HPV Oncoproteins and the Ubiquitin Proteasome System: A Signature of Malignancy?

Human papillomavirus (HPV) E6 and E7 oncoproteins are critical for development and maintenance of the malignant phenotype in HPV-induced cancers. These two viral oncoproteins interfere with a plethora of cellular pathways, including the regulation of cell cycle and the control of apoptosis, which are critical in maintaining normal cellular functions. E6 and E7 bind directly with certain components of the Ubiquitin Proteasome System (UPS), enabling them to manipulate a number of important cellular pathways. These activities are the means by which HPV establishes an environment supporting the normal viral life cycle, however in some instances they can also lead to the development of malignancy. In this review, we have discussed how E6 and E7 oncoproteins from alpha and beta HPV types interact with the components of the UPS, and how this interplay contributes to the development of cancer.


Introduction
Papillomaviridae is a diverse family of small, non-enveloped DNA viruses, approximately 50-60 nm in diameter that infect all homoeothermic vertebrates including humans [1,2]. Interestingly, recent studies have also detected members of Papillomaviridae in fish [3]. Currently, there are known to be approximately 200 different human papillomavirus (HPV) types, which are classified in five genera (alpha, beta, gamma, mu and nu) [4]. The Alphapapillomavirus species (α-HPVs) preferentially infect oral or anogenital mucosa in humans and primates [2]. The α-HPVs are further classified as low-risk (LR) (e.g., HPV-6 and HPV-11) or high-risk (HR) (e.g., HPV-16, HPV-18, and HPV-33), based on their association with human cancers [1]. HPV infections are usually fairly rapidly cleared by the immune system-from a few months up to two years from initial viral entry. However, in some instances, the infections are not neutralized by the immune system and they persist for long periods, which can result in the development of different types of neoplasia [5]. LR HPV types cause self-limiting benign anogenital warts and are only rarely found in squamous intraepithelial lesions, possibly as a part of multiple infections [6,7]. On the other hand, HR HPVs have been recognized as the main causative agent of cervical cancer, with more than 600,000 new cases annually worldwide [1, 2,8,9]. HPV-16 and HPV-18 are associated with approximately 80% of cervical cancer globally, while the remaining 20% are linked to infection by other HR HPVs (e.g., HPV-31, HPV-33, HPV-45, and HPV-58) [1, 8,10].

HPV E6 Oncoprotein and the UPS
The HPV E6 oncoprotein contains around 150 amino acids and has two zinc fingers created by four CXXC motifs [65][66][67]. The integrity of these motifs is crucial for optimal oncoprotein function and they are highly conserved between all HPV E6 oncoproteins identified so far [68,69]. Recent studies have characterized the crystal structure of the intact E6 oncoprotein and also support the fact that E6 forms interactions with a large number of cellular substrates [67,[70][71][72][73]. Recent studies have characterized the crystal structure of the intact HPV-16 E6 oncoprotein and specific domains of HPV-18 E6, HPV-51 E6 and Bovine Papillomavirus 1 (BPV-1) E6, and these data all show that E6 forms interactions with a large number of cellular substrates [70,72,74]. HPV E6 complexes with these substrates through a number of conserved binding motifs. One of these is the so-called PDZ-binding motif (PBM), exclusively present on the C-terminus of HR HPV E6 oncoproteins, through which they bind to numerous PDZ-domain containing proteins [10,61]. Another conserved binding motif on E6 is the LXXLL binding motif. The most notable E6 targets with an LXXLL motif include E6AP, the preferred interacting partner of α-HPV E6 oncoproteins, and MAML-1, interacting with β-HPV E6 oncoproteins [75][76][77][78][79][80]. As mentioned above, E6 oncoproteins from both αand β-HPV types interact with multiple components of the UPS; these interacting partners and their corresponding cellular functions are summarized in Table 1. By interacting with UPS components, HPV E6 oncoproteins can modulate various cellular functions, which are shown in Figure 1 and will be discussed in more detail below.
