Human Papillomavirus Oncoproteins and Ubiquitin Proteasome System

Human Papillomavirus Oncoproteins and Ubiquitin Proteasome System: Comparison

Please note this is a comparison between Version 1 by Josipa Skelin and Version 2 by Peter Tang.

Papillomaviridae is a diverse family of small, non-enveloped DNA viruses, approximately 50–60 nm in diameter that infect all homoeothermic vertebrates including humans. 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.

E6
E7
HPV
cervical cancer
proteasome
UPS
ubiquitin ligases
ubiquitin

1. 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][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.

It is believed that HPVs infect cells in the basal layer of stratified squamous epithelia, exposed as a result of small abrasions and micro-wounds ^[5][6][20,21]. In normal epithelium, epithelial cells in the basal layer are proliferative, while the differentiating cells in the suprabasal layers have exited the cell cycle. The HPV life cycle is dependent upon the replicative machinery of basal cells, which have the potential to proliferate. Following infection, HPV genomes are established as extrachromosomal elements or episomes. Transition to the late stage of HPV infection is followed by the expression of late genes and virion production ^[5][7][20,22].

2. The Ubiquitin Proteasome System

The Ubiquitin Proteasome System (UPS) is the pathway responsible for the majority of intracellular protein degradation in eukaryotic cells. It controls the turnover of thousands of short-lived, regulatory, damaged and misfolded proteins, in order to regulate and maintain various cellular functions and cellular homeostasis ^[8][32]. It is a highly specific process and involves the addition of ubiquitin molecules to cellular substrates, which consequently leads to their modification via various cellular pathways. Fundamental cellular signaling pathways that are regulated by ubiquitination include cell cycle control, cell survival, cell proliferation, transcription, DNA repair, apoptosis, cellular metabolism, protein quality control, and membrane trafficking, as well as ubiquitination being very important in the immune response ^[9][10][11][33,34,35]. Ubiquitination, which involves a complex interplay of ubiquitinating and deubiquitinating enzymes (DUBs), is an essential element of control by the UPS ^[12][13][36,37]. Ubiquitin is transferred to target proteins by the ubiquitination cascade: first, ubiquitin is activated and bound by an E1 ubiquitin-activating enzyme, then transferred to an E2 ubiquitin-conjugating enzyme and, finally, transferred to lysine residues on the target protein by E3 ubiquitin-protein ligase ^[12][13][36,37]. So far, studies have identified two E1 activating enzymes, 40 E2 conjugating enzymes, around 800 E3 ubiquitin-protein ligases and about 100 DUBs ^[14][38]. While all components of the UPS are of great importance for ubiquitination, individual ubiquitin-protein ligases may be valuable viral targets as they are involved in substrate recognition, and hence define the specificity of the system. They are categorized into two main families: the numerous Really Interesting New Gene (RING) finger E3 ubiquitin ligases; and the less common Homologous to E6-associated protein C-Terminus (HECT) domain E3 ligases ^[15][16][39,40]. Polyubiquitin chains attached to substrates via lysine 48 will result in target protein degradation at the proteasome, while polyubiquitin chain attachment to other lysines will modulate proteasome-independent activities ^[17][41]. Proteins can also be mono-ubiquitinated, and this influences processes such as endocytosis and vesicular sorting ^[18][42]. On the other hand, ubiquitin molecules are cleaved from substrates by DUBs ^[19][43], which play essential roles in cellular processes as diverse as DNA replication, gene silencing, and endocytosis, ultimately affecting cell growth and oncogenesis.

