; these are oncoproteins. In HPV associated cancers, continued E6 and E7 expression sustains the continued cancer phenotype. When the HPV early promoter in cervical cancer cell lines is repressed by re-expression of the viral E2 transcriptional repressor or by RNAi mediated repression of viral mRNA expression, cancer cell lines withdraw from the cell cycle and terminally differentiate (Butz et al., 2003; Chen et al., 1995b; Dowhanick et al., 1995; Francis et al., 2000; Goodwin and DiMaio, 2000; Hamada et al., 1996; Hwang et al., 1993; Storey et al., 1991; Tan and Ting, 1995; Thierry and Yaniv, 1987; von Knebel Doeberitz et al., 1994; Yoshinouchi et al., 2003)

In the prototype papillomaviruses (high risk HPV-16 and BPV-1), the three viral oncoproteins transform indicator cells like 3T3 cells, although in other papillomaviruses such as the low risk alpha-group mucosal papillomaviruses (such as HPV-6 or 11) such transforming activity can be unapparent. In BPV-1, the E5 oncoprotein activates receptor tyrosine kinases in the Golgi region of the cell in a ligand independent manner (reviewed in (Talbert-Slagle and DiMaio, 2009)). In contrast, in HPV’s an open reading frame encoding E5 is sometimes not evident, and when E5 is present, it has typically shown poor activity in classic transformation assays (Venuti et al., 2011). The E7 oncoproteins from diverse papillomaviruses have more consistent activities, including associations with cullin ubiquitin ligases and UBR4 (Demasi et al., 2005; Huh et al., 2007; Huh et al., 2005; White et al., 2012b), a large ubiquitin ligase that participates in the N-end rule protein degradation pathway (Besche et al., 2009). Most E7 proteins have an LXCXE binding motif that associates with members of the retinoblastoma family of tumor suppressors, resulting in ubiquitin mediated targeted degradation of the associated RB family members (reviewed in (McLaughlin-Drubin and Munger, 2009)). However, not all papillomavirus E7 oncoproteins associate with RB proteins through LXCXE associations. For example, BPV-1 E7 does not contain a LXCXE motif and, rarely, some papillomaviruses have no E7 gene at all (isolates from domestic pigs, SsPV, and polar bear UmPV, and porpoises (Stevens et al., 2008a; Stevens et al., 2008b; Van Bressem et al., 2007)).

The evolution of E6

Phylogenetic analysis of papillomavirus genomes compared to their host species has shown that each virus coevolved with its host (Bernard, 1994; Chan et al., 1992; Tachezy et al., 2002). Papillomaviruses are classified on the basis of the most conserved L1 open reading frame, but diversity is enhanced in the E6-E7 oncogene region, (Garcia-Vallve et al., 2005). Several papillomaviruses have no E6 gene at all (bovine papillomavirus types 3, 4, 6, and HPV 101 and 103 (Chen et al., 2007; de Villiers et al., 2004; Tachezy et al., 2002; Terai et al., 2002)). Two avian papillomavirus contain an E6 gene with only one zinc-binding region instead of two (Van Doorslaer et al., 2009). This suggests that a progenitor papillomavirus genome with replication and capsid proteins somehow acquired an E7 oncoprotein with a single zinc-binding region. The zinc-binding region of E7 then may have duplicated and subsequently diverged, giving rise to a single zinc finger E6 protein similar to that found in avian species today. A possible additional early duplication of that E6 domain in reptiles (as seen within turtles) resulting in two zinc fingers could have given rise to the E6 protein most commonly observed today (recently reviewed in (Garcia-Vallve et al., 2005; Shah et al., 2010)). The E6 protein sequence and zinc domain fold are distinct and do not resemble described cellular proteins (Nomine et al., 2003).

The high-risk E6 and E7 proteins are typically expressed from a common early promoter. As mentioned above, HrE7 proteins interact with UBR4 and cullin family ubiquitin ligases, and contain an LXCXE peptide-binding motif that binds to members of the retinoblastoma family of proteins that regulate E2F family transcription factors (reviewed in (McLaughlin-Drubin and Munger, 2009)). While hrE7 are themselves oncogenic, E7 from low-risk viruses are weakly oncogenic directly, and only have co-operative activity when co-expressed with additional oncogenes from the high-risk viruses (Halbert et al., 1992). This difference between high and low risk E7 proteins has been explained by the observation that the hrE7 LXCXE motif differs from the low risk LXCXE motif; while both hrE7 and low risk E7 bind to and then target the degradation of the p130 RBL2 protein that regulates G0 to G1 transition in the cell cycle, only the hrE7 proteins also bind to and target the degradation of p105 RB that controls G1 to S transition (Zhang et al., 2006). Further, low risk E7 proteins can be rendered oncogenic if the LXCXE motif of the low risk is mutated to the high-risk sequence (Heck et al., 1992; Sang and Barbosa, 1992). The targeted degradation of p105 RB by oncogenic E7 proteins results in the stabilization of the p53 tumor suppressor protein and thus sensitizes E7 expressing cells to apoptosis (Eichten et al., 2002; Jones et al., 1997; Stoppler et al., 1998). Thus, for the hrE6 proteins the “purpose” of E6 may be to neutralize the untoward consequences of E7 transformation by blocking the function of p53 and inhibiting cell cycle arrest and apoptosis. Consistent with this notion, an examination of numerous hrE6 proteins showed that all targeted the degradation of p53 (Fu et al., 2010). However, the hrE7-RB + hrE6-p53 connection does not explain how most papillomaviruses induce the replication of viral DNA in the spinous layer of papillomas. For example, BPV-1 or the low risk mucosal HPV papillomavirus E6 and E7 proteins induce cell cycle re-entry in the spinous cell layer but do not target p105 RB or p53 for degradation. Similarly, the E7 oncoprotein of cotton tailed rabbit papillomavirus (CRPV) reduces RB expression levels in keratinocytes, but its E6 proteins do not target the degradation of p53, and p53 is still inducible by mitomycin C in the presence of E6 (Ganzenmueller et al., 2008). Thus for most papillomaviruses, how viral oncoproteins induce either the papilloma or the replication of virus within the papilloma is poorly understood.

Quantitative assays for E6 function

Early studies focused upon BE6 and hrE6 proteins because they caused quantitative focus formation or anchorage independent colony formation in immortalized cell lines, while low risk E6 proteins had no quantifiable phenotypes. The first physiologic function for any E6 protein was the transformation of mouse C127 cells in tissue culture by BPV-1 E6 (Schiller et al., 1984) followed shortly thereafter by the transformation of mouse 3T3 and rat-1 cells by high risk E6 and E7 (Bedell et al., 1987). These observations were soon followed by studies demonstrating immortalization of primary keratinocytes by high-risk E6 + E7 (Hawley-Nelson et al., 1989; Hudson et al., 1990; Ma et al., 1987; Munger et al., 1989; Sedman et al., 1991; Woodworth et al., 1989). While the E7 oncoprotein from high risk HPV’s immortalize keratinocytes at low frequency, the E6 oncoproteins alone do not, but the combination of hrE6 and hrE7 immortalizes keratinocytes at high frequency.

The first quantitative in vitro assay for an E6 protein was the association of hrE6 with p53 (Werness et al., 1990) and the targeted degradation of p53 by hrE6 in rabbit reticulocyte lysate (Scheffner et al., 1990). Both the transformation and p53 degradation assays provided quantitative results for studies of E6 mutants and associated proteins. Additional quantitative assays for transcriptional modulation, signal transduction and cell survival have provided opportunities for the study of other HPV and animal papillomavirus E6 proteins. The recent observation that cutaneous E6 proteins repress cellular Notch signaling has provided another system for quantitative analysis of cutaneous E6 biological activity (Brimer et al., 2012; Rozenblatt-Rosen et al., 2012; Tan et al., 2012).

Association of E6 with cellular proteins

We will see that E6 oncoproteins can interact with cellular targets on distinct surfaces of E6, but the primary interaction seen with mucosal and cutaneous HPV E6 and BE6 is to bind an alpha helical acidic LXXLL peptide expressed as part of a cellular target protein.

E6 binding to LXXLL peptides on target cellular proteins

Analysis of p53 degradation by 16E6 led to the identification of a cellular enzyme termed E6AP (E6 Associated Protein, the product of the UBE3A gene), a HECT domain ubiquitin ligase that associates with hrE6 and p53 (Huibregtse et al., 1993a). Mutagenesis of E6AP showed that E6 bound to a 20 amino acid peptide in E6AP that contained a LXXLL sequence. Subsequent work on BE6 identified the BE6 associated protein paxillin (Tong et al., 1998; Tong and Howley, 1997; Vande Pol et al., 1998) and identified LXXLL motifs in paxillin where BE6 bound (Vande Pol et al., 1998). Mutagenesis of the 20 amino acid E6AP peptide that bound 16E6 and mutagenesis of the paxillin peptide that bound BE6 more clearly defined the binding sequence as an acidic LXXLL peptide, shown in Fig. 2 along with additional peptides that interact with BE6 that will be discussed below (Bohl et al., 2000; Chen et al., 1998). The strongest conservation in the LXXLL peptides is observed for the hydrophobic positions L₄, L_7, and (F/L)₈; i.e., LXXLL. Four positions in the motif (3, 5, 6 and 10) show preferences for negative residues. We will generically refer to E6 binding peptides as acidic LXXLL motifs. In the crystal structure of BE6 discussed below, there are contacts between BE6 and the LXXLL peptide over a 10 amino acid peptide with the consensus sequence Φ₁X₂D₃L₄D₅(D/E)_]6L₇(F/L)₈X₉(D/E)₁₀. The recent observation that cutaneous HPV E6 proteins and BE6 also interact with acidic motifs of MAML1 and MAML3 extend the generality of the E6-LXXLL interaction, and the homology to the E6AP LXXLL is striking (Fig. 3), but it is unclear as of yet if the binding of E6 proteins to LXXLL motifs is universal to other animal papillomavirus E6 oncoproteins, especially those with divergent primary structures (such as porpoise E6 with only a single zinc binding domain or cotton tail rabbit papillomavirus with 4 zinc binding domains).

Figure 2.

Known LXXLL binding motifs in E6 associated proteins. Hydrophobic residues are in blue and acidic in red. The number refers to the first amino acid in the motif. The top grouping of LXXLL peptides interacts with BE6 and the bottom two sequences are LXXLL peptides that interact with hrE6. PXN, NP_002850.2; MAML1, NP_055572.1; MAML3, NP_061187.2; UBE3A, NP_570853.1; IRF3, NP_001184051.1

Figure 3.

Ribbon Diagram of 16E6 bound to the LXXLL motif from E6AP. In the left upper panel, the amino-terminal zinc-binding domain (16E6-N in green) is shown at the top and the helical LXXLL peptide (salmon color) is viewed on end from the amino-terminus through its axis. . The LXXLL peptide resides in a deep pocket. The interdomain connecting helix (yellow) is clearly seen to the right side of the right upper panel. The right lower panel is the same view as the right upper panel but with the LXXLL peptide removed from the 16E6 pocket. The left lower panel is rotated to show the dimerization domain that is located within the green 16E6-N domain; the dimerization domain is colored purple (shown only in the lower left image).

E6 docking to LXXLL peptides is essential for BE6 and 16E6 function. First, mutants of E6 that fail to bind LXXLL are functionally defective; BE6 mutants that fail to bind to LXXLL-containing target proteins fail to transform cells, and 16E6 mutants that fail to bind to the E6AP LXXLL docking site fail to target the degradation of p53 (Cooper et al., 2003; Das et al., 2000; Vande Pol et al., 1998; Zanier et al., 2013). Second, deletion of the LXXLL motif in E6 target proteins ablates E6 function; deletion of LXXLL in E6AP both prevents E6 association and E6 directed E6AP-mediated p53 degradation (Huibregtse et al., 1993b), while deletion of the BE6 binding motifs of paxillin prevents BE6 association and transformation (Wade et al., 2008). Third, blocking the LXXLL binding pocked of E6 with an inhibitor blocks E6 function: fusion of a LXXLL motif to the amino-terminus of BE6 binds to BE6 in cis and blocks cellular transformation by BE6, and upon mutation of the LXXLL motif, transformation is restored. This approach avoided potential artifacts due to mutation of BE6 itself (Bohl et al., 2000). Fourth, peptides or drugs that competitively block 16E6 interactions with LXXLL targets inhibit in vitro and in vivo p53 degradation by 16E6 (Baleja et al., 2006; Liu et al., 2004; Sterlinko Grm et al., 2004).