Pathogens 2020, 9, x FOR PEER REVIEW 4 of 29 and they are highly conserved between all HPV E6 oncoproteins identified so far [68,69]. Recent studies have characterized the crystal structure of the intact E6 oncoprotein and also support the fact that E6 forms interactions with a large number of cellular substrates [67,[70][71][72][73]. Recent studies have characterized the crystal structure of the intact HPV-16 E6 oncoprotein and specific domains of HPV-18 E6, HPV-51 E6 and Bovine Papillomavirus 1 (BPV-1) E6, and these data all show that E6 forms interactions with a large number of cellular substrates [70,72,74]. HPV E6 complexes with these substrates through a number of conserved binding motifs. One of these is the so-called PDZ-binding motif (PBM), exclusively present on the C-terminus of HR HPV E6 oncoproteins, through which they bind to numerous PDZ-domain containing proteins [10,61]. Another conserved binding motif on E6 is the LXXLL binding motif. The most notable E6 targets with an LXXLL motif include E6AP, the preferred interacting partner of α-HPV E6 oncoproteins, and MAML-1, interacting with β-HPV E6 oncoproteins [75][76][77][78][79][80]. As mentioned above, E6 oncoproteins from both αand β-HPV types interact with multiple components of the UPS; these interacting partners and their corresponding cellular functions are summarized in Table 1. By interacting with UPS components, HPV E6 oncoproteins can modulate various cellular functions, which are shown in Figure 1 and will be discussed in more detail below. oncoproteins. E6 oncoproteins from α and β types interact with various components of the UPS and as speculated use them to modulate a number of cellular processes. By interacting with the UPS components high-risk (HR) α-type HPV E6s are involved in the regulation of all the processes shown above; low-risk (LR) α-type E6s are involved in regulation of viral life cycle, modulation of E6AP activity, and regulation of the immune response; β-type HPV E6s are involved in regulation of the viral life cycle, modulation of E6AP activity, regulation of the immune response, and E6 oncoprotein stability. Table 1. HPV E6 interactions with ubiquitin-proteasome system components. oncoproteins. E6 oncoproteins from α and β types interact with various components of the UPS and as speculated use them to modulate a number of cellular processes. By interacting with the UPS components high-risk (HR) α-type HPV E6s are involved in the regulation of all the processes shown above; low-risk (LR) α-type E6s are involved in regulation of viral life cycle, modulation of E6AP activity, and regulation of the immune response; β-type HPV E6s are involved in regulation of the viral life cycle, modulation of E6AP activity, regulation of the immune response, and E6 oncoprotein stability. A major ubiquitin-accepting proteasome subunit. Involved in maintaining structural integrity of the 19S regulatory particle. Important in direct and indirect recognition of ubiquitinated substrates of 26S proteasome by interacting with polyubiquitinated proteins and directing them to the proteasome for degradation. A critical controlling factor in regulation of protein degradation at the proteasome.
An E3-HECT domain-containing ubiquitin-protein ligase. It promotes its own degradation in vivo. This imprinted gene is maternally expressed in the brain and biallelically expressed in other tissues. It plays an important role in regulation of the circadian clock and acts as a regulator of synaptic development.

E6 Oncoprotein and Ubiquitin Ligases
HPV E6s interact with a number of cellular ubiquitin ligases. E6AP or UBE3A E3 ubiquitin-protein ligase is the principal ubiquitin ligase that associates with α-HPV E6 proteins. The interaction occurs via an LXXLL binding motif on E6 and leads to stimulation of E6AP ubiquitin ligase activity [81][82][83]. This association forms a stable complex between the viral oncoprotein and the ubiquitin ligase, which then targets a number of cellular substrates for proteasome-mediated degradation, with the p53 tumor suppressor being the most important cellular target [84]. This activity results in modulations of various cellular pathways to optimize the cellular environment for a productive viral life cycle, and in rare cases can initiate the process of carcinogenesis (Figure 1) [75,[84][85][86]. Furthermore, a series of studies have also indicated that the general transcriptional effects of E6 are mostly dependent on the presence of E6AP [87]. This observation was further analyzed and supported by more recent studies, which demonstrated that the stability of α-HPV E6 oncoproteins was strictly dependent on the presence of E6AP [47]. Since E6AP was initially identified through its interaction with HR HPV E6-16 and HPV-18 proteins, it was originally thought that this association was exclusive to HR HPV mucosotropic types [84,85], but further studies showed that a LR HPV type 11 E6 could also complex with E6AP [83,88]. In addition, in vitro proteomic analysis and mass spectroscopy of cutaneous HPV cellular binding partners has shown that E6AP is a pulldown partner of E6 proteins from cutaneous-specific HPV-10 (an α-HPV) and HPV-24 (a β-HPV), indicating that β-HPV types can also interact with E6AP [82]. Furthermore, β-HPV types 24 and 38 E6 were also shown to form a complex with E6AP [77,81,82,88]. Interestingly, the interaction of E6AP with HPV-10 and HPV-24 E6 oncoproteins also increases their cellular protein levels indicating a more general dependence of all HPV types on E6AP for the maintenance of appropriate levels of E6 within the cell [82].