3. HPV and the UPS

Many human pathologies, such as inflammatory and neurodegenerative diseases and certain cancers are, directly or indirectly, promoted by the deregulation of the UPS ^[8][20][32,44]. An intriguing feature of both HPV oncoproteins, E6 and E7, is their ability to direct many of their cellular substrates for proteasome-mediated degradation ^[6][21][21,45]. Those interactions were either shown or confirmed using methods like co-immunoprecipitation, Glutathione-S-transferase (GST)-fusion protein pull-down, affinity chromatography, Yeast Two Hybrid Assay and/or Tandem Affinity Purification and Gaussia princeps luciferase protein complementation assay. HPV E6 and E7 proteins appear to have evolved various strategies to make use of the ubiquitin system to support the viral lifecycle ^[21][22][23][45,46,47]. In particular, to avoid apoptosis, E6 mainly targets p53 while, by targeting the pRb, p107 and p130 pocket proteins, E7 induces cell cycle progression ^[24][25][26][48,49,50]. E6 proteins from high-risk ^[27][51] and low-risk ^[28][29][52,53] HPVs are able to bind p53, and it was further shown that this binding promotes the degradation of p53 via the ubiquitin pathway ^[24][48]. Interestingly, E6 proteins from both high- and low-risk HPVs were also shown to have the capacity to bind p53 but without inducing its degradation ^[30][31][54,55]. Furthermore, a recent study showed that in the case of beta HPVs, HPV17a, HPV-38, and HPV-92 E6s could bind and/or stabilize p53 ^[32][56]. E6 and E7 oncoproteins inactivate the majority of these cellular substrates by interacting with components of the UPS, ultimately inducing their degradation at the proteasome. Among the diverse components of the UPS, the interplay between E6/E7 and ubiquitin ligases is one of the crucial aspects in this process. The best characterized ubiquitin ligases used by the two oncoproteins are E6-associated protein (E6AP) and the cullin-2 ubiquitin ligase complex ^[33][57]. E6AP complexes with E6 and is involved in targeting p53 and some other targets, while E7 uses the cullin-2 ubiquitin ligase complex to target pRb ^[24][34][48,58]. To date, this interaction has only been shown for HPV16 E7 ^[33][57]. There are also additional ubiquitin ligases and other components of the UPS, which are directed for similar activities by the viral oncoproteins ^{[22][35][36][37][38][39]}[46,59,60,61,62,63]. Furthermore, HPV oncoprotein interactions with the UPS are not only critical for successful viral replication, but are also necessary for maintenance of the transformed phenotype ^[21][40][15,45]. Studies have shown that interference with components of the UPS has a down-regulatory effect on the ability of HPV-transformed cells to proliferate indefinitely and to avoid apoptosis ^{[22][23][38][39][41]}[46,47,62,63,64]. Therefore, a better understanding of the interplay between HPVs and the UPS would provide important novel information and greatly improve theour understanding of the viral life cycle and the process of HPV-induced tumorigenesis.

4. 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 ^[42][43][44][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 ^[45][46][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 ^{[44][47][48][49][50]}[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 ^[47][49][51][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 ^[37][52][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 ^{[53][54][55][56][57][58]}[75,76,77,78,79,80]. As mentioned above, E6 oncoproteins from both α- and 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.

Figure 1. The Ubiquitin Proteasome System (UPS) dependent activities of α- and β-HPV E6 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.

	Gene ID		Gene			Protein			Function				β-HPV	Method		Ref.
				LR			HR
	580		BARD1			Breast Cancer 1 Gene (BRCA1)- associated RING domain protein 1			Putative tumor suppressor gene mutated cancers. Homologous to BRCA1 RING motif and BRCT domain. BARD1/BRCA1 heterodimer is disrupted by tumorigenic amino acid substitutions in BRCA1. Heterodimer is required for BRCA1 tumor suppression and increases stability of both proteins.		−	+	?	Yeast-two hybrid, co-IP		Yim et al. (2007)
	672		BRCA1			Breast Cancer type 1 susceptibility protein			Tumor suppressor. The E3 ubiquitin-protein ligase component of BARD1/BRCA1 heterodimer. BRCA1/BARD1 heterodimer coordinates DNA damage repair, ubiquitination and transcriptional regulation to maintain genomic stability.		?	+	?	IP, GST-pull down		Zhang et al. (2005)
	4850		CNOT4			CCR4-NOT transcription complex subunit 4			E3 ubiquitin-protein ligase, promoting degradation of target proteins. Involved in JAK/STAT activation		−	−	+	IP, MS		White et al. (2012)
	1540		CYLD			CYLD lysine 63 (K63) DUB			A lysine 63 (K63) deubiquitinase. Tumor suppressor negatively regulating NF-κB pathway. Ubiquitinated and degraded during Hypoxia-induced NF-κB activation to relieve its inhibition of NF-κB signaling cascade		?	+	?		An et al. (2008)
	8925		HERC1			HECT and RLD domain containing E3 ubiquitin protein ligase family member 1			An E3 ubiquitin-protein ligase accepts ubiquitin from an E2 ubiquitin-conjugating enzyme and then transfers the ubiquitin to targeted substrates. Involved in membrane trafficking.		−	−	+	IP, proximity ligation in situ assay
?

