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Correspondence Address correspondence to: Mingxiong Guo, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan 430072, P.R. China; Ecological Research Center, College of Science, Tibet University, Lhasa 850012, P.R. China. fax: xxx.
Taikang Medical School (School of Basic Medical Sciences), Wuhan University, Wuhan, P.R. ChinaState Key Laboratory of Virology, Hubei Provincial Key Laboratory of Allergy and Immunology, Wuhan, P.R. China
Chronic hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma. However, the function and mechanism of the effect of HBV on host protein ubiquitination remain largely unknown. We aimed at characterizing whether and how HBV promotes self-replication by affecting host protein ubiquitination. In this study, we identified UBXN7, a novel inhibitor for nuclear factor kappa B (NF-κB) signaling, was degraded via interaction with HBV X protein (HBx) to activate NF-κB signaling and autophagy, thereby affecting HBV replication. The expression of UBXN7 was analyzed by Western blot and quantitative reverse transcription polymerase chain reaction in HBV-transfected hepatoma cells and HBV-infected primary human hepatocytes (PHHs). The effects of UBXN7 on HBV replication were analyzed by using in vitro and in vivo assays, including stable isotope labeling by amino acids in cell culture (SILAC) analysis. Changes in HBV replication and the associated molecular mechanisms were analyzed in hepatoma cell lines. SILAC analyses showed that the ubiquitination of UBXN7 was significantly increased in HepG2.2.15 cells compared with control cells. After HBV infection, HBx protein interacted with UBXN7 to promote K48-linked ubiquitination of UBXN7 at K99, leading to UBXN7 degradation. On the other hand, UBXN7 interacted with the ULK domain of IκB kinase β through its ubiquitin-associating domain to facilitate its degradation. This in turn reduced NF-κB signaling, leading to reduced autophagy and consequently decreased HBV replication.
Our results reveal that HBx can promote HBV replication via degradation of UBXN7, thus maintaining high levels of IKK-β to activate NF-κB signaling and NF-κB–dependent autophagy. Our findings suggest that UBXN7 can be targeted for potential new therapies in HBV-related diseases.
Chronic hepatitis B virus (HBV) infection is a major global health problem worldwide. Two hundred fifty-seven million people chronically infected by HBV are at increased risk of death from hepatocellular carcinogenesis.
Antiviral drugs can inhibit HBV replication but cannot completely eliminate HBV. Thus, patients need to take medication for life. Improved understanding of the molecular mechanism of HBV replication may offer novel and better therapies against HBV.
Ubiquitination is a common post-translational modification that plays an essential role in the antiviral mechanism to eliminate viral components.
To clarify the relationship between HBV replication and ubiquitination, we performed stable isotope labeling by amino acids in cell culture (SILAC)-labeling and affinity enrichment of ubiquitinated peptides, followed by high-resolution liquid chromatography with tandem mass spectrometry analysis. Among the proteins found to be differentially ubiquitinated because of HBV was UBXN7.
UBXN7 is a member of the ubiquitin regulatory X (UBX) proteins family, which can interact with a large number of E3 ubiquitin ligases through ubiquitin-associated (UBA) domain at their N termini.
It has also been found that UBXN7 (also called UBXD7) inhibits tumor necrosis factor α–induced NF-κB and human immunodeficiency virus long terminal repeat activities, but the underlying mechanisms are still unclear.
The homologous protein of UBXN7 in Arabidopsis and yeast PUX7/Ubx5 has been identified recently as a selective autophagy receptor for CDC48/p97, which is combined with ubiquitin-interaction motif (UIM)-docking site on ATG8 through its UIM motif. It is worth noting that UBXN7 cannot bind with MAP1LC3a and GABARAP isoforms, the 2 main ATG8 sub-clades, through the UIM-UDS surface in humans.
Thus, the role of UBXN7 in autophagy remains to be determined.