HPV-16 E6 oncoprotein was also shown to interact with HERC2, another putative HECT domain-containing E3 ubiquitin ligase [46,89]. Interestingly, it was found that E6AP physically mediates HPV-16 E6 and HERC2 interaction, which, in turn, can modulate the ubiquitin ligase activity of E6AP [90]. Subsequently, it was also shown that HERC2 can bind to E6AP in the absence of E6 [91].
Follow-up studies have demonstrated that, besides HPV-16 E6, several other HPV E6 oncoproteins (from HPV types 33, 52, 18, 45, 6b and 17a), including HR and LR HPV types, as well E6 oncoproteins from certain cutaneous β-HPV types, also interact with HERC2 [77]. However, the role of HERC2 in the HPV life cycle, and its contribution to the process of HPV-induced malignancy have yet to be elucidated [90].
UBR5/EDD is another HECT domain-containing E3 ubiquitin ligase, alterations in which have been linked to carcinogenesis [92,93]. Studies have shown EDD to be bound strongly by HPV-18 E6, but only weakly by HPV-16 and HPV-11 E6, suggesting that the interaction between E6 and EDD may be restricted to HPV-18 E6. EDD protein alone can bind independently to both E6 and E6AP, and it is also involved in regulating the E6/E6AP complex [94]. It appears that EDD regulation of E6AP expression is independent of E6, but that loss of EDD stimulates the proteolytic activity of the E6/E6AP complex, significantly increasing the ability of the E6/E6-AP complex to direct the degradation of its cellular substrates, particularly p53. Thus fluctuations of EDD protein levels might directly affect the viral life cycle, and influence the development of HPV-induced malignancies [94]. In addition, it has also been shown that the association of EDD with HR HPV E6 is important in destabilizing TIP60; a histone acetyltransferase tumor suppressor, thus ultimately contributing to the development of HPV-induced malignancies [95].
The BARD1 protein is a RING heterodimer that interacts with BRCA1, providing the E3 ubiquitin ligase activity that is required for BRCA1's tumor suppressor function, as well as coordination of ubiquitination to maintain genomic stability [99,100]. BARD1 is a binding partner of HR HPV-16 and -18 E6, and it only interacts with these two HR HPV types, while no association was observed with LR HPV-11 E6 [101]. Furthermore, HR E6 oncoproteins were shown to interact directly with BRCA1 in both in vitro and in vivo conditions [102]. Interestingly, these interactions do not stimulate any degradation of either BRCA1 or BARD1, but the E6-induced increase in hTERT activity was considerably greater in the presence of BRCA1 than in its absence, suggesting that E6 possibly functions in part to antagonize the activity of BRCA1 [102]. However, it still remains to be clarified whether BRCA1 plays a role in the initiation of cervical cancer. In a recent study, Poirson et al. [38] using the Gaussia princeps luciferase protein complementation assay (GPCA), identified a number of cellular ubiquitin ligases or substrates with ubiquitin ligase activity as interacting partners of HPV E6 [38]. These studies assembled and screened a library of 590 cDNAs related to the UPS that covered about 50% of the human ubiquitination system, together with co-immunoprecipitation to confirm novel cell target protein-E6 interaction. Results indicated various new target proteins, including three RING-type Ub ligases MGRN1, LNX3 and LNX4 [38,103]. MGRN1 was shown to interact with E6 proteins from LR HPV-6 and HR HPV types 16, 18 and 33, as well as β-types HPV types 8 and 38. Interaction with LNX4 was restricted to HPV-16 E6, while LNX3 interacted with E6 oncoproteins from HR HPV types 16, 18 and 33 and β-HPV type 8 E6. The other ubiquitin ligases examined in this study were shown to exclusively bind HPV-16 E6, and they include ITCH, TRAF6, TRAF5, UBAC1, VHL, XIAP, RNF25 and RNF40 [38]. These ubiquitin ligases are associated with the regulation of several different cellular pathways, however, biochemical and mechanistic analyses to elucidate their roles in the viral life cycle and in HPV-mediated malignancies are still lacking [38,104].