	IP		Berezutskaya and Bagchi (1997); Ben-Saadon et al. (2004)
	84282		RNF135			ring finger protein 135			An E2-dependent E3 ubiquitin-protein ligase, involved in innate immune defense against viruses.		+, <	+	−	GPCA, co-IP		Poirson et al. (2017)
	57630		SH3RF1			SH3 domain containing ring finger 1			Has an E3 ubiquitin-protein ligase activity. In the absence of an external substrate, it can catalyze self-ubiquitination.		+, <	+	?	GPCA		Poirson et al. (2017)
	92799		SHKBP1

IP


White et al. (2012); Thomas et al. (2013)


51366


UBR5


Ubiquitin- protein ligase E3 component n-recognin 5


Also known as EDD (E3 identified by Differential Display). A HECT domain-containing E3 ubiquitin-protein ligase component of the N-end rule pathway. Involved in coordinating the balance between cell cycle progression and differentiation. A regulator of DNA damage response, acts as a guard against excessive spreading of ubiquitinated chromatin at damaged chromosomes, as well as tumor suppressor. Frequently overexpressed in breast and ovarian cancer.


−		+			+			co-IP, MS		Tomaić et al. (2011); White et al. (2012)
	9958		USP15			Ubiquitin specific peptidase 15			A deubiquitinating enzyme of the ubiquitin specific protease (USP) family. Plays a critical role in ubiquitin- dependent processes through polyubiquitin chain disassembly and hydrolysis of ubiquitin- substrate bonds.		−	+	+	Yeast-two hybrid, GPCA, IP		Vos et al. (2009); Poirson et al. (2017); Yaginuma et al. (2018); Chiang et al. (2018)
	64854		USP46			Ubiquitin carboxyl- terminal hydrolase 46			A deubiquitinating enzyme that plays a role in behavior by regulating GABA action. Has little intrinsic deubiquitinating activity and requires interaction with regulator of deubiquitinating complexes WDR48 (WD repeat-containing protein 48) for high activity.		−	+	?	co-IP		Kiran et al. (2018)
	7428		VHL			Von Hippel-Lindau tumor suppressor			VHL is a component of the protein complex that includes elongin C, elongin B, and cullin-2. An E3 ubiquitin-protein ligase involved in the ubiquitination and degradation of hypoxia- inducible factor (HIF), a transcription factor crucial to oxygen-related gene expression.. HPV16 E6 promotes hypoxia- induced Warburg effect through blocking the association of HIF-1α and VHL		?	+	?	GPCA		Poirson et al. (2017); Guo et al. (2014)
	331		XIAP			X-linked inhibitor of apoptosis			A multi-functional protein that regulates apoptosis, modulates inflammatory signaling and immunity, cell proliferation, cell invasion and metastasis. An E3 ubiquitin-protein ligase regulating NF-κB signaling and other target. An important regulator of innate immune signaling via regulation of Nod-like receptors (NLRs).		?	+	?	GPCA		Poirson et al. (2017)

+ confirmed to interact; +, < confirmed interactions with lower affinity compared to hR-HPV E6; - interactions not detected; ? interactions not tested. Co-IP: co-immunoprecipitation; GPCA: Gaussia princeps luciferase protein complementation assay, IP-MS/MS: immunoprecipitation-mass spectrometry, IP: immunoprecipitation.

5. 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 ^[42][65]. The E7 oncoprotein also contains CR3, a zinc-binding site formed by two CXXC domains that function as a dimerization domain ^[59][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 ^[60][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 ^{[61][62][63][64]}[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 ^[65][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 ^[66][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.

Figure 2. The Ubiquitin Proteasome System (UPS) dependent activities of α- and β-HPV E7 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.

