The interaction of hepatitis B virus with the ubiquitin proteasome system in viral replication and associated pathogenesis

It is well-known that virus-encoded proteins are essential for viral replication. However, multiple studies have shown that the UPS serves as an anti-viral mechanism through selective degradation of viral proteins [18‐23]. Until now, different HBV proteins have been reported to be degraded by the UPS (Fig. 1). For example, Np95/ICBP90-like RING finger protein (NIRF), an E3 ligase, could bind to HBc and promote its ubiquitination and degradation to decrease the levels of HBc protein [18]. Cellular inhibitor of apoptosis protein 2 (cIAP2), which has a carboxy-terminal RING finger domain with the activity of E3 ligase, is able to promote the degradation of HBV Pol protein mediated by the UPS [20].

Different laboratories have investigated the role of HBx on viral replication. Owing to the lack of a natural model of HBV infection previously, the role of HBx in the complete virus life cycle has not been well-established. Given this limitation, most reported studies, including the studies using a plasmid-based HBV replication system and associated animal models, strongly support the importance of HBx for virus replication [24]. Currently, HepG2 cell overexpressing the HBV entry receptor NCTP (HepG2-NTCP) has been established, and was highly susceptible to wide-type HBV infection [25, 26]. In addition, it was found that the virus with a defective mutation of HBx fails to produce the detectable levels of HBV replication after infecting HepG2-NTCP cells [27]. Using the cell model, Ko et al. also investigated the infection kinetics of HBV during long-term culture, and the research showed that the kinetics of intracellular HBx expression was followed the viral cccDNA dynamics and reaches maximum levels earlier than other viral proteins [28]. Together, these preliminary results imply that HBx is essential for the natural course of HBV infection, and could serve as an early viral protein that is needed immediately for viral replication. In general, HBx is found to be produced at a very low level in chronic hepatitis patients with HBV infection, because it was found to be rapidly degraded by the UPS pathway in the host cells [29, 30]. Furthermore, recent studies have indicated that several special cellular proteins as restriction factors limit HBV replication by repressing the expression of HBx via the UPS. For example, the tumor suppressor p53 could induce Ub-dependent proteasomal degradation of HBx [31], through an E3 ligase, seven in absentia homologue 1 (Siah-1) [22]. Ling et al. showed that Id-1 [32], a member of the HLH protein family, was capable of interacting with proteasome subunit C8 (PMSC8), to facilitate the degradation of HBx. In addition, the X-linked tumor suppressor TSPX was found to bind with the proteasome component RPN3 and reduce the stability of HBx via the proteasome-dependent degradation [33]. It was also shown that Hdj1 and phospholipid scramblase 1 (PLSCR1) could promote HBx degradation by ubiquitination and a proteasome-dependent mechanism to repress viral replication [34, 35]. More importantly, six lysines in HBx protein were found to be ubiquitinated [23]. However, how these factors as mentioned above selectly target these ubiquitination sites of HBx is largely unknown. Therefore, further experiments are warranted to elucidate the details related to HBx ubiquitination mediated by different cellular factors.

Host cells could utilize the UPS to degrade viral proteins and restrict viral growth, but the HBV can manipulate many specific proteins to limit the degradation of viral proteins mediated by the UPS pathways. For example, although the turnover of HBx is rapid, the reported studies indicated that the bimodal half-life of HBx was influenced by the intracellular location of the protein [24], and it was speculated that the interaction of HBx with other intracellular proteins in a different cell location may be necessary for the stability of the HBx protein. In addition, several proteins have been reported to control HBV replication via the interaction with HBx to regulate the stability of the protein through UPS-associated mechanisms. For example, damaged DNA binding protein 1 (DDB1) and E3 ligase CUL4 could interact with HBx, forming a HBx-DDB1-CUL4 E3 ligase complex, and protect the viral protein from proteasome-mediated degradation to promote HBV replication [36‐38]. Additionally, Liu et al. showed that the E3 ligase HDM2 (also known as MDM2) promoted the NEDDylation of HBx to enhance HBx stability by blocking its degradation mediated by ubiquitination [39]. The study from Yeom et al. indicated that proteasomal activator 28 gamma (PA28γ) could down-regulate the expression of Siah-1 and p53 to facilitate the inhibition of HBx degradation [40]. Besides, it is found that cellular FLICE inhibitory protein (c-FLIP) could interact with HBx and protect it from Ub-dependent degradation to maintain HBx stability [41]. Saeed et al. showed that parvulin 14 (Par14) and parvulin 17 (Par17) proteins enhanced the stability of HBx via the direct interactions with the viral protein [42]. Fatty acids, including palmitate, oleate, and stearate, were also found to increase the stability of HBx protein by preventing its proteasome-dependent degradation [43]. Su et al. found that deubiquitylating enzyme Ub-specific protease 15 (USP15) directly interacted with HBx to reduce the ubiquitination and proteasomal degradation of the viral proteins [44]. Taken together, these evidence indicate that the UPS could be used to control the degradation or stability of HBc, Pol, and HBx protein. However, the molecular mechanisms associated with expression of viral envelope proteins regulated by the UPS remain unknown and require additional studies to elucidate.