Interestingly, there are also ubiquitin ligases that interact exclusively with β-cutaneous E6 oncoproteins, for example, HPV-17a and HPV-3 E6 which were reported to interact with 10 subunits (CNOT1, CNOT2, CNOT3, CNOT4, CNOT6L, CNOT7, CNOT9/RQCD1, CNOT10, C2orf29, and TNKS1BP1) of the Ccr4-Not multiprotein complex, which has been shown to be associated with various enzymatic activities including a ubiquitin ligase function [77,105]. White et al. detected those interactions by using an unbiased proteomic study including immunoprecipitation and mass spectrometry [77]. In addition, an E3 ubiquitin-protein ligase, UBR4/p600 was characterized as an interacting partner of cutaneous β-HPV-38 E6 [82], which has been shown to promote tumorigenesis in transgenic mouse models [28,106]. Moreover, this association was also observed but not validated in the proteomic analysis of another study [77]. Interaction between E6 and p600 was quite surprising, since p600 had been previously reported only as interacting partner of E7 [62,63], and more recent studies have shown that high risk E7 oncoproteins use p600 to target the PTPN14 tumor suppressor for proteasome-mediated degradation [59,107,108]. Therefore, it would be of a great interest to investigate the possible role of p600 in HPV-38-induced malignancy. Finally, a study by Holloway et al. [60] showed that β-HPV-5 E6 uses a specific and unique mechanism to target the proapoptotic factor BAK by recruiting the HECT domain-containing E3 ubiquitin ligase HERC1, which binds the apoptosis-promoting BAK protein in UV-damaged, E6-expressing cells. HERC1 specifically targets BAK in its active conformation, and only in the presence of E6, suggesting that HPV-5 E6 interacts with HERC1 to redirect its activity towards activated BAK, specifically to abrogate apoptosis of the infected cell and maintain the virus lifecycle [60].

E6 Oncoprotein and Deubiquitinating Enzymes
Apart from ubiquitin ligases, there are also other elements of the UPS, such as DUBs, that have been shown to interact with E6 oncoproteins from αand β-HPV types. One of these is CYLD lysine 63 (K63) deubiquitinase, a negative regulator of the NF-κB pathway, which was shown to form complexes with HR HPV-16 and -18 E6, although there was no evidence for direct interaction [109]. Nonetheless, a study demonstrated that CYLD polyubiquitination and degradation under hypoxic conditions was E6-mediated. This implies that this mechanism, which E6 employs under hypoxic conditions, is likely to contribute to an aggressive microenvironment in tumors caused by HPVs; it also suggests that prolonged hypoxia-induced NF-κB activation may be specific only to HPV-induced cancers, such as cervical and head-and-neck cancers [109].
HPV E6 oncoproteins were further reported to interact with two other DUBs: ubiquitin-specific protease (USP) enzymes, USP15 and USP46 [38]. USP15 was shown to be bound by E6 proteins from α-HPV types 16, 18, 33 and 6, and β-HPV types 8 and 38. Biochemically it was demonstrated that USP15 was involved in the regulation of HPV-16 E6 protein stability [98]. Furthermore, HPV E6 protein forms a ternary complex with USP15 and TRIM25, resulting in the inhibition of immune surveillance and antiviral responses, thereby exhibiting a direct involvement in immune system regulation [98]. Recently, it was demonstrated that HR HPV-16, -18 and -31 E6 bind to USP46, but that no interactions were seen with the LR HPV types [110]. Another study also showed that USP46 is required for the proliferation of HPV-transformed cancers and derived cancer cells, indicating that USP46 can be essential for the survival of HPV-transformed cancers [110].