	Gene ID			Gene			Protein			Function				α-HPV					β-HPV			Method			Ref.
					LR			HR
	8454			CUL1			Cullin-1			A core component of cullin-RING-based SCF E3 ubiquitin- protein ligase complex, which mediates ubiquitination and proteolysis of E7.			+			+			?			IP			Münger et al. (1989); Boyer et al. (1996); Oh et al. (2004)
	8453			CUL2			Cullin-2			A core component of cullin-RING-based ECS E3 ubiquitin- protein ligase complex. Stabilizes APOBEC3A.		Required for E7-induced degradation of pRB contributing to cell transformation by dysregulating G1/S cell cycle checkpoints			−			+			-			co-IP			Huh et al. (2007); Narisawa-Saito and Kiyono. (2007); White et al. (2012); Xu et al. (2016); Westrich et al. (2018)
	8452			CUL3			Cullin-3			A core component of cullin-RING-based BCR E3 ubiquitin- protein ligase complexes, which mediate the ubiquitination and proteasomal degradation of target proteins.			+			+			?			co-IP			White et al. (2012); Poirson et al. (2017)
	90379			DCAF15			DDB1 and CUL4 associated factor 15			May be involved in ubiquitination and degradation through a DBB1-CUL4 E3 protein-ubiquitin ligase.			−			+			−			GPCA, co-IP			Poirson et al. (2017)
	253980			KCTD13			Potassium channel tetramer- ization domain 13			A substrate-specific adapter of a BCR E3 ubiquitin-protein ligase complex required for synaptic transmission.			+			+			+			Holloway et al. (2015)
GPCA, co-IP			Chen et al. (2009); Poirson et al. (2017)			8924
	112939		HERC2				NACC1		HECT and RLD domain containing E3 ubiquitin- protein ligase 2				Nucleus accumbens- associated protein 1		A putative HECT domain E3 ligase. Involved in protein trafficking and degradation pathways regulating ubiquitin-dependent retention of repair proteins on damaged chromosomes. Recruited to sites of DNA damage in response to ionizing radiation. Promotes DNA damage-induced formation of ‘Lys-63’-linked ubiquitin chains.			A transcriptional repressor and transcriptional corepressor in neuronal cells through recruitment of HDAC3 and HDAC4 Required to recruit the proteasome from the nucleus to the cytoplasm and dendritic spines.		<+			?		+			+		<+				?		IP, MS				GPCA		Vos et al. (2009); White et al. (2012)
	Poirson et al. (2017)			83737			ITCH
	9148			NEURL1		E3 ubiquitin- protein ligase Itchy			Neuralized E3 ubiquitin- protein ligase 1		An E3 ubiquitin-protein ligase which is involved in the control of inflammatory signaling pathways. An essential component of an ubiquitin-editing protein complex ensuring the transience of inflammatory signaling pathways by regulating ubiquitin-dependent signaling events. Involved in the cellular antiviral response.			An E3 ubiquitin- protein ligase that activates in vitro ubiquitination of JAG1, inhibiting malignant cell transformation of medulloblastoma cells through the Notch pathway.		?				+		+				+		?			+		GPCA			GPCA, co-IP		Poirson et al. (2017)
	Poirson et al. (2017)			23295			MGRN1			Mahogunin ring finger 1			Has RING- E3 ubiquitin-protein ligase activity in vitro. Involved in regulation of endosome-to-lysosome trafficking. A negative regulator of hedgehog signaling.			+			+			+			GPCA, co-IP			Poirson et al. (2017)
	5700		23024			PDZRN3			PDZ domain containing ring finger 3			A member of the LNX (Ligand of Numb Protein-X) family of RING E3 ubiquitin- protein ligases. Required for vascular morphogenesis and differentiation of adipocytes, osteoblasts and myoblasts.			−			+			+			GST-pull down			Poirson et al. (2017); Thomas and Banks (2015)
	29951			PDZRN4			PDZ domain containing ring finger 4			A member of the LNX family of RING E3 ubiquitin-protein ligases. A suppressor of cell proliferation in human liver cancer cell lines.			−			+			−			GPCA, co-IP			Poirson et al. (2017)