The HBV life cycle involves cell entry, viral genome uncoating, replication, transcription, protein expression, reverse transcription, viral particle maturation, and release (Fig. 1). So far, an abundance of studies, using different experimental systems including the cultured cells transfected with plasmid containing a greater-than-unit-length HBV genome, have indicated that several components of the UPS, such as PJA1 [45] and SMC5/6 [46], could serve as restriction factors to inhibit the replication cycle of the HBV.

However, during HBV infection, virus-encoded proteins have evolved to interact with host UPS, and could regulate the expression or stability of cellular molecules associated with host UPS to meet the requirement for virus replication in various steps of the life cycle (Fig. 1). Based on hepatoma cells or primary hepatocytes transiently transfected with a plasmid encoding the HBV genome expressing HBx, or the plasmid that contains the mutations preventing HBx expression [47], HBx was found to play vital roles in different steps of HBV replication. Furthermore, HBx could affect viral replication via a UPS-dependent pathway [48]. For example, Gao et al. found that HBx could activate and enhance the stability of HBV cccDNA via increasing the expression of MSL2, an E3 ligase that has the capability of reducing the protein level of APOBEC3B [49]. SMC5/6 has been reported to have the capability of inhibiting HBV transcription [46]. Furthermore, PJA1, an E3 ligase, binds to SMC5/6 and facilitates the protein complex to eliminate cccDNA to inhibit HBV replication [45]. As mentioned above, HBx could interact with DDB1 and CUL4 to form a HBx-DDB1-CUL4 E3 ligase complex, and based on the interaction with DDB1 and CUL4, HBx could stimulate viral transcription [37]. Using substrate-trapping proteomics, Murphy et al. identified that SMC5/6 were the substrates of the HBx-DDB1-CUL4 E3 ligase complex, and HBx degraded SMC5/6 through ubiquitination and the proteasomal pathway to enhance HBV replication [27]. In addition, the study from Klundert et al. reported Talin-1 (TLN1) was a viral restriction factor that could repress the transcription of the HBV, but HBx was able to relieve its restriction by inducing the degradation of TLN1 mediated by the proteasome pathway to facilitate HBV replication [50]. In addition, multiple TRIM proteins, which have the function of E3 ligase, including TRIM5, TRIM6, TRIM11, TRIM14, TRIM22, TRIM25, TRIM26, TRIM31, and TRIM41, have been shown to repress HBV transcription [51, 52]. However, HBx could decrease the expression of TRIM22 at the gene level to facilitate the transcription of viral genes [53]. In summary, these mentioned studies demonstrate that HBx could promote the replication of HBV via regulating the expression and function of the components of host UPS.

Apart from HBx, HBc has been shown to play a critical role in viral maturation and release [54], but the precise mechanisms have not been well-assessed. Rost et al. found that HBc could interact with gamma2-Adaptin, an Ub-interacting adaptor, and Nedd4, an E3 ligase, to enhance assembly and particle release of the virus [55]. However, whether the components of the UPS participate in other steps of the viral life cycle, such as genome uncoating during HBV infection, is still unclear.