E6 Oncoprotein and the Proteasome
HPV E6 oncoproteins from αand β-types were also shown to bind directly to the proteasome. In vitro studies using GST-pull down assays have demonstrated that HR HPV-18 E6 interacts with several proteasomal subunits, including PSMC1, PSMC5, PSMD2, PSMC3, PSMC4, and PSMC2, while under the same conditions HR HPV-16 and LR HPV-11 E6 interacts with both subunits PSMD2 and PSMC1 [111]. Intriguingly, these interactions seem to be independent of E6AP, but the presence of E6AP does appear to be required for E6 to interact with PSMD4. This tripartite complex enhances the ubiquitination of PSMD4, an essential subunit of the 19S proteosome regulatory complex [111]. The HPV E6 oncoproteins from many HPV types can interact with diverse proteasome subunits, albeit they differ in their interaction profiles and their preferences for binding different proteasome subunits. These observations were supported by a series of analyses that demonstrated interactions between multiple α-HPV E6 oncoproteins and PSMA3, PSMC2, PSMC3, PSMD1, PSMD2, PSMD3, PSMD14, PSMD11, PSMD7, PSMC4, PSMD13, PSMD6, PSMD8, PSMB7, PSMB9 and PSME4 proteasomal subunits, while a panel of β-HPV E6 oncoproteins interacted only with a few proteasomal subunits, including PSMA3, PSMC2, PSMD1, PSMD2, PSMD3, PSMD11, and PSMD13 [77,112,113]. Those analyses included different methods, including affinity chromatography [113], immunoprecipitation and mass spectrometry [77], as well as the yeast two-hybrid (Y2H) system and tandem affinity purification [112]. Hence, E6 proteins from αor β-HPVs exhibit different affinities of binding to various proteasomal subunits [77]. These observations were additionally confirmed by several other studies [90,[113][114][115][116][117], suggesting the importance of the close association of E6 with the proteasome, probably through E6AP which directly connects E6 to the proteasome [77].

HPV E7 Oncoprotein and the UPS
HPV E7 oncoprotein is a small acidic protein of approximately 100 amino acid residues, having no significant sequence similarities to any cellular protein, except for the LXCXE motif. The amino terminus contains two regions similar to the E1A adenovirus proteins: the conserved region 2 (CR2) and part of CR1 [65]. The E7 oncoprotein also contains CR3, a zinc-binding site formed by two CXXC domains that function as a dimerization domain [118]. The CR3 domains are similar to those of the HPV E6 protein, suggesting that a genetic duplication may have occurred early in HPV evolution [119]. E7 has, in addition, sequences related to those of simian vacuolating virus 40 large tumor antigen (SV40 T), all contributing to the transforming activities of HR HPV E7 oncoproteins [120][121][122][123].
The main function of the HPV E7 oncoprotein is to maintain the infected differentiating cell in a DNA replication-competent state, which it does in part, by targeting the retinoblastoma tumor suppressor (pRb), a critical regulator of cell cycle progression, which controls the G1 to S-phase transition [124]. E7 also binds to the pRb-related members of the pocket protein family, p107 and p130, which assists in driving the cell cycle of the differentiating HPV-infected epithelial cell into an S-phase-like state, an environment suitable for replication of the viral genome [125]. In order to complete these activities, E7 forms interactions with ubiquitin ligases, which are essential elements of the UPS. In addition, E7 was shown to complex with other components of the UPS, and these interactions appeared to be important either for the stability of the oncoprotein or for its role in regulation and modulation of many cellular processes, some of which are summarized in Table 2 and Figure 2. suppressor (pRb), a critical regulator of cell cycle progression, which controls the G1 to S-phase transition [124]. E7 also binds to the pRb-related members of the pocket protein family, p107 and p130, which assists in driving the cell cycle of the differentiating HPV-infected epithelial cell into an S-phase-like state, an environment suitable for replication of the viral genome [125]. In order to complete these activities, E7 forms interactions with ubiquitin ligases, which are essential elements of the UPS. In addition, E7 was shown to complex with other components of the UPS, and these interactions appeared to be important either for the stability of the oncoprotein or for its role in regulation and modulation of many cellular processes, some of which are summarized in Table 2 and Figure 2. oncoproteins. E7 oncoproteins from α and β types interact with various components of the UPS and are thought to use them to modulate a number of cellular processes. By interacting with the UPS components, high-risk (HR) α-type E7s are involved in the regulation of all the processes shown above; low-risk (LR) α-type E7s are involved in cell migration, apoptosis, regulation of the immune response, viral life cycle modulation and E7 protein turnover; β-type E7s are involved in cell migration, apoptosis, endosomal trafficking and regulation of the immune response. Table 2. HPV E7 interactions with ubiquitin-proteasome system components.   