	5684			PSMA3			Proteasome subunit alpha 3				Components of the 20S core proteasome complex involved in proteolysis of most cellular proteins. Associated with two 19S regulatory particles, form the 26S proteasome. Involved in ATP- dependent degradation of ubiquitinated proteins.		SH3KBP1- binding protein 1					SHKBP1 inhibits CBL-SH3KBP1 complex mediated downregulation of EGFR signaling by sequestration of SH3KBP1.		+			+		+			+		+				+IP-MS/MS			White et al. (2012)
			GPCA, co-IP			Poirson et al. (2017)			5695					PSMB7				Proteasome subunit beta 7			+
	7126			TNFAIP1			TNF alpha induced protein 1					A substrate-specific adapter of a BCR E3 ubiquitin-protein ligase complex that mediates the ubiquitination and proteasomal degradation of RhoA, thereby regulating the actin cytoskeleton and cell migration. HPV-16 E7 can modulate the responses of its natural host cell to the closely related cytokines TNF-α.		+				+		−			+		IP-MS/MS				+White et al. (2012)
		GPCA, co-IP			Basile et al. (2001); Poirson et al. (2017)			5698			PSMB9			Proteasome subunit beta 9
	7186			−				TRAF2			TNF receptor associated factor 2			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.		+				−		−			+		IP-MS/MS			+		White et al. (2012)
	GPCA, co-IP			Cheng et al. (2015); Poirson et al. (2017)			5700
	7187		PSMC1			TRAF3		Proteasome 26S subunit 4, ATPase 1			TNF receptor associated factor 3		A component of the 26S proteasome belonging to the heterohexameric ring of AAA proteins (ATPases associated with diverse cellular activities). Unfolds ubiquitinated target proteins.			?			+			?			GST-pull down			Regulates pathways leading to activation of NF-κB and MAP kinases, and plays a central role in the regulation of B-cell survival. An essential constituent of several E3 ubiquitin-protein ligase complexes. Overexpression of TRAF3 enhances p53 and pRb expression		Tomaić et al. (2013)
	?			+			?			GPCA			Poirson et al. (2017); Zhang et al. (2018)			5701			PSMC2			Proteasome 26S subunit 7, ATPase 2			Components of the 26S proteasome.			+			+			+			IP-MS/MS			White et al. (2012); Tomaić et al. (2013)
	5702			PSMC3			Proteasome 26S regulatory subunit 6A, ATPase 3			+			+
	9618			TRAF4			TNF receptor associated factor 4			An adaptor protein and signal transducer linking members of the TNFR family to different signaling pathways. Plays a role in the activation of NF-κB and JNK, and in the regulation of cell survival and apoptosis. May interact selectively and non-covalently with E3 ubiquitin- protein ligase enzymes.			?			+			?			GPCA						Poirson et al. (2017)		−			IP-MS/MS, GST-pull donw			White et al. (2012); Tomaić et al. (2013)
	7188			TRAF5			TNF receptor associated factor 5			An adaptor protein and signal transducer linking members of the TNFR family to different signaling pathways.		May interact selectively and non-covalently with E3 ubiquitin-protein ligase enzymes.			+			+			+						GPCA, co-IP			Poirson et al. (2017)			5704			PSMC4			Proteasome 26S subunit 6B, ATPase 4
	10346			TRIM22			tripartite motif containing 22		+				An interferon- induced antiviral protein involved in innate immunity. May have E3 ubiquitin-protein ligase activity. Activated by integration of E6/E7 genes.		+			?		−			+					IP-MS/MS			White et al. (2012)
	?			GPCA			Pett et al. (2006); Poirson et al. (2017)			5705
	22954		PSMC5				TRIM32		26S proteasome regulatory subunit 8			?			tripartite motif containing 32		+				An E3 ubiquitin-protein ligase. It ubiquitinates DTNBP1 and promotes its degradation.			+?			GST-pull down				+			+Tomaić et al. (2013)
		GPCA, co-IP			Poirson et al. (2017)			5707			PSMD1			26S proteasome non-ATPase regulatory subunit 1			+			+			+			IP-MS/MS			+			+			+			IP-MS/MS, GST-pull down			White et al. (2012); Tomaić et al. (2013)
	PSMC1			26S proteasome regulatory subunit 4			A component of the 26S proteasome.			−			+
	493829	White et al. (2012)