In vitro E6 LXXLL binding assays are difficult, possibly because bacterially expressed E6 preparations contain aggregated E6 proteins that have high non-specific binding. Even in vitro translated protein has high non-specific binding, prompting the addition of non-ionic detergents that (in our hands at least) degrades specific interactions between hrE6 and LXXLL peptides. LXXLL interactions with E6 are easily performed by yeast 2-hybrid assays (Cooper et al., 2003; Elston et al., 1998; Vande Pol et al., 1998). In contrast to hrE6, BE6 is not inhibited by non-ionic detergents, which has allowed for robust in vitro binding assays (Das et al., 2000). LXXLL binding assays with hrE6 proteins are possible, but require careful preparation and purification of hrE6 proteins (Nomine et al., 2001).

Why do divergent E6 proteins bind acidic LXXLL peptides?

The low risk Alpha genus HPV-11 E6 (11E6) also binds the LXXLL motif of E6AP (Brimer et al., 2007). Recent studies of BE6 (Delta genus), HPV-1 E6 (Mu genus) and HPV-8 E6 (Beta genus) reveal that all three of these E6 proteins bind to the same acidic LXXLL motif of the MAML1 co-activator (discussed below). Is there some common underlying biological reason for the interaction of E6 proteins with acidic LXXLL peptides? Is there some commonality to the acidic LXXLL of E6AP and the acidic LXXLL of paxillin or MAML1? LXXLL peptides are used as docking sites for the interaction of nuclear hormone receptor receptors with their co-activators and co-repressors (reviewed in (Savkur and Burris, 2004)). The LXXLL motifs that associate with nuclear hormone receptors are typically basic (Heery et al., 1997), while the E6 associated LXXLL motifs are acidic. Further, while nuclear hormone receptors interact with a 6 amino acid peptide, E6 proteins interact with an extended 10 amino acid sequence containing a central LXXLL motif as we shall see below. The conservation of acidic LXXLL BE6 binding motifs implies a possibly conserved biological significance that is currently unappreciated (See Table I for a summary of known E6-interacting proteins that contain LXXLL motifs).

Table I.

LXXLL based interactions with Alpha, Beta, or BPV E6s

Gene Name	Other Name(s)	Method	Alpha (low)	Alpha (high)	Beta	BPV	References
UBE3A	E6-AP	IP/AP-MS/GPL/Y2H	+	+			(Brimer et al., 2007; Huibregtse et al., 1991, 1993a; Thomas et al., 2013; White et al., 2012a)
MAML1	MAML1	IP/AP-MS			+	+	(Brimer et al., 2012; Rozenblatt-Rosen et al., 2012; Tan et al., 2012; White et al., 2012a)
PXN	Paxillin	GST/IP/AP-MS			+ (92)	+	(Tong and Howley, 1997; Vande Pol et al., 1998; White et al., 2012a)
AP1G1	AP1	GST/AP/MS				+	(Tong et al., 1998)
RCN2	E6-BP/ERC-55	GST/IP		+		+	(Chen et al., 1998; Chen et al., 1995a)
IRF3	IRF3	GST		+			(Ronco et al., 1998)
TSC2	tuberin	Y2H/GST		+			(Lu et al., 2004)

GST:GST pulldown; IP: Immunoprecipitation; Y2H: Yeast 2-Hybrid; GPL: Gaussian princeps luciferase assay; AP-MS: Affinity Purified-Mass spectrophotometry. “+” means that interaction has been noted in the literature. If no “+” the interaction has not been published. The number in parenthesis indicates the specific HPV type in which the interaction has been observed/demonsrated. If no number, the interaction has been observed/demonstrated in more than one type of the indicated group.

What are the biological consequences of E6 interaction with LXXLL motifs on cellular proteins?

When E6AP expression is reduced by RNAi in cervical cancer cell lines, E6 half-life is dramatically reduced (Tomaic et al., 2009b). Similarly, when 16E6 is expressed in E6AP null cells, 16E6 expression levels are augmented by either co-expression of E6AP or co-expression of just an LXXLL peptide that binds to 16E6 (Ansari et al., 2012). Thus 16E6 and 18E6 are unstable in vivo in the absence of binding to a suitable LXXLL peptide. It was further observed that 16E6 binding to a LXXLL peptide could also restructure 16E6 to interact with p53 in the absence of the entire E6AP protein (Ansari et al., 2012). Thus, LXXLL peptide interactions stabilize and restructure 16E6.

For cutaneous type E6 proteins that interact with MAML1, the transcriptional activation of MAML1 is repressed upon binding to the E6 protein. It is as yet unknown if these E6 proteins are restructured upon binding to LXXLL to then interact with additional cellular proteins as is observed with 16E6 first binding to LXXLL and then recruiting p53 (discussed below).

The structure of E6 proteins bound to LXXLL peptides

When expressed in bacteria and concentrated, E6 proteins become insoluble (Zanier et al., 2007). However, when an LXXLL peptide from paxillin is fused to the amino-terminus of BE6, the fused peptide binds to BE6 in cis, blocks transformation by BE6 (Bohl et al., 2000), and solubilizes BE6. 16E6 solubility requires both provision of the LXXLL peptide of E6AP, mutation of non-conserved cysteines, and mutation of a dimerization surface in the amino-terminus of 16E6 to obtain concentrated and soluble protein preparations (Zanier et al., 2012). These efforts recently resulted in the crystallization of both BE6 and 16E6 in complex with LXXLL peptides of paxillin and E6AP respectively (Figs 3–6 and (Zanier et al., 2013)).

Figure 6.

Divergent E6 proteins have conserved folds. 16E6 (green) and BE6 (red) structures are superimposed in the frontal view as shown in Figures 3 and 4. The image shows both the extra alpha helix at the amino-terminus of 16E6 compared to BE6 but also the close conservation of the protein fold. The paxillin and E6AP LXXLL peptides are colored with their respective E6 proteins. Alignments performed in PYMOL software.

BE6 and 16E6 contain two zinc-binding domains with a conserved fold that are connected to each other by a helical linker

Both the amino-terminal E6 zinc-binding domain and the carboxy-terminal zinc-binding domain have a conserved overall fold in the crystal to what was previously solved by solution NMR for the isolated 16E6 C-terminal motif (Nomine et al., 2006; Zanier et al., 2012). The two zinc domains, together with an alpha-helix tube that connects them, form a deep pocket into which the LXXLL peptide makes close contacts (Figs 3–5). The LXXLL peptides (MDDLDALLAD from paxillin and ELTLQELLGEE from E6AP) adopt an amphipathic alpha-helical conformation: the hydrophobic leucine side chains are oriented to one side and face into the base of the hydrophobic pocket, opposite from the negatively charged aspartic and glutamic acids of the LXXLL peptides, which face out of the pocket and make charge interactions with E6 and solvent. The alpha helix that connects the N-terminal to C-terminal zinc binding domains is anchored at each end of the helix to each of the zinc-binding motifs like a rigid connecting tube. This inter-domain connecting helix also forms part of the binding pocket for the LXXLL peptide. Specific contacts between LXXLL peptide and E6 are discussed in (Zanier et al., 2013).

Figure 5.

Space filling views of the 16E6 protein bound to the LXXLL peptide. Colors and orientation are as in Fig 3., and the right panel shows the same view but in space-filling form.

BE6 lacks sequence corresponding to the first 14 amino acids of 16E6 that is a conserved feature of both the Alpha and Beta genus proteins, and within the crystal structure of BE6, the first 10 amino acids of BE6 are not observed, indicating that this sequence had too much motion to be resolved in the crystal structure (Zanier et al., 2013). Thereafter however, the BE6 and 16E6 structural folds are remarkably similar and superimpose nicely despite the limited overall sequence conservation (24%) and evolutionary divergence (Fig. 6 and (Zanier et al., 2013)).

The contacts between the N-terminal and C-terminal zinc binding domains are not likely strong enough to hold the domains in the conformation observed in the LXXLL-bound E6 crystal, so E6 could adopt a quite different overall conformation in the absence of a bound LXXLL peptide. Another way to state this is that LXXLL-peptide interactions may cause E6 to adopt its final conformation. As noted above, there is evidence in vivo for this, in that 16E6 is unstable in cervical cancer cells when its preferred binding partner, E6AP, is not present. However, there is evidence that the hrE6 proteins have E6AP independent functions; while E6AP is required for the induction of cervical cancer in mice (Shai et al., 2010), 16E6 retains oncogenic activity in the skin of E6AP null mice, indicating an important non-E6AP function for 16E6 or other associations in skin that stabilize16E6 (Shai et al., 2007).

16E6 contains a dimerization region in the amino-terminal zinc-binding domain (amino acids 23-24 and 39-47, shown in purple in the left lower portion of Fig. 3). Lipari and co-workers found that the E6 amino-terminal zinc-binding domain could be expressed alone in soluble form and dimerized in vitro (Lipari et al., 2001). This domain has been delineated by mutagenesis and NMR spectroscopy and was shown to be required for the degradation of p53 by 16E6 (Zanier et al., 2012) (discussed below).

At the carboxy-terminus of mucosal hrE6 proteins there is a short peptide sequence for the interaction with a specific set of PDZ domain containing proteins that will be discussed further below. This peptide motif is unstructured in its unbound state (Nomine et al., 2006), but the peptide has been resolved in both a crystal structure and NMR structure when bound to MAGI1 (Charbonnier et al., 2011; Zhang et al., 2007).

E6* proteins

E6* is an Amino-terminal portion of Alpha genus hrE6 protein that is produced by splicing within the E6 ORF. The splice donor site is highly conserved, expressing the first 42 or 44 amino acids of 16E6 or 18E6 respectively and then a few amino acids of non-conserved sequence derived after variable the splice acceptors. E6* contains the first CXXC zinc-binding motif of E6. Functions for this polypeptide in the context of the complete viral infectious cycle remain obscure, because mutation of the splice donor site in the context of the viral genome could alter expression of E7 and E1. E6* is unlikely to interact with LXXLL. When E6* is expressed as a glutathione S-transferase (GST) fusion protein, it binds to in vitro translated E6AP, 16E6, 18E6 and inhibited in vitro E6-mediated p53 degradation (Pim et al., 1997). Similarly, E6* isoform antagonizes the effect of the full-length E6 protein upon procaspase 8, stabilizing it rather than accelerating its degradation (Tungteakkhun et al., 2010). Overexpression of 18E6* alone promotes proteasome dependent degradation of a varied proteins that are the target of full length E6 (such as DLG1) and additionally alters the expression of proteins that are not the target of full length E6 (such as AKT) (Pim et al., 2009). Retrovirally transduced16E6* and 18E6* target the degradation of the TIP60 acetyltransferase in keratinocytes (Jha et al., 2010). The mechanism behind these observations remains obscure, and as yet no direct and validated cellular interaction targets of E6* are described. E6* has not been described in the low risk Alpha HPVs, but a cDNA from BPV-1 encodes a BE6-BE7 fusion protein that has not been characterized (Yang et al., 1985).

What are the main cellular targets of E6 that interact through LXXLL binding?

Because E6 proteins are expressed at very low levels, identification of E6-associated cellular proteins in the past relied upon overexpression of tagged E6 with immune-purification of complexes, yeast two-hybrid screens, and in vitro binding assays. Although many proteins have been thus identified, some doubts have persisted as to the validity of some. As the sensitivity of mass spectrometry has improved, it has become possible to immunopurify epitope-tagged E6 proteins stably expressed in keratinocytes and identify associated proteins by mass spectrometry. This has identified new interactors and confirmed some previously identified interactors.