Regulates the activation of NF-κB and JNK and plays a central role in the regulation of cell survival and apoptosis. An essential constituent of several E3 ubiquitin-protein ligases. HPV16 E6/E7 switch cells from apoptotic to proliferative fates under TWEAK/Fn14 interaction, possibly by favoring Ras and TRAF2 activation and modulating TNF receptor expression. An E3 ubiquitin-protein ligase, which self-ubiquitinates in cooperation with E2 enzyme UBE2D2/UBC4. Serves as a targeting signal for proteasomal degradation.  Also known as p600. An E3 ubiquitin-protein ligase that recognizes proteins with specific destabilized N-terminal residues, leading to their ubiquitination and degradation. May mediate some pRBindependent transforming activities of HPV-16 E7, but is not sufficient for cellular transformation as interactions were also found with low-risk HPV E7 oncoproteins. It is speculated that UBR4 could potentially play a role in viral replication.

E7 Oncoprotein and Ubiquitin Ligases
A large number of ubiquitin ligases have been reported to be HPV E7 interactors, a principal one being cullin 2 (CUL2), a core component of the cullin-RING-based ESC (ElonginBC-Cullin-SOCS-box) E3 ubiquitin-protein ligase complex [126,127]. This interaction occurs via the E7 CR1 domain, as well as through its C-terminal sequences, and drives cell cycle progression by degradation of pRb and upregulation of CDK2 and cyclins A and E [58,128]. In fact, interactions between E7 and CUL2 complex were validated for 17 different HPV types from both the α-(HPV-16, -18, -31, -33, -45, -6b, -55, -74, -2a, and -57) and β-genus (HPV-8, -25, -98, -17a, -38, -76, and -92) HPV types. Furthermore, it has been shown that HPV-16 E7-expressing cells require ZER1 (or Zyg11BL), a substrate-specificity factor for a CUL2-RING ubiquitin ligase [57,129]. ZER1 is required for HPV-16 E7 to bind CUL2 and destabilize pRb, suggesting that HPV-16 E7 exploits the CUL2-ZER1 complex for pRb degradation. Interestingly, the ZER1 association appears to be specific only to HPV-16 E7. This may be related to the fact that ZER1 contains BC and a CUL2-binding site and acts in a complex with elongin B, elongin C, and CUL2, while HPV-16 E7 interacts with CUL2, and elongin C is required specifically for the binding of ZER1 to CUL2 [58,130]. Thus, all three proteins interconnect through a number of possible protein interactions.
E7 oncoprotein, similarly to E6, generally has a short half-life, approximately one hour [124]. It has been shown that E7 is ubiquitinated by the UBE2L3/Cullin 1 (CUL1) complex, followed by degradation at the proteasome and, to date, this has been observed only with HPV-16 E7 (122)(123)(124). CUL1 is a core component of multiple cullin-RING-based SKP1-CUL1-F-box proteins (SCF) E3 ubiquitin-protein ligase complexes, which were previously described not only to degrade E7 itself, but also to cause destabilization and degradation of the p130 pocket protein through its interactions with E7 [131][132][133][134][135]. Therefore, E7 interacts with the SCF ubiquitin ligase complex containing CUL1 and Skp2 and can be ubiquitinated by the CUL1-containing ubiquitin ligase in vitro and in vivo [131]. Finally, the half-life of E7 was found to be significantly longer in Skp2 (-/-) mouse embryo fibroblasts (MEFs) than in wild-type MEFs. Interactions were also observed between cullin 3 (CUL3) and E7 from HPV types 16, 18, 6b, 55 and 8, with a suggestion that the E7 and CUL3 may not interact directly, but probably via some BTB proteins [57]. In addition, E7 protein turnover is shown to be driven by ubiquitin-dependent proteolysis and the process itself is regulated by the two E2 ubiquitin-conjugating enzymes, UbcH7 and UBE2A [38,131]. While there are no solid reports about UbcH7 functions, it is known that UBE2A plays roles in a number of different biological processes and its binding partners include KCMF1 and UBR4/p600 [136]. Their roles in HPV E7 modulations of various cellular functions will be discussed in more detail further in the review.