Here, we showed that HBx encoded by HBV interacts with UBXN7 through aa 55-136 and promotes its ubiquitin-dependent degradation. We further revealed that UBXN7 could interact with IκB kinase β (IKK-β) through its UAS domain to promote IKK-β ubiquitination and subsequently degradation of IKK-β, leading to inactivation of NF-κB signaling and inhibition of autophagy and HBV replication. Our results indicated that HBx promoted HBV replication via degradation of UBXN7 to enhance IKK-β level for activation of NF-κB signaling.
Hepatitis B Virus Reduces UBXN7 Protein Level
To investigate the effect of HBV infection on the ubiquitination of host proteins in hepatocytes, we analyzed the ubiquitylome and proteome in HBV-expressing stable cell line HepG2.2.15 and the corresponding parental cell line HepG2. We performed SILAC labeling and affinity enrichment of ubiquitinated peptides, followed by high-resolution liquid chromatography with tandem mass spectrometry and bioinformatics analyses (Figure 1A).
Among thus identified proteins was UBXN7, a member of the ubiquitin regulatory X proteins family. In response to HBV infection, UBXN7 protein level was reduced by half in HepG2.2.15 cells (Figure 1B, left). In addition, the ubiquitination of the 2 lysine residues in UBXN7 (K84 and K99) were both up-regulated by more than 2-fold in HepG2.2.15 compared with HepG2 (Figure 1B, right). To verify the SILAC findings, we analyzed the mRNA and protein levels of UBXN7 in HepG2 and HepG2.2.15 cells by real-time polymerase chain reaction (PCR) and Western blot, respectively. Consistent with the SILAC results, Western blot showed that UBXN7 protein level was significantly lower in HepG2.2.15 cells compared with HepG2 cells (Figure 1C, bottom). However, there was no significant difference in UBXN7 mRNA levels between the 2 cell lines (Figure 1C, upper), suggesting that HBV regulates UBXN7 protein level post-transcriptionally. To further investigate the regulation of UBXN7 protein level by HBV, we transfected Huh7 or HepG2 cells with pHBV1.3 or a control vector and determined the mRNA and protein levels of UBXN7. Again, the transfection of pHBV1.3 in these 2 cell lines did not affect the mRNA level of UBXN7 but significantly reduced its protein level (Figure 1D). Then, we treated HepAD38 cells with tetracycline, which inhibited the production of HBV in HepAD38 cells. After tetracycline treatment, the RNA level of UBXN7 in cells did not change significantly, but the protein level increased significantly (Figure 1E). Our results indicated that HBV infection significantly reduced UBXN7 protein level but had no effect on its mRNA level. Thus, we suggested that HBV down-regulates UBXN7 protein expression at the post-transcriptional level.
Hepatitis B X Protein Suppresses UBXN7 Protein Level via the Proteasome Pathway
There are 2 main pathways for protein degradation, proteasome pathways and the lysosome pathways. To determine UBXN7 degradation pathway induced by HBV, we studied the effects of the proteasome inhibitor MG132 and the lysosomal inhibitor NH4Cl and CQ. We found that HBV reduced UBXN7 protein levels dose-dependently in Huh7 (Figure 2A, left) and HepG2 (Figure 2B, left). Importantly, treatment with MG132 blocked this reduction, whereas treatment with NH4Cl and CQ treatment had no effect (Figure 2A, middle and right, Figure 2B, middle and right). Similar results were obtained in HepAD38 cells with or without tetracycline treatment (Figure 2C). These results indicated that HBV down-regulated UBXN7 protein level through the proteasome pathway.
To determine which HBV protein might function to reduce the protein level of UBXN7, we transfected plasmids expressing different viral proteins into hepatic cells. As shown in Figure 2D, HBx, but not HBc, HBs, or HBp, significantly reduced UBXN7 protein level. Consistently, when pHBV1.2-Δx plasmid, an HBV plasmid in which the HBx gene was deleted, was transfected into cells, it failed to reduce UBXN7 protein level, unlike that parental wild-type (WT) pHBV1.2 plasmid (Figure 2E). Furthermore, like HBV plasmid, transfection of HBx also dose-dependently reduced UBXN7 protein level but not its mRNA level (Figure 2F).