The most intriguing result of these recent studies is an apparent dichotomy between those HPVs that replicate in cutaneous compared to squamous mucosal epithelia. For papillomavirus types that replicate in skin, the E6 proteins examined so far interact with LXXLL motifs found in MAML family transcriptional co-activators; for HPV types that replicate in squamous mucosa (Alpha types), their E6 proteins associate with the LXXLL motif in E6AP (Brimer et al., 2012; Rozenblatt-Rosen et al., 2012; Tan et al., 2012; White et al., 2012a). Although dual recognition can be observed in overexpression, in vitro binding, or yeast two-hybrids, there is not yet compelling evidence that E6 proteins associate at normal expression levels with both MAML1 and E6AP in vivo. The converse result also holds: while Alpha group E6 proteins interact with E6AP, they do not interact with MAML1, and while Beta group E6 proteins interact with MAML1 they interact poorly with E6AP (Brimer et al., 2012; White et al., 2012a). Further examination of other genus and animal papillomaviruses will determine if this is due to mucosal versus cutaneous biology or reflects an early evolutionary divergence in replication strategy.

Other experimental approaches come to somewhat different conclusions about cellular E6 interactors as is seen in the comparison of E6 interactors that are identified by yeast 2-hybrid screens and those identified by IP/MS. Two recent high-throughput analysis of multiple cutaneous and mucosal E6 types identified largely non-overlapping sets of interacting proteins compared to those identified by IP/MS (Neveu et al., 2012; Rozenblatt-Rosen et al., 2012), despite the further validation in one of those studies of the interactions by mammalian high-throughput protein complementation assay (based on Gaussia princeps luciferase, GPL methodology (Neveu et al., 2012)). Although some targets are common to both data sets, most are not; there is currently a lack of consensus on how to interpret these disparate results.

A critical tool in the analysis of both E6 interactors and E6 biological effects are mutations in E6. Until the structure of E6 was solved, it was difficult to discern if E6 mutants were selectively defective for a particular function, such as LXXLL peptide binding, or were globally defective because the mutation disrupted the E6 protein fold. For most mutants, this type of analysis has not been performed. Since E6 interaction with LXXLL peptides requires proper folding for most of the E6 sequence, truncation or in-frame deletion mutants of E6 are for the most part untrustworthy, and will not be considered further here. An exception is the linear PDZ binding motif at the carboxy-terminus of E6, which can be deleted without compromising the E6 pocket. Table V is a compilation of 16E6 point mutants with associated phenotypes.

Table V.

Phenotypes of Point mutations in 16E6

Mutanta	Binds LXXLLb	Binds E6APc	Binds p53d	Degrades p53e	Other phenotypes and notes.	Reference
16E6 WT	++++	++++	++++	++++	Extends HEK life span with E7f Immortalizes mammary epithelial cells (MEC). Immortalizes keratinocytes with E7. Induces telomerase.	(Nakagawa et al., 1995), (Band et al., 1991; Klingelhutz et al., 1996)
F2V	+++	++++	−	−	Immortalizes MEC. Does not support episomal replicationof genome. Degrades Ada3 and blocks p53 acetylation.	(Liu et al., 1999; Park and Androphy, 2002) (Shamanin et al., 2008)
F2L		+++		+	Supports episomal replication of genome.	(Liu et al., 1999; Park and Androphy, 2002)
D4G	++++	++++	−	−		(Cooper et al., 2006)
P5R	++++		−	−		(Cooper et al., 2006)
Q6A	++++		++	++		(Cooper et al., 2006)
E7A	++++		++++	++++		(Cooper et al., 2006)
R8Q	+++		−	−		(Cooper et al., 2006)
8S/9A/10T			−	−	Induces telomerase. Does not immortalize keratinocytes with E7. Does not support episomal genome replication.	(Klingelhutz et al., 1996) (Foster et al., 1994; McMurray nd McCance, 2004; Mietz et al., 1992; Park and Androphy, 2002)
R10A	++			++++		(Zanier et al., 2013)
K11E	++		−	−		(Cooper et al., 2006)
L12S	++++		+/−			(Cooper et al., 2006)
P13L	+++		+/−	−		(Cooper et al., 2006)
C16R	−	+/−	−	−		(Cooper et al., 2006)
C16S	+++	++++	+++	++		(Cooper et al., 2006)
I23V	++++	++++	+	+		(Cooper et al., 2006)
H24L/I27V		+++	+++	+++	Immortalizes MEC.	(Dalal et al., 1996)
Y32A	++++					(Zanier et al., 2013)
Y32G	+++			++++		(Zanier et al., 2013)
K34E		+++	+++	+++	Immortalizes MEC.	(Dalal et al., 1996)
Q35R		+++	+++	+++	Immortalizes MEC.	(Dalal et al., 1996)
L37F			−	+	No HEK lifespan extension with E7	(Nakagawa et al., 1995)
L37S		+/−	+	+++ at 25C, − at 37C	Does not degrade p53 early passage. Degrades p53 in vivo late passage. Does not degrade ADA3. Induces p21 in UV irradiated cells.	(Liu et al., 1999) (Shamanin et al., 2008) (Thomas and Chiang, 2005)
E39G			++++	+++	No HEK lifespan extension with E7.	(Nakagawa et al., 1995)
E42G			+++	+++	No HEK lifespan extension with E7.	(Nakagawa et al., 1995)
Y43G			−	++	No HEK lifespan extension with E7	(Nakagawa et al., 1995)
D44G			−	+++	Extends HEK life span with E7.	(Nakagawa et al., 1995)
F45Y			++++	+++		(Crook et al., 1991),
F47Y			++++	+++		(Crook et al., 1991),
D49H			++++	+++		(Crook et al., 1991),
31E6F45Y,F4 7Y, D49H					Fails to support HPV-31 replication.	(Thomas et al., 1999)
F45Y, F47Y, D49H			−	−	Blocks induction of p21 in UV irradiated cells. Does not induce telomerase	(Crook et al., 1991; Foster et 1994; Thomas and Chiang, 2005) (Klingelhutz et al., 1996)
F47L	−	−	+	++	No HEK lifespan extension with E7.	(Nakagawa et al., 1995)
E49G			+++
L50G	−	−	−	−	Binds CBP but not E6AP or p53. Does not degrade ADA3.	(Zimmermann et al., 1999) (Shamanin et al., 2008)
L50A	−			−		(Zanier et al., 2013)
L50E				−		(Zanier et al., 2013)
I52T	++	++	+	+		(Cooper et al., 2006)
V53G			+++	+++	No HEK lifespan extension with E7.	(Nakagawa et al., 1995)
Y54D		+			Induces telomerase but does not degrade p53 in vivo. Is defective in p53 degradation and inhibits p53 acetylation, stabilization, and growth arrest induced by p14ARF. Degrades Ada3	(Liu et al., 1999; Shamanin et al., 2008) (Shamanin and Androphy, 2004)
Y54H		+++	+	+++ at 25C, − at 37C	Immortalizes MEC. Does not support episomal replication of genome.	(Liu et al., 1999; Park and Androphy, 2002)
R55A	++			+++		(Zanier et al., 2013)
Y54S			++	+	No HEK lifespan extension with E7.	(Nakagawa et al., 1995)
R55G			++	++	Extends HEK life span with E7.	(Nakagawa et al., 1995)
N58G			+++	+++	Extends HEK life span with E7.	(Nakagawa et al., 1995)
C63H			+++		Mutation in CXXC.	(Crook et al., 1991),
C63R					Supports episomal replication of genome.	(Park and Androphy, 2002)
C66G			−	−	Mutation in CXXC.g	(Foster et al., 1994)
S71A	++			+++		(Zanier et al., 2013)
Y79N	−	−	−	−		(Cooper et al., 2006)
Y84C		+++	+++	+++	Immortalizes MEC.
I101V		++	+++	+++	Immortalizes MEC.	(Liu et al., 1999)
R102A	−			+		(Zanier et al., 2013)
C103R	−	−	−	−	Mutation in CXXC.	(Cooper et al., 2006)
C103G			++++		Mutation in CXXC.	(Crook et al., 1991),
Q107R		+/−	+/−	+++ at 25C, + at 37C	Immortalizes MEC.	(Liu et al., 1999)
L110P			+++	+	No HEK lifespan extension with E7.	(Nakagawa et al., 1995)
L110Q		−	−	−	Does not degrade p53 early passage. Degrades p53 in vivo late passage. Does not support episomal genome replication. Does not degrade ADA3.	(Liu et al., 1999) (Park and Androphy, 2002) (Shamanin et al., 2008)
C111R	−	+	−	−		(Cooper et al., 2006)
E114G			+	+++		(Nakagawa et al., 1995)
E114A		+	+++	+++	Immortalizes MEC. Degrades p53 in vivo.	(Liu et al., 1999)
K115E			+++	+		(Nakagawa et al., 1995)
R117G			+	+++	Extends HEK life span with E7.	Nakagawa et al., 1995)
H118D		+/−	+	+++	Immortalizes MEC. Degrades p53 in vivo.	(Liu et al., 1999)
H118N		++	++	+++	Degrades p53 in vivo.	(Liu et al., 1999)
L119R		+++				(Liu et al., 1999)
D120G			++++	++++	Extends HEK life span with E7.	(Nakagawa et al., 1995)
D120A		+++				(Liu et al., 1999)
D120T		+++				(Liu et al., 1999)
R124T		+++				(Liu et al., 1999)
F125L	−	+++	++	−	Induces telomerase	(McMurray and McCance, 2004) (Cooper et al., 2006; Liu et al., 1999)
F125V		−	++	+++ at 25C, − at 37C	Immortalizes MEC. Degrades p53 in vivo.	(Liu et al., 1999)
N127G			+	++	Extends HEK life span with E7.	(Nakagawa et al., 1995)
N127K		+++				(Liu et al., 1999)
I128T		+/−	++	+++ at 25C, − at 37C	Immortalizes MEC. Degrades p53 in vivo.	(Liu et al., 1999)
R129A	++++					(Zanier et al., 2013)
R129G				+++		(Zanier et al., 2013)
G130R			+		Extends HEK life span with E7.	(Nakagawa et al., 1995)
G130V		−			Immortalizes keratinocytes with E7. Induces telomerase Does not degrade ADA3.	(McMurray and McCance, 04) (Liu et al., 1999; Shamanin et al., 2008)
R131A	−			++		(Zanier et al., 2013)
W132R		+/−	−	−	Does not immortalize MEC.	(Dalal et al., 1996)
G134V		+/−	+	+++ at 25C, − at 37C	Immortalizes MEC. Immortalizes keratinocytes with E7. Does not induce p21 in UV irradiated cells.	(Liu et al., 1999; McMurray and McCance, 2004; Thomas and Chiang, 2005)
C136H			++		Mutation in CXXC.	(Crook et al., 1991),
C136G			−	−	Mutation in CXXC.	(Foster et al., 1994)
Δ145-151 (ΔPDZ)	++++	++++	++++	++++	Induces telomerase. Does not induce telomerase Defective for binding and degradation of PDZ proteins	(Klingelhutz et al., 1996) (Kiyono et al., 1998) see Table III

^aPoint Mutations in 16E6. Bold underlined text denotes residues that make contact with the E6AP LXXLL peptide in the crystal structure of 16E6 (Zanier et al., 2013).

^bBinding to LXXLL peptides assayed in yeast.

^cBinding to E6AP as reported by the authors of the study, being in vitro or by immune precipitation and western blotting.

^dIn vitro degradation assays.

^fExtends life span of human epithelial kidney cells when co transfected together with 16E7.

^gAlthough CXXC mutations are point mutations, they are predicted to disrupt the overall fold of E6.

Alpha group E6 proteins associate with E6AP

As mentioned above, hrE6 proteins associate with E6AP (Huibregtse et al., 1993a). It was determined that this leads to the recruitment of p53 and the transfer of ubiquitin from a thio-ester cysteine bond in the E6AP ubiquitination domain to p53 (Scheffner et al., 1993). Although rabbit reticulocyte lysate supported the degradation of p53, wheat germ lysate did not unless supplemented with E6AP. The carboxy-terminal ubiquitination domain was found present in a family of similar ubiquitin ligases now termed HECT domain ubiquitin ligases (for Homologous to E6AP Carboxy-Terminus) of which E6AP is the prototype (Huibregtse et al., 1995). Mutation of the cysteine that conjugates with ubiquitin creates a dominant negative form of E6AP that can bind to E6 and p53 but fails to result in p53 degradation. E6AP expression is imprinted, and loss of E6AP or mutation with loss of ubiquitin ligase activity is the cause of Angleman syndrome, a complex neuro-developmental disorder (Kishino et al., 1997; Matsuura et al., 1997). How loss of E6AP ubiquitin ligase activity results in the Angelman syndrome remains poorly understood.