The HPV-16 E7 protein binds to eleven POZ/BTB (Pox virus and Zinc finger/Bric-a-brac Tramtrack Broad complex) domain-containing proteins (BTBD15, KCTD13, NAC-1/NACC-1, TNFAIP1, SHKBP1, ZBTB9, ZBTB20, ZBTB32, ZBTB42, ZBTB43 and ZBTB48) [38]. The functions of these BTB proteins are largely unknown, and a number of different functional roles have been reported for the POZ/BTB domain, including transcriptional repression [137,138], cytoskeleton regulation [139,140], and protein ubiquitination [141][142][143]. POZ/BTB proteins interact with the CUL3-SCF-like E3 ubiquitin ligase complex, allowing the POZ/BTB domain to recruit substrate molecules for ubiquitination by the CUL3 component of the SCF-like complex [126,144,145]. Furthermore, some BTB domain-containing proteins have been identified as substrate adaptors of certain CUL3-RING Ub ligases, such as tumor necrosis factor α-induced protein 1 (TNFAIP1) and the homologous KCTD13 [146]. In addition, some BTB proteins also have a C2H2-type zinc finger (ZBTB9, ZBTB32, BTBD15, ZBTB48), which is frequently found in transcriptional activators, and this is consistent with E7 being a major transcriptional regulator [147]. The majority of these UPS proteins have only been characterized as E7 interacting partners, and importantly, the biological consequences of these interactions have yet to be investigated ( Table 2). For example, NAC-1 is a POZ/BTB domain-containing transcriptional repressor protein related to tumor recurrence, and is essential for tumor growth and survival. The interaction between the BTB/POZ domains of NAC-1 is critical for tumor cell proliferation [143,148]. NAC-1 has been reported to be highly expressed in a number of human carcinomas, and, especially interestingly, NAC-1 is overexpressed in oral squamous cell carcinoma cells from different oral lesions [149,150]. Furthermore, HPV infection induces cytokine production, with TNF-α and related cytokines inducing various signaling pathways leading to growth arrest, proliferation, or cell death, but can also cause the expression of TNFAIP1, another POZ/BTB domain-containing protein. HPV-16 E7 oncoprotein was found to bind TNFAIP1, leading to TNF-α-mediated apoptosis [151].
Moreover, HPV E7 was found to interact with TNF receptor-associated factors (TRAFs), a family of E3 ubiquitin ligases associating with the TNF receptor superfamily, and contributing to the activation of NF-κB and MAP kinases [152]. TRAF2, an anti-apoptotic protein and an essential constituent of several E3 ubiquitin-protein ligases complexes, was shown to interact with HR HPV types 16, 18 and 33 E7 oncoproteins and also with β-HPV types 8 and 38, while no interaction was detected for LR HPV E7 oncoproteins [38]. A later study determined that HPV-16-infected keratinocytes exhibited an increase in TRAF2 expression and, more specifically, TNF receptor profile changes from type 1 to type 2, as well as the cells being more resistant to TNF-α induction of apoptosis [153]. This is probably responsible, at least in part, for the switch from an apoptotic to a proliferative fate, seen in HPV-16 infected keratinocytes [153]. Conversely, a unique adaptor protein and ubiquitin ligase, TRAF3, was found to interact only with HR HPV types 16 and 18 E7 oncoproteins [38]. A further study showed that increased expression of TRAF3 enhances p53 and pRb expression and decreases HPV E6 expression in HPV-positive cells, thus inhibiting cell growth, colony formation, migration, and enhances susceptibility to TNF-α and cisplatin-induced cell death [154]. These findings suggest that TRAF3 might have a tumor suppressor role, modulating established cancer hallmarks in HPV-infected cells [154]. Although interactions of TRAF4 and TRAF5 with E7 were detected, their biological effects on the host cell are yet to be studied [38]. TRAF5 is also an interacting partner of HR E6, as well as interacting with the E7 of all HPV types examined, including HPV-16, -18, -33, -6, -8 and -38.