To confirm whether the inhibition of UBXN7 by HBx is also through the proteasome pathway, we used the 3 inhibitors as above. Again, we found that HBx reduced UBXN7 protein level in a dose-dependent manner in the presence of NH4Cl and CQ but not MG132, indicating the involvement of the proteasome pathway (Figure 2G). In addition, when we used cycloheximide to block new protein synthesis, we found that the protein level of UBXN7 decreased gradually with the increasing of treatment time, with a half-life of 9 hours (Figure 2H, left). Upon overexpressing HBx, half-life of the UBXN7 protein in cells treated with cycloheximide was only 6 hours (Figure 2H, right). Similar results were obtained in HepG2 cells (Figure 2I). In addition, cells transfected with pHBV1.2 plasmid also had reduced half-life of UBXN7 protein compared with cells transfected with pHBV1.2-Δx plasmid (Figure 2J). These results suggest that HBx shortens the half-life of UBXN7.
Hepatitis B X Protein Promotes K48-Linked Ubiquitination of UBXN7
To determine whether HBV promotes UBXN7 degradation by catalyzing its polyubiquitylation, we first examined the effect of HBV on UBXN7 in an in vivo ubiquitylation assay. Hemagglutinin (HA)-ubiquitin, Flag-UBXN7, and HBV or control vector were co-transfected into Huh7 cells, and UBXN7 proteins were isolated by Flag antibody pull-down, followed by Western blot analysis of the immunoprecipitated proteins. The results showed that HBV significantly up-regulated UBXN7 ubiquitination in the presence of MG132 (Figure 3A, left). Similar results were obtained in HepAD38 cells in the absence of tetracycline (Figure 3A, right). We further investigated which HBV protein played a role in promoting the ubiquitination of UBXN7. As shown in Figure 3B, transfection with HBx expression plasmid increased UBXN7 ubiquitination, whereas transfection with plasmid expressing HBs, HBc, or HBp did not. Next, we showed by coimmunoprecipitation that UBXN7 interacts with HBx, but not with HBs, HBc, or HBp (Figure 3C and D).
UBXN7 contains 4 domains: UBA domain, ubiquitin-associating (UAS) domain, UIM, and UBX domain. To determine the region of UBXN7 important for its interaction with HBx, we constructed a series of plasmids expressing Flag-tagged mutants UBXN7 (Figure 3E, up panel). Coimmunoprecipitation assay showed that the amino acids 55 to 136 in UBXN7 were required to interact with HBx (Figure 3E, down panel).
There are 2 types of polyubiquitination, one through K48 and the other through K63 in ubiquitin. To determine whether HBV promotes UBXN7 degradation through K48 or K63 ubiquitination, we transfected plasmid expressing HA-tagged WT ubiquitin (Ub) plasmid (HA-Ub-WT) or mutants in which all lysine residues except K48 or K63 were replaced with arginine (HA-Ub-K48 and HA-Ub-K63). Immunoprecipitation followed by Western blot analyses showed that HBV/HBx promoted the K48-linked ubiquitination of UBXN7 (Figure 3F and G). In addition, SILAC ubiquitylome results showed that HBV caused an increase of ubiquitination at 2 lysine residues on UBXN7 (K84 and K99) (Figure 1B). To validate these results, we constructed two UBXN7 mutants in which lysine 84 or 99 was replaced with arginine (UBXN7-K84R or UBXN7-K99R). We transfected various FLAG-UBXN7 (WT, K84R, K99R), HA-Ub (K48), and MYC-HBx plasmids into Huh7 cells, and the results showed that HBx promoted the K48-linked ubiquitination of the UBXN7-K84R mutant but not UBXN7-K99R mutant (Figure 3H). These results indicated that HBV via HBx promoted K48-linked ubiquitination at lysine 99 of UBXN7.