The host innate immune response is the first line of defense against viral infection. Via recognizing viral pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), including NOD like receptors (NLRs), toll-like receptors (TLRs), nucleic acid sensing PRR includes the stimulator of interferon genes (STING) and RIG-I like receptors (RLRs), the host cells trigger special signals to activate intracellular pathways and induce the production of interferon (IFN) and cytokines to clear the virus [11, 56]. However, during infection with HBV, the virus does not trigger or only triggers a very limited innate immune response to cause persistent infection [57]. The exact mechanisms of the inhibition of the innate immune response mediated by the HBV are not well-understood. Until now, several studies from different laboratories found that the HBV had evolved to use the UPS to interfere with the expression of PRRs-associated proteins (Fig. 1), to act against the antiviral immune response. For example, Khan et al. demonstrated that the HBV was able to induce Parkin, an E3 ligase, which had the capability of recruiting the linear Ub assembly complex (LUBAC) to mitochondria, bonding to mitochondrial antiviral signaling (MAVS), and accumulating unanchored linear polyubiquitin chains on MAVS protein through LUBAC, to block the synthesis of IFN-β [58]. In addition to these, current researches demonstrate that, among the HBV proteins, HBx could utilize the UPS to inhibit the innate immune response induced by the virus. For example, Jiang et al. indicated that HBx could inhibit the ubiquitination of multiple proteins that associate with PRRs, such as IRF3, IRF7, RIG I, RIG I-2CARD, TRAF3, and IKKi, to inhibit the production of IFN and facilitate viral replication [59]. In addition, in HBV-infected cells, HBx has been shown to interact with MAVS and promote the degradation of MAVS mediated by the proteasome, to prevent the production of IFN-β [60]. HBx also reduces the expression of TIR-domain-containing adaptor inducing interferon-beta (TRIF) protein via the proteasomal pathway to inhibit the activation of TLR signals [61]. Apart from HBx, HBV Pol protein was also found to interact with STING, and disrupt the ubiquitination of STING to inhibit the activation of STING-stimulated IRF3 as well as the induction of IFN-β [62].

IFN, an immune molecule that could be induced by PRRs, has been successfully used for clinical treatment of patients with HBV infection. However, the molecular mechanisms of the anti-HBV effect mediated by IFN are not completely understood. It is well-accepted that, upon binding to the cellular receptors IFNAR, IFN could initiate intracellular Janus kinase/signal transducer and activator of transcription (JAK/STAT) signal pathways to induce the expression of numerous IFN-stimulated genes (ISGs) to clear the virus [8]. Furthermore, current studies indicate that IFN could inhibit HBV replication at different steps of the viral life cycle in various ways [57]. Specifically, Robek et al. showed that the inhibitors of the proteasome can block the antiviral effect mediated by IFN [63]. Further study demonstrated that TRIM5, TRIM6, TRIM14, TRIM22, TRIM25, TRIM26, and TRIM31, which have the activity of E3 ligase that could inhibit HBV transcription, are ISGs [52, 53, 64, 65]. In addition, Tan et al. showed that TRIM14 could limit HBV replication via binding the C-terminal of HBx and blocking HBx-mediated degradation of Smc5/6 that is dependent on the HBx-DDB1-CUL4 E3 ligase complex [64]. Taken together, these reported results indicate that IFN-induced anti-HBV immune response is related to the component of the UPS.

Although IFN could eradicate the HBV, the antiviral function of IFN could be compromised by the virus. The exact mechanism for the inhibition of IFN mediated by HBV remains not well clear. However, it is estimated that interfering with the function of ISGs that are associated with the UPS contributes to the anti-IFN function of the virus. For example, HBV could repress the expression of TRIM25 to facilitate viral infection [66]. ISG15/USP18 is also an ISG, and it could conjugate to Ub, incorporate into Ub chains, and negatively regulate the turnover of ubiquitylated proteins [67]. In addition, the reports of Kim and Li et al. showed that ISG15/USP18 could promote HBV replication [68‐70].

The components of the UPS and cellular proteins regulated by the UPS are involved in several cellular processes, including the cell cycle, proliferation, invasion, and apoptosis [15, 16, 71]. Therefore, the abnormal expression of cellular proteins mediated by the HBV through disrupting the function of the UPS is responsible for the abnormal biological function of HBV-related hepatocytes or hepatoma cells. For example, ISG15/USP18 promotes the proliferation and inhibits the apoptosis of HBV-associated HCC cells [72]. In addition, the HBV stimulates the gene expression of the E3 ligase Parkin and disrupts mitochondrial dynamics toward fission and mitophagy to decrease the virus-induced apoptosis in hepatocytes [73]. Liu et al. showed that HBe and its precursors could interact with NUMB, which could enter in a complex with p53 and the E3 ligase HDM2, and impair the stability of p53 mediated by Ub-dependent proteasomal degradation, to promote proliferation and inhibit apoptosis of HCC cells [74]. In addition, Hsieh et al. demonstrated that the pre-S2 LHBS mutant induces the Ub-dependent proteasomal degradation of cyclin-dependent kinase inhibitor p27 (Kip1) through interacting with the Jun activation domain-binding protein 1 (JAB1) to promote the proliferation of HCC cells [75].