Expression of 16E6 from the Keratin 14 promoter (K14-16E6) in mice produces skin hyperplasias and cervical cancers with prolonged latency when the mice are also treated with estrogen; in this system, K14E6 enhances the tumorigenicity of estrogen treatment upon cervical and vaginal neoplasms, and loss of E6AP ablated this enhancement (Shai et al., 2010). K14-16E6 mice null for E6AP have enhanced incidence of cancer compared to estrogen treated animals without E6 (Shai et al., 2010). Cell cycle arrest in K14-16E6 irradiated mouse skin is ablated by E6, but surprisingly this phenotype did not require E6AP, 16E6 can ablate p53 function without E6AP despite the increase of p53 expression in K14-16E6-E6AP−/− mice compared to K14-16E6 mice (Shai et al., 2010). 16E6 has been reported to target p53 degradation in E6AP null mouse cells by an unknown mechanism but and another group has not observed E6AP independent p53 degradation in the same cells (Ansari et al., 2012; Massimi et al., 2008).

Estrogen is essential in the development of cervical cancers in mice that express hrE6 and hrE7. In 1999, Nawaz et al. demonstrated that E6AP could serve as a co-activator for estrogen, gluccocorticoid, androgen, thyroid hormone and retinoic acid receptors in transient transfection assays. E6AP is recruited to the androgen-responsive PSA promoter (Khan et al., 2006), and to the estrogen-responsive pS2 promoter in a hormone responsive manner (Reid et al., 2003). E6AP was shown to be able to target the degradation of the estrogen receptor and progesterone receptor (Li et al., 2006). The role of E6AP as a nuclear receptor co-activator has been recently reviewed (Ramamoorthy and Nawaz, 2008).

E6AP associates with a second ubiquitin ligase, HERC2, that is isolated in association with high and low risk E6 proteins in a high molecular weight nuclear complex (Martinez-Noel et al., 2012; Rozenblatt-Rosen et al., 2012; Vos et al., 2009; White et al., 2012a). HERC2 is a HECT domain ubiquitin ligase that through association with E6AP can stimulate the ubiquitin ligase activity of E6AP and my thus regulate E6AP activity (Kuhnle et al., 2011).

Early in vitro binding assays showed enhanced avidity of bacterially expressed GST-16E6 for E6AP compared to GST-18E6, GST-11E6 or GST-6E6 (Huibregtse et al., 1993a), but the comparative in vivo affinities of different alpha E6 proteins for E6AP and what consequences such affinity differences could confer in vivo is unknown. Low risk Alpha group E6 proteins, such as HPV types 6 or 11 interact with E6AP in vivo and activate E6AP ubiquitin ligase activity, yet have not been found to target the degradation of p53 (Brimer et al., 2007). E6AP is also found in association with low-risk E6 types in IP/MS experiments (Rozenblatt-Rosen et al., 2012; White et al., 2012a). Substrates for the low risk Alpha HPV E6 proteins that are analogous to p53 for the hrE6 proteins have not yet been identified.

Secondary substrates of Alpha group E6 proteins that associate with E6AP: p53

How does p53 associate with the E6-E6AP complex? As noted above, the association of E6 with E6AP (Scheffner et al., 1993), or even a peptide similar to the LXXLL of E6AP (Ansari et al., 2012) alone is sufficient to restructure 16E6 to interact with p53 in yeast hybrid analysis. Many mutations made in E6 ablate the ability of E6 to interact with LXXLL, and thus also ablate the interaction with p53. However, mutations in the very amino terminus of E6 and others elsewhere in the N-terminal zinc domain retain the capacity to interact with E6AP, yet lose the capacity to interact with p53 or target the degradation of p53 (Cooper et al., 2003; Kao et al., 2000; Liu et al., 1999).

The interaction of 16E6 with E6AP induces the dimerization and ubiquitination of E6AP (Nuber et al., 1998). 16E6 mutations of the dimerization surface disrupt both E6 dimerization and p53 in vitro degradation; thus, the dimerization of E6 is functionally linked to the initiation of degradation of p53 (Zanier et al., 2012). When a mutant in the dimerization domain was expressed in HeLa cells (an HPV-18 high risk E6 expressing cell line) it induced senescence, presumably through a dominant negative interaction with E6AP and p53 (Ristriani et al., 2009). The E6-E6AP-p53 complex requires the ability of E6 multimerize via self-association of the amino-terminal domain of E6 (Zanier et al., 2012) to initiate the transfer of ubiquitin from a carboxy-terminal thioester in the HECT domain of E6AP to p53 (Scheffner et al., 1993).

In vitro and yeast expression binding experiments have shown that E6+E6AP or E6+LXXLL peptide associates with the core DNA binding domain of p53 when p53 is in a native conformation, but does not associate with the DNA binding domain of most (but not all) p53 cancer associated mutants (Ansari et al., 2012; Scheffner et al., 1992). A second modality of E6 association with p53 was defined using bacterially expressed E6 proteins; GST-E6 protein from both high and low risk papillomavirus types associate in vitro with the p53 oligomerization domain at the carboxy-terminus of p53 (Li and Coffino, 1996). It remains controversial if this is a biologically meaningful result or an artifact of bacterially expressed and detergent treated E6.

The fact that the interaction of 16E6 with E6AP induces the dimerization and ubiquitination of E6AP may explain the observation that E6AP expression and half-life are reduced in cervical cancer cell lines (Kao et al., 2000). In contrast, in K14-16E6 transgenic mice there is no reduction of E6AP in tissues expressing E6 compared to non-E6 expressing cells (Shai et al., 2010). A related question is how does E6 escape being the target of E6AP ubiquitination and degradation? E6 immunopurified from cell lysates is in a complex with the ubiquitin specific protease USP15 (Vos et al., 2009). RNAi knockdown of USP15 resulted in the reduction of E6 expression but there was no induction of p53 in cervical cancer cell lines, indicating that further development of this area is an important research goal.

While hrE6 targets p53 degradation, residual p53 often remains, yet checkpoint control and p53-induced apoptosis is blocked. Low risk E6, and Beta-papillomavirus E6 (both of which fail to target p53 degradation) block some p53-induced transcription (Giampieri et al., 2004); one important mechanism involves modulation of protein acetylation (discussed below). HrE6 degradation of p53 is blocked by inhibitors of nuclear export indicating that p53 degradation occurs in cytoplasmic and not nuclear proteasomes (Freedman and Levine, 1998; Hietanen et al., 2000; Stewart et al., 2005).

Because hrE6 proteins target the degradation of p53, other E6 proteins have been examined for the same property without success. However, it is premature to dismiss roles of low-risk E6 in manipulating p53 because degradation has yet to be observed. Low risk Alpha E6 proteins are reported to block the activation of p53 by blocking the acetylation of p53 (Thomas and Chiang, 2005), and to block the transcriptional induction of pro-apoptotic genes after DNA damage (Giampieri et al., 2004). Interestingly, p53 co-immunoprecipitates with HPV38 and HPV92 E6, and these two E6 proteins as well as additional Beta genus E6 types stabilize p53 in vivo (White et al., 2012a); HPV-38 E6 and E7 together induce deltaNp73 which acts as a repressor of p53 function resulting in the loss of UV checkpoint control (Accardi et al., 2006; Dong et al., 2008) Since these E6 proteins associate with MAML1 and not E6AP, the role of these p53 associations is as yet unknown.

Other secondary substrates of the high risk E6 + E6AP complex identified by IP/Mass Spectrometry

Proteasome subunits have been found in association with high and low risk Alpha type E6 proteins in two studies utilizing immune precipitation and mass spectrometry (Rozenblatt-Rosen et al., 2012; White et al., 2012a). In both studies, 16E6 mutant I128T, which reduces E6AP association with E6, also greatly reduced proteasome association with E6, suggesting that proteasome subunits associate with E6 through association with E6AP, which has been previously described (Besche et al., 2009; Wang et al., 2007).

P300/CBP

Alone among the Alpha group E6 proteins, HPV16 E6 associated with CBP/p300 in a IP/MS experiment (White et al., 2012a) which was in agreement with earlier studies that identified p300 by hypothesis-directed in vitro binding and that proposed to thereby effect NfkB and p53 transactivation (Patel et al., 1999; Zimmermann et al., 1999) (Thomas and Chiang, 2005). The mechanism of 16E6 association with p300 is as yet unresolved; it could be a direct association or it could depend upon prior association with E6AP and/or p53.

Other secondary associated proteins not identified by IP/MS

The cellular E6TP1 protein (SIPA1L1 gene product) was isolated by yeast two-hybrid, and is a Ran-Gap protein with a PDZ domain that is targeted by high risk E6 + E6AP for degradation, although not through PDZ-mediated association with E6 (Gao et al., 2002; Gao et al., 1999). E6TP1 has not been identified in IP/MS experiments associated with E6, but was a common target of cutaneous and mucosal types in yeast hybrid and mammalian GPL interaction analysis (Neveu et al., 2012). Tuberin has been reported as targeted for degradation by high risk E6 but also in a later report as a target of E6AP in the absence of E6 (Lu et al., 2004; Zheng et al., 2008); one other study that examined tuberin did not see enhanced degradation of tuberin within E6 expressing keratinocytes (Spangle and Munger, 2010). NFX-91 was isolated as an E6 + E6AP associated protein by yeast two hybrid, and was found to be targeted for degradation by 16E6 and to be a regulator of telomerase expression (see subsequent discussion of telomerase (Gewin et al., 2004)). Additional secondary targets of E6 proteins are listed in Table II.

Table II.