UBXN7 Inhibits the Nuclear Factor Kappa B Signaling via IκB Kinase β
Earlier studies have suggested that UBXN7 might negatively regulate tumor necrosis factor-α–induced NF-κB responsive promoter activity.
We hypothesized that in the presence of HBV, UBXN7 also affects the activity of NF-κB pathway. To test this hypothesis, we performed dual-luciferase assays of NF-κB activity in Huh7 cells transfected with pHBV1.3 plasmid plus control vector or UBXN7 expression plasmid; the result showed that the transfection of Huh7 cells with UBXN7 expression plasmid decreased HBV-induced NF-κB responsive promoter activity (Figure 4A). To further evaluate the molecular mechanisms that UBXN7 inhibits NF-κB signaling in the presence of HBV, we first examined the binding of UBXN7 to several NF-κB signaling proteins in Huh7 cells. The results showed that UBXN7 interacted with IKK-β, but not with IκBα, p65, TAK1, and IKK-α (Figure 4B and C). IKK-β is a key upstream kinase in NF-κB signaling. To determine whether the interaction between UBXN7 and IKK-β affects the function of IKK-β in HBV-induced NF-κB signaling, we transiently overexpressed UBXN7 in the presence of HBV. The results showed that UBXN7 reduced the protein level and phosphorylation of IKK-β. Moreover, IκBα was increased, and phosphorylation of IκBα was reduced (Figure 4D, left). Similar results were found in HepG2.2.15 (Figure 4D, right). When UBXN7 was knocked down by using siRNA, the opposite effects were observed (Figure 4E). These results collectively suggest that UBXN7 inhibited the HBV-induced NF-κB signaling via interacting with IKK-β.
UBXN7 Inhibits Hepatitis B Virus Transcription and Replication via Nuclear Factor Kappa B Signaling
Earlier studies have found that UBXN7 could significantly inhibit some retroviruses and lentiviruses, but it is unclear whether UBXN7 can inhibit HBV replication. To investigate the effect of UBXN7 on HBV replication, we co-transfected pHBV1.3 and UBXN7 plasmid or UBXN7 siRNA into Huh7 cells. As shown in Figure 5A, overexpression of UBXN7 reduced the levels of secreted viral protein HBs and HBe and viral RNA level in a dose-dependent manner. Similar results were found in HepG2 (Figure 5B) and HepG2.2.15 cells (Figure 5C). Consistently, knocking down UBXN7 had the opposite effects (Figure 5D–F). Then, we analyzed HBV RNA level in Huh7, HepAD38, and HepG2 cells by Northern blot (Figure 5G and H), and the results showed that HBV RNA levels in cells with HBx mutant were similar to those in UBXN7 overexpressing cells, which were lower than those in WT HBV replicon cells, indicating that UBXN7 inhibited HBV transcription. Consistently, knocking down UBXN7 had the opposite effects (Figure 5I). Finally, we analyzed HBV DNA level in Huh7 cells by Southern blot (Figure 5J). These results collectively showed that UBXN7 reduced NF-κB signaling to inhibit HBV replication.
Ubiquitin-Associating Domain of UBXN7 Is Crucial for Its Effect on Hepatitis B Virus–Induced Nuclear Factor Kappa B Signaling and Autophagy
To investigate whether UBXN7 reduced IKK-β protein level through ubiquitination-dependent proteasome degradation, we co-transfected HA-Ub-WT or HA-Ub-K48 and HA-Ub-K63 mutants with UBXN7 and IKK-β in Huh7 cells. As shown in Figure 6A, UBXN7 up-regulated ubiquitination of IKK-β with Ub-WT and Ub-K48 in the presence of MG132 to block proteasome activity (Figure 6A). On the other hand, UBXN7 did not affect K63-linked ubiquitination of IKK-β (Figure 6A). These results suggested that UBXN7 promoted IKK-β degradation by enhancing its K48-linked ubiquitination.