		TRIM72			tripartite motif containing 72			A muscle-specific protein that plays a central role in cell membrane repair by nucleating the assembly of the repair machinery at injury sites. May be involved in proteasome- mediated, ubiquitin-dependent protein catabolic processes.			−			+			+			GPCA, co-IP			Poirson et al. (2017)			5708			PSMD2
	114088			TRIM9		Proteasome 26S subunit 2, non-ATPase 2			tripartite motif containing 9		Component of the 26S proteasome, binds to the intracellular domain of tumor necrosis factor type 1 receptor (TNFR1); the binding domain of TRAP1 and TRAP2 resides outside the death domain of TNFR1.			An E3 ubiquitin- protein ligase, which self-ubiquitinates in cooperation with E2 enzyme UBE2D2/UBC4. Serves as a targeting signal for proteasomal degradation.			?			+			?			GPCA			Poirson et al. (2017)			5710			PSMD4
	7314		26S proteasome non-ATPase regulatory subunit 4		UBB				ubiquitin BA 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.			?			+				Targets cellular proteins for degradation by the 26S proteasome. Involved in the maintenance of chromatin structure, the regulation of gene expression, and the stress response.		?			IP-MS/MS			?		Tomaić et al. (2013)
	+			?			GPCA			Poirson et al. (2017)			5709			PSMD3			Proteasome 26S subunit 3, non-ATPase 3
	7319			Components of the 26S proteasome.				UBE2A			Ubiquitin- conjugating enzyme E2A			Accepts ubiquitin from the E1 complex. In association with the E3 enzyme, UBE2A plays a role in transcription regulation by catalyzing the ubiquitination of histone H2B.		+				+		+			+		+			?		IP-MS/MS			White et al. (2012)
	GPCA						Poirson et al. (2017)			9861			PSMD6
	7332						UBE2L3		Proteasome 26S subunit, non-ATPase 6			Ubiquitin- conjugating enzyme E2L3		+			Specifically acts with HECT-type and RBR family E3 ubiquitin- protein ligases. Accepts ubiquitin from the E1 complex and catalyzes its covalent attachment to other proteins including E7.		+			−			IP-MS/MS			White et al. (2012)
	?						+			?			IP			Reinstein et al. (2000)			5713			PSMD7			Proteasome 26S subunit, non-ATPase 7			+
	23352						UBR4			Ubiquitin- protein ligase E3 component n-recognin 4		+				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 pRB- independent 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.			+		−			+		IP-MS/MS			White et al. (2012)		+
	IP						Huh et al. (2005); De Masi et al. (2005); White et al. (2012)			5714			PSMD8
	8237			USP11		Proteasome 26S subunit, non-ATPase 8				Ubiquitin carboxyl- terminal hydrolase 11		+			Inhibits degradation of target proteins by the proteasome. Plays a role in the regulation of pathways leading to NF-κ-B activation. Augments HPV-16E7 activity in modulating downstream target genes, such as pRb, Bcl-2, and Cdc-2, suggesting that this interaction may contribute to cell transformation by HPV-16E7.		+			?		−				+		IP-MS/MS			?		White et al. (2012)
	IP			Lin et al. (2008); Poirson et al. (2017)			5719
	83844		PSMD13			26S proteasome non-ATPase regulatory subunit 13			+			USP26		+				Ubiquitin carboxyl- terminal hydrolase 26			Involved in the ubiquitin-dependent proteolytic pathway in conjunction with the 26S proteasome.+				+			+		IP-MS/MS			White et al. (2012)
	+			GPCA, co-IP			Poirson et al. (2017)			10213			PSMD14			26S proteasome non-ATPase regulatory subunit 14			+			+			−
	57663			USP29			Ubiquitin carboxyl- terminal hydrolase 29			A thiol-dependent hydrolyser of ester, thioester, amide, peptide and isopeptide bonds formed by the C-terminal Gly of ubiquitin.			−		IP-MS/MS				+			−		White et al. (2012)
		GPCA, co-IP			Poirson et al. (2017)			23198			PSME4			Proteasome activator subunit 4