Secondary E6 Interactors

Gene Name	Other Name(s)	Method	Alpha (low)	Alpha (high)	Beta	BPV	References
BAK1	Bak	GST/HA	+	+	+		(Thomas and Banks, 1998, 1999; Underbrink et al., 2008)
BARD1	BARD1	Y2H/FL		+			(Yim et al., 2007)
BRCA1	Brca-1	GST/GPL		+			(Neveu et al., 2012; Zhang et al., 2005)
CASP8	Procaspase 8	HA/FL					(Filippova et al., 2007; Tungteakkhun et al., 2010)
CREBBP	CBP	GST/FL/HA/AP-MS		+(16)	+		(Howie et al., 2011; Muench et al., 2010; Patel et al., 1999; White et al., 2012a; Zimmermann et al., 1999; Zimmermann et al., 2000)
CYLD	CYLD1	HA		+(16)			(An et al., 2008)
EP300	P300	GST/FL/HA/AP-MS	+	+(16)	+	+	(Howie et al., 2011; Muench et al., 2010; Patel et al., 1999; White et al., 2012a; Zimmermann et al., 1999; Zimmermann et al., 2000)
FADD	FADD	GST/GPL		+	+		(Neveu et al., 2012; Tungteakkhun et al., 2010)
GPS2	Gps2	Y2H/GST/IP	+	+			(Degenhardt and Silverstein, 2001b)
HERC2	SHEP1	TAP-MS/AP-MS	+	+			(Vos et al., 2009; White et al., 2012a)
KAT5	Tip60	GST	+	+			(Jha et al., 2010)
MCM7	Mcm7	Y2G/MBP	+	+			(Kukimoto et al., 1998)
MGMT	MGMT	IP	+	+			(Srivenugopal and Ali-Osman, 2002)
MYC	c-Myc	IP		+(16)			(Veldman et al., 2003)
NFX1	NFX1-91	Y2H/IP		+(16)			(Gewin et al., 2004)
PML	PML	GST	+	+(18)			(Guccione et al., 2004)
NOTCH1	NOTCH1	AP-MS			+ (some)		(White et al., 2012a)
NOTCH2	NOTCH2	AP-MS			+ (some)		(White et al., 2012a)
SIPAIL1	E6TP1	Y2H/GST/GPL		+			(Gao et al., 1999; Neveu et al., 2012)
SLC12A8	CCC9	AP-MS		+	+ (92)		(White et al., 2012a)
TADA3	Ada3	Y2H/IP/GPL	+	+			(Kumar et al., 2002; Neveu et al., 2012; Zeng et al., 2002)
TERT	TERT	GST/AU1-IP		+(16)			(Liu et al., 2009)
TNFRSF1A	TNF R1	Y2H/HA-IP					(Filippova et al., 2002)
TP53	P53	GST/IP/AP-MS	+	+	+(38,92)		(Scheffner et al., 1990; Werness et al., 1990; White et al., 2012a)
TYK2	Tyk2	GST/FL		+(18)			(Li et al., 1999)
USP15	USP15	AP-MS		+(16)			(White et al., 2012a)
XRCC1	XRCC1	Y2H/IP		+	+		(Iftner et al., 2002)
ZYX	Zyxin	Y2H/GST/MY	+(6)				(Degenhardt and Silverstein, 2001a)
HPV-17a and 38
AMBRA1	DCAF	AP-MS			+		(White et al., 2012a)
MX2	MXB	AP-MS			+		(White et al., 2012a)
NEK1	SRPS2	AP-MS			+		(White et al., 2012a)
UHMK1	KIST	AP-MS			+		(White et al., 2012a)
MTA1	MTA1	AP-MS			+		(White et al., 2012a)
UBR4	P600	AP-MS/GST			+		(Thomas et al., 2013; White et al., 2012a)
KCMF1	DEBT91	AP-MS			+		(White et al., 2012a)
TNKS1BP1	TAB182	AP-MS			+		(White et al., 2012a)
C2orf29	C2orf29	AP-MS			+		(White et al., 2012a)
CNOT1-10	Ccr-Not	AP-MS			+		(White et al., 2012a)
C21orf2	YF5/A2	AP-MS			+(17a)		(White et al., 2012a)
UBA5	UBE1DC1	AP-MS			+(17a)		(White et al., 2012a)
UFM1	UFM1	AP-MS			+(17a)		(White et al., 2012a)
JMJD1C	TRIP8	AP-MS			+(17a)		(White et al., 2012a)
TANK1	PARP5A	AP-MS			+(17a)		(White et al., 2012a)
TJAP1	PILT	AP-MS			+(17a)		(White et al., 2012a)
HPV-92 Specific
AAMP	AAMP	AP-MS			+ (92)		(White et al., 2012a)
ARNT	HIF1β	AP-MS			+ (92)		(White et al., 2012a)
AZI1	CEB131	AP-MS			+ (92)		(White et al., 2012a)
CAMSAP3	NEZHA	AP-MS			+ (92)		(White et al., 2012a)
CEP152	CEP152	AP-MS			+ (92)		(White et al., 2012a)
CEP44	KIAA1712	AP-MS			+ (92)		(White et al., 2012a)
CEP63	CEP63	AP-MS			+ (92)		(White et al., 2012a)
DUSP3	DUSP3	AP-MS			+ (92)		(White et al., 2012a)
HIF1A	HIF1α	AP-MS			+ (92)		(White et al., 2012a)
JUB	JUB	AP-MS			+ (92)		(White et al., 2012a) [
SLC12A8	CCC9	AP-MS		+	+ (92)		(White et al., 2012a)

GST:GST pulldown; HA: HA tag pulldown; MY: myc tag pulldown; FL: Flag tag pulldown; IP: Immunoprecipitation; Y2H: Yeast 2-Hybrid; TAP: Tandem affinity purification; GPL: Gaussian princeps luciferase assay; AP-MS: Affinity Purified-Mass spectrophotometry; MT: Maldi-Tof; MBP: Maltose binding protein. “+” means that interaction has been noted in the literature. If no “+” the interaction has not been published. The number in parenthesis indicates the specific HPV type in which the interaction has been observed/demonsrated. If no number, the interaction has been observed/demonstrated in more than one type of the indicated group.

Beta, Delta and Mu genus cutaneous E6 proteins associate with MAML family proteins

In contrast to the Alpha genus E6 proteins that interact with E6AP, BE6 and HPV E6 proteins from the Beta and Mu Genus associate with MAML1 (BPV and HPVs) and MAML3 (BPV-1 E6), binding a LXXLL motif near the carboxy-terminus of MAML and repressing the activity of the Notch transcriptional activation complex as will be discussed below (Fig. 2) (Brimer et al., 2012; Rozenblatt-Rosen et al., 2012; Tan et al., 2012; White et al., 2012a). As would be expected, the subunits of the Notch transcription complex (RBPJ and Notch1) were also detected with MAML1 in association with these E6 proteins. Additional proteins were found in association with the Beta genus E6 proteins, but restricted to species within the genus and it is unclear if these associations are direct with E6 or dependent upon the prior binding of E6 to a LXXLL protein, presumably MAML1. The CCR4-Not complex was found in association with Beta-species 2 (HPV-92), associated with HIF13/HIF13 and centrosome localized proteins. Beta genus species 1 (HPV-8 prototype) have strong association with CBP/p300 (Howie et al., 2011; Rozenblatt-Rosen et al., 2012; White et al., 2012a). P300/CBP have LXXLL docking sites and could serve as a primary docking site via LXXLL interactions, but this has not yet been demonstrated and those sites do not closely resemble the LXXLL sites of E6AP, MAML1, and paxillin. Interestingly, UBR4, a large ubiquitin ligase that participates in the N-end rule protein degradation pathway and is a primary binding protein of HPV E7 oncoproteins (Demasi et al., 2005; Huh et al., 2005; White et al., 2012b), was found to associate with 17E6 and 38E6 (Thomas et al., 2013; White et al., 2012a).

BE6 associates with multiple cellular binding proteins via LXXLL interactions

BE6 was found to associate with paxillin by IP/MS in transiently transfected cells (Tong and Howley, 1997), and yeast two-hybrid (Vande Pol et al., 1998). BE6 binds to a LXXLL motif similar to that of E6AP (Fig. 2), and BE6 mutants that discriminate in binding between E6AP and paxillin suggested that BE6 transformation was more closely related to paxillin association than E6AP (Das et al., 2000). Paxillin knockout cells are not transformed by BE6 unless reconstituted with paxillin, indicating that paxillin is required for BE6 transformation or alternatively that paxillin might be generally required for anchorage independent cell proliferation (Wade et al., 2008). BE6 also associates with the AP1 adaptor complex for clathrin endocytosis (Tong et al., 1998); that association was not clearly linked to transformation by BE6. As discussed above, BE6 is associated with MAML1 and MAML3 and represses notch signal transduction, but dominant negative MAML1 does not transform cells that are transformed by BE6 (Brimer et al., 2012). It may be that multiple interactions by BE6 with LXXLL motifs on multiple proteins are required for full transformation by BE6.

After binding to LXXLL, how does E6 interact with secondary associated proteins?

Binding experiments in vitro and in yeast demonstrated that the first 8 amino acids of 16E6 could be deleted, ablating p53 binding but without ablating E6AP association [Kao, 2000 #663]; thus, while a central core region of E6 (corresponding to BE6 amino acids 11-132) is required for LXXLL interactions, additional amino-terminal sequences seem to be for other interactions. Much work remains to understand these interactions, since in only a single instance (cancer associated HPV E6 and its p53 interaction) have any such interactions been demonstrated and mapped to the amino-terminal surface of E6 (Cooper et al., 2003).

In the case of BE6, there are 10 amino acids that must be deleted before the ability of BE6 to bind to LXXLL motifs is abolished. For most papillomaviruses, the amount of “extra” amino-terminal sequences beyond that required to interact with LXXLL peptides is more substantial than BE6, ranging from 23–25 amino acids for the cancer associated E6 proteins, to 34 amino acids for the cutaneous HPV-5 E6, and an entire additional zinc-binding domain for the long form of CRPV E6 (Meyers et al., 1992). These amino-terminal sequences are candidates for secondary E6 associated proteins.

The crystal structure of LXXLL-bound 16E6 reveals numerous clefts and surfaces that could mediate other protein-protein interactions. Unfortunately, 16E6 deletion mutants typically used to delineate binding sites are predicted to ablate the overall fold of 16E6, making such mutants undesirable to map protein-protein interactions, and most 16E6 point mutants have not been well enough characterized for LXXLL interactions, stability, and retention of secondary function to inspire confidence. The recent structure should allow a new generation of mutants to be characterized for the mapping of biological functions and associations on the surface of E6.

A PDZ ligand on hrE6 interacts with cellular PDZ containing proteins implicated in signal transduction and polarity

PDZ domains (named for the proteins PSD95, DLG, and ZO1) are small domains that bind to peptide ligands on target proteins. PDZ peptide ligands can be internal, but are most typically carboxy-terminal peptide ligands with a consensus sequence XX(S/T/Y)X(V/L/M). Adapter proteins often contain multiple PDZ domains, resulting in large complexes built through the association of multiple PDZ domain proteins and their binding partners. Affinities of PDZ-ligand interaction are typically in the low micro-molar range, and can be modulated by phosphorylation of the PDZ ligand or the PDZ domain (reviewed in (Lee and Zheng, 2010)).

DNA tumor viruses that target p105RB by viral oncoproteins such as Adenovirus E1A, SV40 TAg, or high risk HPV E7 also produce proteins that either associate with cellular proteins containing PDZ domains, or target cell polarity (reviewed (Javier, 2008; Tomaic et al., 2009a)). For example, Adenovirus E1A interacts with RB, and the E4ORF4 protein associates with cellular PDZ proteins. High risk papillomavirus E7 targets RB for degradation and hrE6 associates with a subset of PDZ proteins via an 8 amino acid PDZ ligand at its carboxy-terminus. Different hrE6 proteins vary in the sequence of the PDZ ligand and consequently target somewhat different sets of PDZ domain proteins (Thomas et al., 2005). Similarly to the high risk HPVs, SV40 TAg associates with RB and small t antigen disrupts the integrity of cellular tight junctions (Nunbhakdi-Craig et al., 2003). In contrast to hrE6, Low-risk Alpha E6 proteins do not have a carboxy-terminal PDZ ligand, nor do Beta genus or BE6. Interestingly, the rhesus monkey papillomavirus E7 protein both targets RB and has a PDZ ligand at the carboxy-terminus that can interact with scribble and PAR3, PDZ binding proteins that also complex with hrE6 (Tomaic et al., 2009a).

The PDZ ligand of 16E6 alters differentiation of the skin or eye in transgenic mice (Nguyen et al., 2003a; Nguyen et al., 2003b; Simonson et al., 2005. Under low expression conditions, the PDZ ligand of E6 reduces growth factor dependence in human keratinocytes [Jing, 2007 #1318). In SV40 immortalized keratinocytes, the E6 PDZ ligand function promotes epithelial to mesenchymal transitions (Watson et al., 2003), and in human keratinocytes the E6 PDZ ligand promotes co-operation with ras and anchorage independent colony formation (Spanos et al., 2008a). In the context of the entire hrHPV genome, deletion of the E6 PDZ ligand causes loss of the viral plasmid upon cell passaging (Lee and Laimins, 2004).

So which PDZ associations with E6 mediate these phenotypes? Described interactions are shown in Table III. Although there are hundreds of cellular proteins with PDZ domains only a handful associate with high risk E6 proteins through PDZ interactions. It is as yet unclear if one or more than one of these interactions is critical for the virus life cycle or cancer. All of these associations were published as resulting in the targeted degradation of the PDZ protein, usually in an E6AP and proteasome dependent manner, analogous to the previously described and widely replicated targeted degradation of p53 by high risk E6 proteins. Like p53, such degradation is supported in vitro from reticulocyte-translated proteins. However, the targeted overall degradation of some of the PDZ proteins by E6 in vivo has been challenged (Kranjec and Banks, 2010). DLG1, which is targeted for degradation by hrE6 in vitro, does not show reduced expression or re-localization in the context of E6 expressed from episomal genomes in primary keratinocytes (Lee and Laimins, 2004). Some studies have found that only certain subcellular fractions of hrE6-associated PDZ proteins are degraded (Massimi et al., 2004; Massimi et al., 2006; Narayan et al., 2009) but again, these experiments involve expression levels presumably higher than produced by episomal genomes. While multiple PDZ domain proteins have been described after affinity isolation or yeast two-hybrid identification, only scribble, PDZ11 and the tyrosine phosphatases PTPN3 and PTPN13 were isolated by IP/MS of E6 in stably expressing keratinocytes (White et al., 2012a). Interestingly, PDZ11 and PTPN13 were also associated with some Beta genus E6 proteins even though they do not have a classic carboxy-terminal PDZ binding motif (White et al., 2012a). Adding further complication, the E6* protein (produced by internal splicing of high risk E6 proteins) reduces the half-life of DLG1 and other PDZ proteins despite having no PDZ ligand with which to associate with PDZ proteins (Pim et al., 2009).