To determine the regions through which IKK-β interacted with UBXN7, we generated a series of IKK-β deletion constructs (Figure 6B, up panel) and co-expressed them with UBXN7. We analyzed the binding to UBXN7 by coimmunoprecipitation and found that the ubiquitin-like domain (ULD) of IKK-β was crucial for its interaction with UBXN7 (Figure 6B, down panel). To determine the domain in UBXN7 important for its binding to IKK-β, different UBXN7-deletion constructs were co-expressed with IKK-β. As shown in Figure 6C, IKK-β interacted with the UAS domain of UBXN7. It is worth noting that IKK-β represented the first interacting protein for the UAS domain ever discovered.
To determine whether the UAS domain of UBXN7 functions in HBV-induced NF-κB signaling, we transfected UBXN7 or UAS domain deletion-mutant (UBXN7-ΔUAS) plasmid into HepG2.2.15 cells. Western blot results showed that UBXN7-ΔUAS had no inhibitory effect on NF-κB signaling (Figure 7A). It has been shown that HBV promotes its own replication by activating NF-κB pathway to induce autophagy. To test whether UBXN7 suppresses HBV replication by inhibiting autophagy, we transfected GFP-LC3, an autophagy marker, and UBXN7 or UBXN7-ΔUAS expressing-plasmids into HepG2.2.15 cells. Confocal microscopy analysis revealed that UBXN7 suppressed HBV-induced autophagy, whereas UBXN7-ΔUAS had no effect (Figure 7B). Enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR analyses demonstrated that the UAS domain of UBXN7 is essential for UBXN7 to reduce HBsAg and HBeAg secretion and HBV RNA level (Figure 7C and D).
Then, we transfected pHBV1.2, pHBV1.2-Δx, and FLAG-IKK-β plasmids in Huh7 cells. Northern blot results showed that after transfection of HBx-deleted replicons into cells, UBXN7 was no longer inhibited by HBx, whereas overexpression of IKK-β increased HBV transcription in cells. Combined with our previous results, these results indicated that IKK-β–mediated NF-κB signaling is critical for the function of HBx to regulate viral transcription by inhibiting UBXN7 (Figure 7E).
To examine the relevance of these findings to humans, we carried out studies in primary human hepatocytes (PHHs). ELISA and real-time quantitative PCR showed again that HBsAg secretion and HBV pgRNA level in HBV-infected PHH cells were decreased after overexpressing UBXN7. On the other hand, the UAS domain deletion-mutant (UBXN7-ΔUAS) had no such inhibitory effect, indicating that the UAS domain of UBXN7 is required to inhibit HBV replication in human cells as well (Figure 7F and G). Furthermore, consistent with the result in Figure 7A, Western blot results indicated that the UAS region in UBXN7 was crucial for its suppression of LC3expression in PHHs (Figure 7H). Similar results were obtained in HepG2-NTCP cells (Figure 7I and J). These results indicated that UBXN7 suppressed HBV-induced NF-κB signaling to inhibit autophagy via its UAS domain, thereby reducing HBV replication.
UBXN7 Suppresses Hepatitis B Virus Replication in Vivo
To examine whether UBXN7 can inhibit HBV replication in vivo, we introduced pHBV1.3 and UBXN7 or control plasmids into mice through hydrodynamic injection and killed the mice 4 days later. ELISA results showed that UBXN7 significantly reduced the secretion of HBsAg and HBeAg in plasma (Figure 8A). Real-time quantitative PCR, immunohistochemical, and immunofluorescence analyses of the liver samples showed that UBXN7 suppressed HBV RNA level and core protein level (Figure 8B–D). Western blot showed that UBXN7 inhibits the IKK-β protein level in vivo (Figure 8E). Taken together, these results indicate that UBXN7 suppressed HBV replication through the NF-κB pathway in vivo (Figure 8F).