	23032			USP33			Ubiquitin carboxyl- terminal hydrolase 33		A proteasome component that specifically recognizes and promotes ATP- and ubiquitin-independent degradation of acetylated core histones during DNA damage response to double-strand breaks.			A deubiquitinating enzyme involved in centrosome duplication, cell migration and beta-2 adrenergic receptor/ADRB2 recycling.		−				+		+			+		−			IP-MS/MS			White et al. (2012)
	+			GPCA, co-IP			Poirson et al. (2017)			64320			RNF25			Ring finger protein 25
	90850			ZNF598			Zinc finger protein 598		A RING finger- dependent E3 ubiquitin- protein ligase that mediates ubiquitination and stimulates transcription mediated by NF-κB.			An E3 ubiquitin- protein ligase required for terminal stalling of ribosomes during translation of poly(A) sequences by mediating ubiquitination of 40S ribosomal protein.		?				?+			?		+				?		GPCA			GPCA		Poirson et al. (2017)
	Poirson et al. (2017)			9810			RNF40			Ring finger protein 40			A component of the RNF20/40 E3 ubiquitin- protein ligase complex; forms a H2B ubiquitin ligase complex in cooperation with the UBE2A or UBE2B. Supports maintenance of tumorigenic features and inflammatory signaling by promoting nuclear NF-κB activity.			?			+			?			GPCA			Poirson et al. (2017)
	85456			TNKS1BP1			Tankyrase-1-binding protein			A subunit of the smaller 1-MDa core of Ccr4-Not complex. Ccr4-Not is an mRNA deadenylase and has a ubiquitin-protein ligase function.			<+			<+			+			IP-MS/MS			White et al. (2012)
	7188			TRAF5			TNF receptor associated factor 5			Tumor necrosis factor receptor-associated factor (TRAF) protein family. An adapter protein and signal transducer linked to various signaling pathways by association with the receptor cytoplasmic domain and kinases. Involved in cytokine signaling and mediates activation of NF-κB and JNK. It is also involved in apoptosis.			?			+			?			GPCA			Poirson et al. (2017)
	7189			TRAF6			TNF receptor- associated factor 6			An E3 ubiquitin-protein ligase that mediates the synthesis of ‘Lys-63’- linked-polyubiquitin chains conjugated to target proteins and ubiquitination of unanchored poly- ubiquitin chains. Induces activation of NF-κB and JUN. An adaptor protein and signal transducer linking TNFR proteins to different signaling pathways. Plays a role in signal transduction initiated via TNF receptor, IL-1 receptor and IL-17 receptor.			?			+			?			GPCA			Poirson et al. (2017)
	7706			TRIM25			Tripartite motif containing 25			An E3 ubiquitin-protein ligase involved in innate immune defense against viruses. Crucially involved in the interferon response to viral infection.			+			+			+			co-IP			Chiang et al. (2018)
	10422			UBAC1			UBA domain containing 1			A ubiquitin-protein ligase required for poly-ubiquitination and proteasome-mediated degradation of CDKN1B during G1 phase of the cell cycle.		A non-catalytic subunit of the KPC Kip1 ubiquitination- promoting complex.			?			+			?			GPCA			Poirson et al. (2017)
	7337			UBE3A			Ubiquitin- protein ligase E3A			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.			<+			+			+			Yeast-two hybrid, co-crystal x-ray crystalography, co-IP			Poirson et al. (2017); Storey et al. (1998); Kao et al. (2000); Brimer et al., 2007; Thomas et al. (2013)
	23352			UBR4			Ubiquitin- protein ligase E3 component n-recognin 4			An E3 ubiquitin-protein ligase, a component of the N-end rule pathway. Recognizes and binds to proteins bearing a specific N-terminal leading to ubiquitination and subsequent degradation. Forms meshwork structures involved in membrane morphogenesis and cytoskeletal organization and regulates integrin-mediated signaling.			−			−			+

+ confirmed to interact; +, < confirmed interactions with lower affinity compared to HR-HPV E7; - interactions not detected; ? interactions not tested. Co-IP: co-immunoprecipitation; GPCA: Gaussia princeps luciferase protein complementation assay, IP-MS/MS: immunoprecipitation-mass spectrometry, IP: immunoprecipitation.