Table III.

PDZ Interactors with High Risk E6s

Gene Name	Other Name(s)	Method	Comments on Protein	References
DLG1	hDlg	GST/MY	MAGUK cell polarity/junction protein	(Gardiol et al., 1999; Kiyono et al., 1997; Massimi et al., 2004)
GOPC	CAL	GST/MT/HA/MS	Vessicular Trafficking	(Jeong et al., 2006; White et al., 2012b)
INADL	PATJ	Y2H/GST/FL/MY	Tight junction associated	(Latorre et al., 2005; Storrs and Silverstein, 2007)
MAGI1	MAGI-1	GST/HA/GPL	MAGUK cell polarity/junction protein	(Glaunsinger et al., 2000; Neveu et al., 2012)
MAGI2	MAGI-2	GST	MAGUK cell polarity/junction protein	(Thomas et al., 2002)
MAGI3	MAGI-3	GST	MAGUK cell polarity/junction protein	(Thomas et al., 2002)
MUPP1	MUPP1	GST	MAGUK cell polarity/junction protein	(Lee et al., 2000)
PTPN13	PTPN13	IP	Phosphatase; Degradation involved in anchorage independent growth and invasive properties; Potentiates MAP kinase signaling	(Hoover et al., 2009; Lee et al., 2000; Spanos et al., 2008b)
PTPN3	PTPN3	IP/TAP	Phosphatase; Degradation reduces growth factor requirements	(Jing et al., 2007)
SCRIB	Scribbled	IP/GPL/MS	MAGUK cell polarity/junction protein	(Nakagawa and Huibregtse, 2000; White et al., 2012a)
YWHAC	14-3-3zeta	GST/HA	Contributes to steady-state levels of E6 by regulating phosphorylation by PKA	(Boon and Banks, 2013)

GST:GST pulldown; HA: HA tag pulldown; MY: myc tag pulldown; FL: Flag tag pulldown; IP: Immunoprecipitation; Y2H: Yeast 2-Hybrid; TAP: Tandem affinity purification; GPL: Gaussian princeps luciferase assay; MS: Mass spectrophotometry; MT: Maldi-Tof

Studies as to the mechanism by which E6 may reduce expression of PDZ proteins, have differed with most showing E6AP dependence (Handa et al., 2007; Jing et al., 2007; Kuballa et al., 2007), but others observing neither ubiquitin nor proteasome dependence (Ainsworth et al., 2008; Grm and Banks, 2004). The E6 PDZ ligand can be phosphorylated (Massimi et al., 2001), resulting in association of E6 with 14-3-3 proteins to the exclusion of PDZ proteins (Boon and Banks, 2013). Thus, particular culture conditions in vivo for PDZ interactions with E6 may be necessary for modulation of phosphorylation to occur before phenotypes are observed, which could account for some discordant observations in the literature.

The crystal structure of the PDZ domains of DLG1 and MAGI1 in association with the PDZ ligand of E6 has been solved (Zhang et al., 2007), as has the solution structure of the second PDZ domain of MAGI1 in the presence and absence of the E6 PDZ ligand (Charbonnier et al., 2011).

Biological Functions of E6

The previous sections focused mainly on E6 structure and the mechanisms by which different E6’s interact with cellular proteins. A wealth of information exists on how E6 and E7 affect various aspects of transformation, cell differentiation, metabolism, immune response, and virus replication. Some of these topics have already been touched upon earlier in this review because they fit well with the discussion on E6-interacting proteins. Here, we will discuss other topics in more detail to convey a wider appreciation of the biological functions that have been attributed to E6.

Transformation and immortalization

It should be emphasized that E6 and E7 are expressed together in HPV infected and transformed cells. There is value, however, in dissecting the functions of E6 and E7 by expressing them individually. As mentioned in the introduction, early studies focused on the ability of hr-HPV to transform 3T3 cells (Yasumoto et al., 1986) and then E6 and E7 to transform rodent cells and immortalize human keratinocytes (Bedell et al., 1989; Durst et al., 1987; Phelps et al., 1988; Pirisi et al., 1987; Sedman et al., 1991; Storey et al., 1988). HrE6 is effective in combination with oncogenic Ras in transforming baby rat kidney (BRK) cells; mutants of hrE6 that were unable to cause the degradation of p53 still had some transforming potential, indicating p53 independent functions (Pim et al., 1994; Storey and Banks, 1993). HPVs infect keratinocytes, and ideally, examination of how the different proteins affect function should be done in this type of cell. Full-length E6 from high-risk types such as 16, 18, and 31 can extend the lifespan of keratinocytes, but E7 in combination with E6 is required for efficient immortalization frequency (Hawley-Nelson et al., 1989; Hudson et al., 1990; Munger et al., 1989; Sedman et al., 1991; Woodworth et al., 1989). A caveat to this is that hrE6 can immortalize epithelial cells (e.g. mammary epithelial cells) that have an aberrant RB pathway (through down-regulation of the cdk/cyclin inhibitor p16) (Band et al., 1991; Dalal et al., 1996; Foster et al., 1998). Low-risk mucosal E6s have little transformation function in keratinocytes (Band et al., 1993; Halbert et al., 1992). Of the cutaneous Beta HPVs only a subset are able to transform primary human keratinocytes. Expression of E6/E7 in combination from HPV-5, -8, -24, -36, and -38 extends the lifespan of human keratinocytes (Bedard et al., 2008) with occasional subpopulations of cells emerging that are immortal, particularly in HPV-38 and 49 E6/E7 cultures (Bedard et al., 2008; Cornet et al., 2012). Immortalization by CRPV and HPV-38 E6 involves the inhibition of p53 dependent apoptosis via the association of E6 with p300 and blocking the acetylation of p53 (Muench et al., 2010).

As discussed in previous sections, transgenic mice that express high risk E6 and E7 develop cancer (Lambert et al., 1993), a phenotype that is primarily due to E7 expression (Riley et al., 2003), but high-risk mucosal HPV-16 E6 has modest transforming functions when expressed as a transgene from a keratin specific promoter in the epithelium of mice; this activity was lost upon deletion of the PDZ domain of E6 or mutation of I128T which greatly decreases E6AP association with 16E6 (Nguyen et al., 2002; Nguyen et al., 2003a; Riley et al., 2003; Simonson et al., 2005; Song et al., 1999).

The vast sequencing of HPV-16 genomes has shown associations between certain polymorphisms within E6 (particularly L86V) and the relative risk of developing cancer, but the reasons for this remain poorly understood (Cornet et al., 2013).

E6 and Telomerase

E6s from high-risk mucosal HPVs and from certain cutaneous HPVs are capable of activating telomerase, the enzyme complex that adds telomere repeats to the ends of chromosomes (Klingelhutz et al., 1996). The activation of telomerase was found not to be dependent on the ability of E6 to target p53 for degradation since the 16E6-8S9A10T mutant could still activate telomerase but not degrade p53 and conversely, the 3118-122 mutant that has partial ability to target p53 could not activate telomerase (Kiyono et al., 1997; Klingelhutz et al., 1996). Most studies indicate that E6s activate telomerase through transcriptional up regulation of TERT, the reverse transcriptase component of telomerase (Gewin and Galloway, 2001; Oh et al., 2001; Veldman et al., 2001A). A recent study demonstrated that there was a strong correlation between the ability of E6 of certain HPV types to activate the TERT promoter and the association of those types with cancer (Van Doorslaer and Burk, 2012). The mechanism by which this occurs is not entirely clear but appears to involve E6AP binding (Gewin and Galloway, 2001; Oh et al., 2001). The 16E6 L50G mutant is defective in binding E6AP and does not activate telomerase, and knockdown of E6AP by shRNA abrogates the ability of 16E6 to up regulate TERT (Gewin et al., 2004). The PDZ binding domain of 16E6 is dispensible for telomerase activation ((Klingelhutz et al., 1996). One model proposes that E6 and E6AP bind to a repressor of TERT transcription called NFX1-91 which binds to the mSin3a/HDAC complex that causes deacetylation of histones (Gewin et al., 2004; Katzenellenbogen et al., 2009; Xu et al., 2008). Interaction with E6 causes the ubiquitination of NFX1-91, degradation, and release of transcriptional repression at the TERT promoter. The NFX1 locus also codes for a splice variant called NFX1-123 which apparently stabilizes TERT transcripts in HPV-16 E6 expressing cells by binding to poly-(A) binding proteins (Katzenellenbogen et al., 2007; Katzenellenbogen et al., 2009). Another model indicates that E6 and E6AP bind to c-myc and that this somehow causes c-myc to be a better transcriptional activator of TERT (Veldman et al., 2003). Mutations of the E box in the TERT promoter affects the ability of E6 to activate TERT in experiments using TERT-promoter luciferase constructs (Au Yeung et al., 2011; James et al., 2006a; Veldman et al., 2003). In the context of E6 expression, the c-myc protein may displace the inhibitory USF transcriptional repressor from the E box in the TERT promoter (McMurray and McCance, 2003). These two models are not mutually exclusive and other mechanisms are possible. Interestingly, hrE6 has been shown to bind directly to the TERT protein but the consequences of this in telomerase activation are not entirely clear (Liu et al., 2009). The observation that Beta HPV types, such as HPV-5 and HPV-8 that are associated with skin cancer, can also activate telomerase brings an added complication (Bedard et al., 2008). Although it has been shown that the Beta E6 proteins can associate with E6AP in vitro and in transient expression assays (Thomas et al., 2013), in stable expression IP/MS experiments, E6AP binding was not observed (White et al., 2012a). Thus, the mechanism of telomerase activation by the Beta types may be different.

It is clear that different culture conditions (such as culturing in serum and fibroblasts feeder cells compared to serum-free low calcium media formulations) affect the induction of telomerase in keratinocytes raising the possibility that much of the ultimate effects of E6 upon telomerase expression could be rather indirect (Fu et al., 2003). The reason HPV activates telomerase is unknown. It would not appear to be essential for replication since a large number of types do not activate telomerase. One possibility is that telomerase activation allows an extension of keratinocyte lifespan to provide an advantage for replication. However, immortalization of cells is not necessary for HPV replication and low-risk types that do not activate telomerase are certainly able to replicate. Adding to the confusion, telomerase mutants defective for enzymatic activity immortalize keratinocytes in combination with hrE7 proteins (Miller et al.). It is possible that TERT has other functions besides telomere elongation, such as inhibition of apoptosis, and certain HPVs could be taking advantage of this to increase replication or cell survival to allow replication (Saretzki, 2009). A consequence is that infection with these types provides a higher likelihood of malignant conversion.

P53 regulation by high risk E6

To summarize the material presented so far on how hrE6s target p53 for degradation, 16E6 is unstable upon translation in vivo but is stabilized upon binding the LXXLL peptide on E6AP (Tomaic et al., 2009b), and changes its conformation to one that interacts with p53 (Ansari et al., 2012). The E6-E6AP-p53 complex requires the ability of E6 multimerize via self-association of the amino-terminal domain of E6 (Zanier et al., 2012) to initiate the transfer of ubiquitin from a carboxy-terminal thioester in the HECT domain of E6AP to p53 (Scheffner et al., 1993). This then leads to the degradation of p53 through the proteasome.

While hrE6 targets p53 degradation, p53 is often not completely degraded in hrE6 expressing cells. Despite residual p53 expression, p53 dependent transcription, checkpoint control and p53-induced apoptosis are blocked. Low risk E6, and Beta-papillomavirus E6 (both of which fail to target p53 degradation) block some p53-induced transcription (Giampieri et al., 2004); one important mechanism involves modulation of protein acetylation (discussed below). HrE6 degradation of p53 is blocked by inhibitors of nuclear export (Freedman and Levine, 1998), indicating that p53 degradation occurs in cytoplasmic and not nuclear proteasomes.