Herein, we discovered a novel HBV replication inhibitor, UBXN7, a E3 ubiquitin ligase adaptor. We showed that HBx interacted with UBXN7 and increased its K48 ubiquitination to promote its proteasome-dependent degradation. Furthermore, our results demonstrated that UBXN7 interacted with the ULD domain of IKK-β to increase IKK-β K48 ubiquitination for its degradation, leading to the inhibition of the NF-κB signaling and autophagy. Together, our results indicated that HBx binds UBXN7 to promote its degradation, thus blocking UBXN7-induced IKK-β degradation to maintain NF-κB action, which in turn induces autophagy to help HBV replication.
HBx, a key viral oncoprotein, plays crucial roles in HBV replication.
A number of studies have shown that HBx acts as a transcriptional transactivator, suggesting that HBx directly or indirectly modulates a large number of cellular genes for HBV replication by interacting with transcription factors.
demonstrated an interaction between HBx and DDB1 (a CUL4 adaptor). Furthermore, Adrien et al and Christopher et al independently identified HBx-induced HBV replication via DDB1-CUL4-ROC1 (CRL4) E3 ligase to target the structural maintenance of chromosomes (Smc) complex Smc5/6 for degradation.
In addition, Neetu et al showed that HBx directly interacted with the F box region of Skp2 and destabilized the CUL1-SKP2 E3 ligase complex, resulting in blockage of the ubiquitination degradation of MYC, PAX8.
In this study, our unbiased global SILAC analysis revealed that HBV enhanced UBXN7 ubiquitination. Further studies showed that HBx interacted with UBXN7 to induce K48 ubiquitination of UBXN7 for its degradation via the proteasome pathway, suggesting that HBx modulated UBXN7 at the post-translational level in a role distinct from a viral transactivator.
UBXN7, a member of the UBA-UBX family, can interact with a large number of E3 ubiquitin ligases and serves as the substrate-binding adaptor
and contains 4 domains: UBA domain, UAS domain, UIM motif, and UBX domain. Earlier studies have identified binding partners for UBA, UIM, and UBX domains. Here, we discovered that the UAS domain can bind to IKK-β and enhance its K48 ubiquitination.
NF-κB plays a central role in HBV-relative hepatocarcinogenesis. In unstimulated cells, NF-κB is bound to IκBα (the inhibitory protein of NF-κB) and remains in the cytoplasm. Extracellular stimuli lead to activation of the IκB kinase (IKK) complex and phosphorylates IκBα for its degradation, which results in p65/p50 release from IκBα and subsequent translocation from the cytoplasm to the nucleus. Although previously a number of reports suggested that HBx and large HBs activate NF-κB to induce HBV replication,
On the other hand, Jing et al found that HBx could not co-precipitate with the subunits p65 or p50 and the suppressor IκBα and suggested that HBx affected NF-κB signaling pathways through regulating the Notch signaling pathway in L02 cells.
hinted that HBx physically interacts with p22-FLIP and NEMO and potentially forms a ternary complex to induce NF-κB activation. Our findings here reveal a novel mechanism by which HBx regulates NF-κB activation via regulating the stability of IKKs kinase. Our results showed that HBx interacted with UBXN7 to promote ubiquitination and degradation of UBXN7. This in turn prevents UBXN7-induced destabilization of IKK-β, leading to NF-κB activation under HBV infection. Our results appear to differ from those of Hu et al,
who reported that UBXN1, N9, and N11, which are the same family of UBXN7, blocked the canonical NF-κB pathway by binding to Cullin1 to inhibit IκBα degradation for re-activating quiescent human immunodeficiency virus from latent viral reservoirs in chronically infected individuals. In addition, our results showed that UBXN7 selectively binded to IKK-β via ULD domain but not to IκBα, p65, TAK1, and IKK-α, suggesting that IKK-β may be a natural target of UBXN7-mediated ubiquitination in the absence of HBV infection. Furthermore, our mutational studies suggested that the UAS domain of UBXN7 was essential for binding to IKK-β and its effect on HBV replication. Therefore, UBXN7 is a crucial regulator in HBV viral transcription and subsequent viral protein production. In fact, reduction of IKK-β protein level by UBXN7 only occurred in the absence of HBx expression, suggesting that the effect of UBXN7 on IKK-β protein levels is also dependent on HBx, most likely via the interaction between HBx and DDB1-containing E3 ubiquitin ligase. Thus, UBXN7 treatment may lead to destabilization of IKK-β, which may contribute to reduced activates NF-κB signaling. In addition, because NF-κB signaling is involved in hepatocellular carcinogenesis, reducing NF-κB signaling may also reduce hepatocellular carcinogenesis in patients with chronic hepatitis B. Further investigations into this are clearly warranted.