HBx is considered as a multifunctional regulator with a vital role in the development of HCC [12]. Several studies have demonstrated that transgenic mice with a high HBx level could develop HCC [76]. Additionally, although HBx is detected at low levels in the liver tissue of CHB patients, it is detected at high frequency in HBV-associated HCC patients [77]. These results indicate that high-level hepatic expression of HBx is a key for the development of HBV-related HCC. Based on HBx over-expression assays in HCC cells, the protein was demonstrated to regulate the expression of cellular proteins that are mediated by the UPS to modulate a variety of biological processes (Fig. 2). For example, HBx increases TRIM52 expression via the NF-κB signal pathway [78], and elevates MSL2 based on YAP/FoxA1 signaling [49], to promote cellular proliferation. Upon binding with DDB1 and CUL4A [79, 80], HBx affects the cell cycle and induces genetic instability to favor HCC development. Plk1 is a molecule that participates in cell cycle progression. Zhang et al. revealed that HBx increased the expression of Plk1 in favor of mediating the ubiquitination of SUZ12 and ZNF198 to promote the progress of HCC [81]. c-Myc is an oncoprotein that contributes to the proliferation of HCC cells [82]. The stability of c-Myc is regulated by Skp/cullin/F-box (SCF) E3 ligase. Lee et al. found that HBx bound to SCF E3 ligase and inhibited the ubiquitination and proteasomal degradation of c-Myc [83, 84]. In addition, HBx reduces the stability of neuregulin receptor degradation protein 1 (Nrdp1) that is regulated by the proteasome to increase the expression of ErbB3 and further enhance the proliferation of HCC cells [85]. Current studies showed that HBx mutants also have been implicated in the development of HCC [86], and the components of the UPS are involved in the development of HCC mediated by HBx mutants. For instance, Huang et al. indicated that HBx with mutations in the core promoter region deregulated S phase kinase-associated protein 2 (SKP2), a member of the F-box family that acts as a substrate-specific adaptor in the SCF E3 ligase complex, to decrease the stability of its target protein p21 [87], and induce the changes in the cell cycle to facilitate the proliferation of HCC cells. Besides, Qian et al. showed that the downregulation of USP16 mediated by carboxyl-terminal truncated HBx mutants promotes the proliferation of HCC cells [88].

The protooncogene pituitary tumor-transforming gene 1 (PTTG1) is related to tumor invasiveness. In HBV-related HCC, HBx could induce the accumulation of the protein via disrupting the interaction of PTTG1 with the SCF E3 ligase complex to repress PTTG1 degradation [89]. In addition, HBx stabilizes amplified in breast cancer 1 (AIB1) protein through preventing it from proteasome-dependent degradation and cooperates with this molecule to enhance the invasiveness of HCC cells [90]. β-catenin is a critical molecule associated with epithelial-mesenchymal transition (EMT) [91], a process that contributes to the invasion of HCC cells. However, the mechanism of β-catenin upregulation in HBV-related HCC is unclear. Jung et al. showed that HBx affected the expression of β-catenin via Ub-dependent proteasome pathways that rely on p53 status. In the presence of p53, HBx increased the ubiquitination of β-catenin via the activation of a p53-Siah-1 proteasome pathway. In the absence of p53, HBx attenuated the ubiquitination of β-catenin protein via the inhibition of the glycogen synthase kinase-3β (GSK-3β) pathway [92]. E-cadherin is an adhesion molecule that could suppress EMT. Kim et al. showed that HBx is able to increase the expression of the transcriptional repressors E12/E47 by inhibiting Ub-dependent proteasomal degradation to repress E-cadherin protein and induce EMT to favor invasion of HCC [93]. In addition, CUL4A, an E3 ligase, also interacts with HBx and promotes EMT to enhance invasion of HCC cells [80].

Apart from the role in regulating the cell cycle, proliferation, and invasion, HBx also has been reported to sensitize hepatocytes to apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL) via inhibiting E3 Ligase A20 [94]. Stabilizing hypoxia-inducible factor-1α (HIF1-α) protein from proteasome-dependent degradation, HBx could induce angiogenesis [95]. In addition, early studies revealed that HBx enhanced the expression of MDM2 by directly binding with MDM2 and inhibiting its Ub-directed degradation to enhance the stem-like properties in HCC cells [96]. Recently, it has been demonstrated that HBx utilized the UPS to mediate the degradation of insulin receptor substrate 1 (IRS1) via ubiquitination to impair insulin signaling in hepatocytes [97].