Effects of E6 on Transcription

p53-dependent and p53-independent alterations of global cellular transcription by hrE6 proteins has been observed in transduced keratinocytes (Duffy et al., 2003; Garner-Hamrick et al., 2004; Kuner et al., 2007; Mendoza-Villanueva et al., 2008). E6 effects upon cellular signal transduction by hrE6 could in part explain these effects (such as through the effects of E6 upon cellular PDZ proteins), however, several specific interactions of E6 with cellular transcription complexes is the likely cause. Histone acetyltransferases (HATs) are components of eukaryotic transcription complexes. Apart from acetylating histones to enable chromosomal remodeling, several HATs (p300, CBP, PCAF, TIP60, and hMOF) acetylate p53 and other transcription factors and function as p53 co-activators. HrE6 proteins target the degradation of Ada3 and Tip60 acetyltransferases, and interact with p300.

Ada3 (for the yeast alteration/deficiency in activation protein) is a component of yeast HAT complexes, and mammalian Ada3 is a transcription co-activator for p53 and other cellular transcription facts such as estrogen receptor and RXR-alpha that are targeted for degradation by 16E6 and E6AP (Balasubramanian et al., 2002). RNAi knockdown of hAda3 blocks the acetylation of lysine 382 in p53, inhibits p53 stabilization, and attenuates p14ARF-induced senescence (Hu et al., 2009; Kumar et al., 2002; Meng et al., 2004; Nag et al., 2007; Sekaric et al., 2007; Shamanin et al., 2008; Zeng et al., 2002). Thus the E6 mediated degradation of Ada3 blocks p53 transcription and could modulate estrogen effects in HPV infected cervical cells. The association of cutaneous E6 proteins with Ada3 has also recently been found by enzyme complementation analysis in mammalian cells (Neveu et al., 2012).

Both low and high risk mucosal HPV E6s are able to interact with the acetyltransferase TIP60 (Jha et al., 2010). Among other substrates, TIP60 can acetylate p53 at residue K120. The interaction of E6 with TIP60 destabilizes p53 complexes and affects regulation of p53 responsive genes. It is interesting to note, however, that the effects are more specific to genes involved in regulating apoptosis than to than those involved in cell cycle arrest, such as p21. Thus, E6’s interaction with TIP60 apparently fine-tunes its regulation of p53, which could be more important for the low-risk types since the high-risk types would effectively degrade p53. However, it was demonstrated that E6’s affect on TIP60 did not depend on binding to E6AP or p53 and, in fact, only the first 43 amino acids of E6 (in the E6* splice variant) were necessary for the effect. Correlating with the above study, EP400, a component of the NuA4/TIP60 histone acetyltransferase complex, was identified in a genome wide RNAi scan for factors that are necessary for E2-mediated repression of the high risk HPV 16 early promoter, implicating E6 in a feed-forward regulation of both basal transcription from the early promoter and possibly E2 mediated repression (Smith et al., 2010).

HrE6 also modulates the function of other chromatin modifiers including CARM1, PRMT1 and SET7 to negatively regulate their activity, inhibit p53 activation of transcription and enhance the degradation of p53 by hrE6 (Hsu et al., 2012).

In addition to targeting RB family members, Adenovirus E1a modulates transcription through association with p300, prompting a search for similar interactions in papillomaviruses. In vitro translated E6 proteins associate in vitro with GST fusions of p300 fragments (Patel et al., 1999) and GST-E6 proteins associate in vitro with partially purified p300 preparations (Zimmermann et al., 1999). Similar in vitro binding experiments showed association of p300 with BE6 (Zimmermann et al., 2000); in all of these studies, the association was related to the inhibition of p53 transcriptional activation independent of p53 degradation, and loss of p53 acetylation. In vitro reconstituted chromatin templates demonstrated that both high and low risk E6 proteins could repress p53 transcription through inhibition of p300 dependent histone acetylation, thus converting p53 transcription complexes from transcriptional activators to repressors (Thomas and Chiang, 2005).

p300 has been isolated by IP/MS from keratinocytes stably expressing multiple Beta-type E6’s, but among the Alpha E6s only associated with 16E6 (White et al., 2012a). CRPV E6 and the Beta-papillomavirus HPV 38 E6 association with p300 correlated with the blockage of p53 acetylation by p300; mutants that failed to associate with p300 were defective for tumor formation (Muench et al., 2010). In the case of Beta type HPV 5 and 8, a role for this interaction is implicated in degradation of p300, activating AKT kinase, inhibiting differentiation (Howie et al., 2011), and mediating the ATR response to UV-induced DNA damage (Wallace et al., 2012). For HPV-38, the interaction of E6 with p300 appears to be essential for immortalization of keratinocytes by HPV-38 E6/E7 (Muench et al., 2010).

E6 Functions in Replication

Determining the role of the different viral proteins in the HPV lifecycle has been somewhat difficult due to the technical problems associated with establishing HPV replication in vitro. Since E6 is important for the extension of normal keratinocyte lifespan (which are notorious for having a short replicative lifespan in vitro), removing E6 or mutating it can result in abrogated immortalization function. The low-risk HPVs do not readily extend the lifespan of keratinocytes. Thus, it is difficult to know whether the effects that one observes are due to the lack of an important function in replication or the lack of the ability to extend the lifespan of the cells. Several groups have been able to get HPVs to replicate in immortal cells, the caveat being that the immortal cells generally have active telomerase already and may have a defect in the p53 pathway. Nevertheless, it does appear that E6 is important for replication (Oh et al., 2004; Thomas et al., 1999). For HPV-16, loss of E6 or mutations that result in loss of p53 degradation result in loss or poor maintenance of HPV genomes (Park and Androphy, 2002). Similar results were found for HPV-11 with missense mutations in E6 (Oh et al., 2004). Lack of E6 results in accumulation of p53 and a reduction in genome amplification (Wang et al., 2009a). The role of E6 in any function should always be viewed in the context of E7 expression since the two are expressed together in cells.

E6, Notch, MAML, and Keratinocyte Differentiation

Since HPV infects keratinocytes and their life cycle is closely associated with differentiation, it would be expected that HPV proteins would affect differentiation. There is considerable evidence that hrE6 can modulate keratinocyte differentiation (Alfandari et al., 1999; Sherman et al., 1997). Microarray analysis indicates that expression of HPV-16 E6 causes down regulation of specific genes that are involved in keratinocyte differentiation (Duffy et al., 2003; Muench et al., 2010). HrE6’s ability to down regulate differentiation specific genes might delay differentiation until enough genomes have been replicated for subsequent production of infectious virions. Part of this effect on differentiation may have to do with E6’s ability to down regulate the Notch pathway, which is a key player in regulating keratinocyte differentiation.

As discussed above, BE6 and Beta genus HPV cutaneous E6 proteins interact with an acidic LXXLL peptide on MAML1 and MAML3, precipitating a complex containing the DNA binding subunit RPB-J and Notch1 and repressing Notch dependent transcriptional activation (Brimer et al., 2012; Meyers et al., 2013; Rozenblatt-Rosen et al., 2012; Tan et al., 2012). The MAML1 coactivator is most well known for its function in Notch signaling. Notch signaling between adjacent cells affects the developmental fates of those cells, linking the differentiation fate of a given cell to that of its adjacent neighbor. Notch1 and 2 genes are expressed in the first spinous cell layer and the Notch ligand, Jagged2, is expressed in the basal layer; signaling to Notch1 in the spinous cell layer then drives early and late squamous epithelial differentiation (Blanpain et al., 2006; Rangarajan et al., 2001b) (and reviewed in (Watt et al., 2008)). Upon canonical Notch signaling, the Notch receptor is cleaved by the intramembranous gamma-secretase protease, liberating the Notch intracellular domain that forms a complex with the RBP-J DNA binding protein. This displaces a repressor-histone-deacetylase complex and recruits the MAML1 coactivator, thus converting the RBP-J complex from a transcriptional repressor to an activator (Figure 8 and reviewed in (Tanigaki and Honjo, 2010)).

Figure 8.

Notch signaling in squamous epithelium. Notch signaling is initiated when basal cells that express Notch ligands engage Notch that is expressed in suprabasal cells, resulting in proteolytic cleavages of Notch and liberation of the Notch Intracellular Domain (ICD). The first cleavage within the extracellular domain is mediated by TACE, while the second cleavage is mediated by the 3-secretase activity of the presenilin protein complex. The cleaved ICD translocates to the nucleus where it associates with MAML transactivator proteins RBP-Jk, a DNA-binding subunit, displacing HDAC. This converts the RBP-J complex from a transcriptional repressor to a transcriptional activator, activating transcription of basic helix-loop-helix transcription repressor factors that are the Notch effectors. Notch signaling occurs both at the basal-spinous cell junction and in the upper spinous layer where it drives terminal differentiation of the granular layer.

Because Notch signaling is central to squamous differentiation, all papillomaviruses must have developed strategies that in some way manipulate Notch signaling. Complete disruption of Notch signaling in the squamous epithelium of transgenic results in the loss of differentiation and squamous cell cancers, as seen tissue specific Notch deletion (Dotto, 2008; Nicolas et al., 2003), skin specific expression of a dominant negative MAML1 (Proweller et al., 2006), or epithelial deletion of RBP-J (Blanpain et al., 2006) This demonstrates that Notch signaling is a tumor suppressor in squamous epithelium. The role of Notch signaling in hrHPV transformation has been controversial. Activated Notch can cooperate with high risk E6 + E7 oncoproteins to transform immortalized HaCat cells (Rangarajan et al., 2001a). Recent deep sequencing of hrHPV positive and HPV negative squamous cell head and neck cancers revealed a high frequency of amino-terminal mis-sense mutations of Notch1 in both cancer types (Agrawal et al., 2011; Stransky et al., 2011). This suggests that Notch signaling continues to be a tumor suppressor pathway in hrHPV cancers and raises the question as to how the Alpha genus HPVs circumvent the effects of Notch signaling. Terminal differentiation in the squamous superficial layer is necessary to ensure a competent epithelial barrier, since loss of barrier function would predictably result in microbial infections and immune cell infiltration of the papilloma. How papillomaviruses repress and delay spinous differentiation yet allow for terminal corneal differentiation is as yet unclear.

hrE6 can promote the growth of colonies of keratinocytes that fail to stratify when cell cultures are switched from low to high calcium media (Sherman et al., 2002; Sherman et al., 1997; Sherman and Schlegel, 1996). The failure to stratify is not evident when E6 transduced colonies are pooled and passaged or when grown in organotypic cultures. Although a quantitative and intriguing phenotype, more work is needed to understand what interactions with E6 are responsible for this phenomenon.

E6, Autophagy, and Metabolism

E6 is able to stimulate protein synthesis by increasing cap-dependent translation through enhancement of 5′ mRNA cap translation initiation complex via activation of mTORC1 (Spangle and Munger, 2010). Analysis of E6 mutants indicated that preservation of the overall E6 fold to interact with LXXLL motifs was required, and cutaneous E6s were unable to activate cap-dependent translation (Spangle et al., 2012). 16E6 also effects PDK1 and mTORC2 to activate Akt, causing subsequent activation of the mTORC1 pathway. It may be that increasing energy metabolism could enhance HPV replication since HPVs replicate in terminally differentiating cells that are likely to have low a nutrient supply.

Regulation of miRNAs by E6

E6s can regulate the expression of miRNAs in cells (Martinez et al., 2008; McKenna et al., 2010; Wald et al., 2010; Wang et al., 2009b). HrE6 down regulates miR-34a, which is involved in targeting cell cycle control genes (Wang et al., 2009b). There is also evidence that down regulation of miR-218 by hrE6 is important for regulating expression of LAMB3 (Martinez et al., 2008) and that this may play a role in cervical cancer cell growth. Down regulation of miR-23b by hrE6 may also be important for regulating cell migration by causing the up regulation of urokinase plasminogen activator gene (Au Yeung et al., 2011). It is unknown how E6 regulates miRNAs but it seems likely that it is through its interaction with transcriptional factors and signaling proteins.