Some other ubiquitin ligases that are involved in pathogen recognition and which associate with E7 are members of the TRIM family [38]. This comprises a number of proteins involved in many biological and antiviral processes, and E7 oncoproteins from many different HPV types interact with TRIM9, TRIM22, TRIM32 and TRIM72; some of these interactions have been further characterized and confirmed by co-immunoprecipitation, including that between E7 and TRIM32 [38]. TRIM32 is a RING finger Ub ligase implicated in innate immunity and has potential to bind E7 from HR α-HPV-16, -18 and -33, LR HPV-6, and also β-HPV types 8 and 38. TRIM72 has demonstrated the potential to bind E7 oncoproteins from certain HR α-HPVs (HPV-16, -18, and -33) and β-HPVs (HPV-8, and -38), but not a LR HPV-6 E7 oncoprotein [38]. Only TRIM22 has been fully characterized and seems to be downregulated in HPV-16-and HPV-18-positive cervical cancers [155].

E7 Oncoprotein and Deubiquitinating Enzymes
E7 oncoprotein also interacts with some DUBs; amongst these are USP26 and USP33, which are ubiquitin carboxyl-terminal hydrolases known to be involved in the ubiquitin-dependent proteolytic degradation of proteins via the 26S proteasome. Both USP26 and USP33 were identified as interactors of HR HPVs (HPV-16, -18 and -33), but not of β-HPVs (HPV-8 and -38), and have not so far been investigated in detail [38]. The USP29, a DUB implicated in thiol-dependent hydrolysis, also interacts with HR HPV-16, -18 and -33 E7 oncoproteins [38]. USP11, which belongs to a class of DUB that cleaves polyubiquitin chains, and thus inhibits the proteasome-mediated degradation of target proteins, is on the other hand, the only USP component so far shown to be bound by E7. USP11 increases the steady state levels of HPV-16 E7 by attenuating E7 ubiquitination and degradation, thus protecting E7 as well as influencing its effects on cell proliferation [156]. This interaction results in an increased stability and prolonged half-life of E7, but also suggests its requirement for modulation of downstream target proteins, subsequently affecting the biological function of E7, as well as abrogating its contribution to cell transformation.

E7 Oncoprotein and Proteasome Components
Recent studies have shown that the levels of E7 protein in cancer cells are regulated by ubiquitin-dependent proteolysis via the 26S proteasome [132,135,157]. Furthermore, E7 seems to interact with the 26S proteasome subunit 4 (S4) ATPase via E7's carboxyl-terminal zinc binding motif; the interaction thus being independent of the E7-pRb interaction. Therefore, E7 might target pRb for degradation by directly interacting with the 26S proteasome through S4. Moreover, E7 increases the ATPase activity of S4 and this same pathway was shown to be used by E7 in degrading pRb, with tumorigenic effect [135,157].

Concluding Remarks
The interplay between E6/E7 oncoproteins from various HPV types and the UPS has been shown to be one of the critical strategies used by these viruses to create an optimal environment in which they can successfully replicate. However, this process, which is generally highly regulated and controlled through the various stages of the viral life cycle, can in some instances be perturbed, initially causing an imbalance in cellular homeostasis, and ultimately resulting in the transformed cell phenotype. As can be seen from Figures 1 and 2, by interacting with the components of the UPS, viral oncoproteins modulate numerous cellular functions, which are also necessary for initiation and/or maintenance of the transformed cellular phenotype. Interestingly, of the large number of αand β-HPV types that infect humans, only a small proportion are actually associated with human malignancies. Intriguingly, as indicated in Tables 1 and 2, it appears that E6/E7 oncoproteins from HR HPVs, which are associated with various human cancers at different anatomical sites, have been reported to interact with a vast number of the UPS components, including a number of ubiquitin ligases. In contrast, E6/E7 from β-HPVs, which are characterized as co-factors in causing skin cancers under specific conditions, interact with significantly fewer components of the UPS, while E6/E7 from LR types interact with only a few components of the UPS. All of this suggests that HR HPVs may be more efficiently adapted to evade