E6 involvement in regulation of apoptosis and immune response

Like most viruses, HPV can repress the natural response of a host cell to infection. This is mainly through inhibition of apoptosis and inhibition of the immune response. The mechanisms are diverse both in the different pathways that are affected by the same type and in the ways that different types inhibit apoptosis and immunity. While there is evidence that viral proteins such as E2 are involved in modulating the immune response, most studies have focused on E6 and E7. E6 appears to play a significant role.

NF-κB

NF-κB activation is a frequent occurrence in squamous cell carcinomas and there is strong evidence that it is important for transformation of epithelial cells (Huber et al., 2004). High-risk E6s are capable of activating NF-κB (D’Costa et al., 2012; Havard et al., 2005; James et al., 2006b; Nees et al., 2001; Yuan et al., 2005). The mechanism is not entirely clear although there is evidence that it may interact with the PDZ binding motif (James et al., 2006b). Another study indicated that hrE6 inactivates a deubiquitinase called CYLD that causes activation of NF-κB, particularly in conditions of hypoxia (An et al., 2008), and finally hrE6’s activate NF-κB through interaction with NFX1-91 (Xu et al., 2010). There is also evidence that both E6 and E7 from the cutaneous HPV-38 can activate NF-κB (Hussain et al., 2011). The activation of NF-κB leads to up regulation of cIAP2, an inhibitor of apoptosis, which would be expected to confer some resistance to certain DNA damaging agents (James et al., 2006b; Wu et al., 2010).

The consequences of NF-κB activation by E6 are apparently complex and may depend on cell type. A recent study indicated that NF-κB activation by E6 in ectocervical cells increases proliferation, whereas it may be inhibitory to growth of cells that are derived from the transformative zone, where most cervical cancers develop (Vandermark et al., 2012).

E6 effects upon apoptosis

Transgenic expression of HPV-16 E6 and E7 in the mouse eye lens induces tumor formation while expression of E7 induces apoptosis of the developing lens; apoptosis induced by 16E7 is only partially ablated by p53 null status, demonstrating that 16E6 possessed both p53 dependent and p53 independent anti-apoptotic functions (Griep et al., 1993; Pan and Griep, 1995). Both low and high risk mucosal E6s can bind to the pro-apoptotic protein Bak to cause its degradation (Thomas and Banks, 1999). Bak has homology to Bcl-2 and acts at the mitochondria but has an opposite effect of Bcl-2 with regard to apoptotic activation (Shamas-Din et al., 2011). Bak causes the release of cytochrome c and activation of the apoptotic caspase cascade. Bak is generally sequestered by Mcl-1 and Bcl-XL but is released upon DNA damage. E6’s ability to degrade Bak is independent of its p53 degradation function, although p53 activation is known to activate Bak. A more recent study indicated that E6s from multiple Beta papillomavirus types also interact with and cause the degradation of Bak (Underbrink et al., 2008). In the latter case, the degradation was found to be dependent on E6AP, however another study, also using Beta-papillomaviruses, did not observe a similar dependency (Simmonds and Storey, 2008). Proteomic studies have not detected specific interaction of E6 with Bak (White et al., 2012a). The conserved targeting of BAK by Alpha and Beta papillomaviruses implies a conserved structural feature of these E6 proteins that has yet to be delineated.

High risk HPV E6s can bind to procaspase 8 which can prevent E6-expressing cells from responding to apoptotic stimuli (Tungteakkhun and Duerksen-Hughes, 2008). 16E6 binding to procaspase 8 leads to a change in the ability of procaspase 8 to bind to itself or to FADD (Filippova et al., 2007). Interestingly, the small E6* isoform can also bind to procaspase 8, which seems to have an opposite effect of stabilizing it rather than accelerating its degradation; the full-length and E6* forms bind to different sites of procaspase 8 (Tungteakkhun et al., 2010).

E6 and Immune Response

Interferon treatment of HPV associated lesions has resulted in mixed results (Beglin et al., 2009). HPV proteins can modulate the response to interferon and in cells in which HPV has integrated, E6 and E7 are expressed at higher levels (i.e. in higher grade lesions) and are more resistant to the effects of interferon. Both E6 and E7 have been implicated in causing resistance to interferon (Beglin et al., 2009; Nees et al., 2001). The E6 proteins from both low and high-risk mucosal types are able to inhibit the interferon response. E6 causes down regulation of multiple interferon responsive genes (Nees et al., 2001). Both low and high risk mucosal E6s can bind to Tyk2 of the Jak-Stat pathway (Li et al., 1999). HrE6 binds to IRF-3 and inhibits its ability to activate interferon-responsive genes (Ronco et al., 1998). E6’s ability to interact with p53 and p300/CBP is also likely to play a role in interferon response regulation (Hebner et al., 2007). The cutaneous Beta HPV type 38 also interferes with the interferon pathway (Cordano et al., 2008) apparently by down modulating STAT-1 expression. Both E6 and E7 are involved in this process.

Other biological functions of the low risk E6

IP/MS experiments have shown the Alpha group low-risk E6 proteins interact with E6AP (Brimer et al., 2007) and proteasome subunits (Rozenblatt-Rosen et al., 2012; White et al., 2012a). Earlier reports have described cellular binding partners for low-risk E6 proteins, such as zyxin (Degenhardt and Silverstein, 2001a), GPS2 (Degenhardt and Silverstein, 2001b), MCM7 (Kuhne and Banks, 1998; Kukimoto et al., 1998). In addition, there are discordant observations that high-risk and low-risk forms of E6 can bind to p73 (Marin et al., 1998; Park et al., 2001). As discussed above, low risk E6 is required for episomal replication of low risk HPV-11 genomes (Oh et al., 2004).

GFP fusions to low risk Alpha E6 localize to the cytoplasm (Tao et al., 2003). There is evidence that low-risk E6’s bind to p53 but do not target its degradation. Transiently expressed low risk E6-GFP fusion proteins associate with p53 in the cytoplasm and retain p53 there (Sun et al., 2010) and induce p53 dependent apoptosis in HEK 293 or MCF7 cells. In contrast to these observations, examination of low-risk condylomas show abundant p53 expression in the nucleus and not the cytoplasm that co-localizes with p21Cip (Giannoudis and Herrington, 2000; Lassus and Ranki, 1996; Lyman et al., 2008). Recent proteomic analysis indicates that HPV-6b E6 interacts with p53 (White et al., 2012a). Thus, how interaction of low-risk E6’s with p53 affects its function is not entirely clear but may have to do with regulation of p53 acetylation as has been discussed.

Several recent publications have elucidated functions of the long-enigmatic Beta E6 proteins. Beta group HPV’s are cutaneous, and in normal persons are ubiquitous and produce unapparent cutaneous lesions. In the severely immune-compromised or in persons with the autosomal recessive condition epidermodysplasia verruciformis, Beta genus HPV produce visible flat warts that progress to squamous cell carcinomas in sun exposed areas (reviewed in (Orth, 2006)). This has given rise to a hypothesis that the Beta HPVs may predispose to the development of cutaneous squamous cell carcinomas by preventing the loss of UV damaged cells that harbor the virus. Correlating with this hypothesis, various Beta group E6 proteins have been shown to target the degradation of Bak, abrogate ATR activation, and block protein acetylation through the degradation of p300, all of which results in the persistence of UV induced DNA damage (Bedard et al., 2008; Giampieri et al., 2004; Howie et al., 2011; Jackson et al., 2000; Jackson and Storey, 2000; Simmonds and Storey, 2008; Underbrink et al., 2008; Wallace et al., 2012). Unlike squamous cell carcinomas caused by high risk Alpha HPVs, the genomes of the Beta HPVs are not typically found in cutaneous squamous cell cancer cell lines, and the ubiquity of the virus has made the many studies that detect Beta HPVs by PCR in cancers problematic.

Concluding Remarks

E6 proteins appear to be extraordinary: how can such a small protein do so much? Many protein interactions have been reported, all of which purport to connect in some way to altered cell physiology, but certainly such a small protein cannot have so many direct interaction partners! Sorting out which of the effects of E6 are direct, indirect, or fantasy is a challenge. A critical tool for the analysis of E6 phenotypes and interactions is E6 mutants, but many E6 mutants used in the past (such as deletion mutants) will turn out to be globally defective for core functions of E6 such as LXXLL interactions, making conclusions drawn from the use of these mutants now subject to new interpretation. Extending those observations to indirect E6 interactions that may underlie its protean phenotypes is the challenge for the future. The solved structure of BE6 and 16E6 will now allow a precise mapping of the functions of E6 to its structure, and also opens the door to the development of small molecule inhibitors.

Figure 4.

Ribbon diagram of BE6 bound to an LXXLL motif from paxillin. In the left panel, the amino-terminal zinc-binding domain (green) is shown at the top, the BE6-C domain in blue, and the helical LXXLL peptide (salmon color) is viewed on end through its axis. The interdomain connecting helix (yellow) is clearly seen in the middle panel, where the LXXLL peptide has been removed. The right panel is the same view as the middle panel but with the LXXLL peptide residing in the pocket.

Figure 7.

Diagram of the functional domains of E6AP. The locations of the nuclear receptor co-activation region, E6 LXXLL binding domain (aa 409 and another similar motif at 662) and HECT domain are illustrated. See text for details and references.

Table IV.

Biological Phenotypes of Alpha E6 Proteins

Biological Process	High-Risk HPV E6 (HPV 16, 18, or 31)	Low-Risk HPV E6 (HPV 6 or 11)	References
Maintenance of Viral Genomes	+	+	(Oh et al., 2004; Park and Androphy, 2002; Thomas et al., 1999)
Genome Amplification upon Differentiation	+		(Wang et al., 2009a)
Inhibition of p53 Transactivation	+	+	(Crook et al., 1991; Lechner and Laimins, 1994; Pim et al., 1994)
Inhibition ofp53 Acetylation	+	+	(Jha et al., 2010; Patel et al., 1999; Thomas and Chiang, 2005)
Bypass of Growth Arrest Upon DNA Damage	+		(Havre et al., 1995; Kessis et al., 1993; Song et al., 1998)
Induction of Genetic Instability	+		(Duensing and Munger, 2002; Liu et al., 2007; Plug Demaggio and McDougall, 2002; Schaeffer et al., 2004)
Immortalization of Human Cells (with Rb Inactivation)	+		(Hawley-Nelson et al., 1989; Hudson et al., 1990; Munger et al., 1989; Sedman et al., 1991; Woodworth et al., 1989)
Induction of Hyperplasia/Cancer in Transgenic Mice	+		(Nguyen et al., 2002; Riley etal., 2003; Shai et al., 2007; Simonson et al., 2005; Song et al., 2000)
Epithelial to Mesenchymal Transition/Invasion	+		(Krishna Subbaiah et al., 2012; Lopez-Ocejo et al., 2000; Watson et al., 2003)
NF-kappaB Activation	+		(An et al., 2008; James et al., 2006b; Nees et al., 2001; Yuan et al., 2005)
Telomerase Activation			(Gewin and Galloway, 2001; Gewin et al., 2004; Kiyono et al., 1998; Klingelhutz et al., 1996; Veldman et al., 2001; Xu et al., 2008)
Inhibition of Keratinocyte Differentiation	+	+/−	(Alfandari et al., 1999; Duffy et al., 2003; Nees et al., 2000; Sherman et al., 1997; Sherman and Schlegel, 1996)
c-Myc Activation	+		(Veldman et al., 2003)
Wnt Activation	+		(Bonilla-Delgado et al., 2012) (Lichtig et al., 2010)
Inhibition of Interferon Response	+	+/−	(Cordano et al., 2008; Nees et al., 2001; Ronco et al., 1998)
mTORC Activation	+	+	(Spangle et al., 2012; Spangle nd Munger, 2010)
miR Regulation	+		(Au Yeung et al., 2011; Martinez et al., 2008; McKenna et al., 2010; Wald et al., 2010)

Research Highlights.

1. E6 oncoproteins have two zinc-structured domains connected by an alpha helix.

2. The E6 protein fold is highly conserved in evolution.

3. E6 makes a pocket that binds to LXXLL peptides on target proteins.

4. E6 biological activities are mediated by E6-LXXLL interaction.