In summary, we have shown that HBx interacts with UBXN7 to promote K48-linked ubiquitination of UBXN7 for its degradation. Our results reveal that IKK-β is the first reported binding partner of the UAS domain of UBXN7 and that its interaction with UBXN7 leads to increased IKK-β ubiquitination and proteasomal degradation. We further show that this UBXN7-induced degradation of IKK-β decreases the activation of NF-κB signaling and autophagy, thereby inhibiting HBV replication. Moreover, we have shown that HBx can promote HBV replication via degradation of UBXN7, thus maintaining high levels of IKK-β to activate NF-κB signaling and NF-κB–dependent autophagy (Figure 8F). Our findings suggest that UBXN7 can be targeted for potential new therapies in HBV-related diseases.
Materials and Methods
HEK293, Huh7, and HepG2 cells were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum. HepAD38 and HepG2-NTCP were maintained in the same medium but with 50 μg/mL Collagen Type 1 Rat tail spreading on the bottom in advance. Tetracycline can suppress HepAD38 to replicate HBV. HepG2.2.15 was cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum and 400 μg/mL G418. PHHs were purchased, cultured, and infected as described.
All cells were maintained at 37°C in humidified CO2 (5%) incubators.
Plasmids and Transfection
The pHBV1.3 plasmid was provided by Guangxia Gao (Chinese Academy of Sciences, China). pEF-UBXN7 and pEF-IKK-β were generated by PCR cloning into pEF vector. UBXN7 mutants with an arginine substitution at residue lysine 84 (K84R) and lysine 99 (K99R) residues were generated by PCR-based mutations on pEF-UBXN7. Multiple truncated mutants of UBXN7 (Δ-UBA, Δ-UAS, Δ-UIM, Δ-UBX, 1-261, 1-305, and 136-489) and IKK-β (1-680, 1-400, 1-308, 308-756, and 400-756) were generated by PCR-based mutations on pEF-UBXN7 or pEF-IKK-β. NF-κB luciferase reporter plasmid, pEF-IκBα, pEF-P65, pEF-TAK1, pEF-IKK-α, pRL-TK, pRK-Ub, and pRK-Ub mutant plasmids were generated by PCR cloning into pEF or pRK vector as described.
Cyclohexamide (C7698), chloroquine (C6628), ammonium chloride (A9434), MG-132 (M7449), and protease inhibitor cocktail (P1860) were purchased from Sigma-Aldrich (St Louis, MO). Tetracycline (MB5564) was purchased from Meilunbio (Dalian, China). Anti-phospho-IKKα/β (#2697), anti-total-IKKβ (#2684), anti-phospho-IκBα (#9246S), anti-total-IκBα (#4812S), and anti-LC3I/II (#4108) antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-β-tubulin (10094-1-AP), anti-GAPDH (10494-1-AP), anti-Myc-Tag (16286-1-AP), anti-Flag-Tag (20543-1-AP), and anti-HA-Tag (51064-2-AP) antibodies were purchased from Proteintech (Wuhan, China). Anti-HBcAg (MAB16990) antibody was purchased from Millipore (Temecula, CA). Anti-UBXN7 (NBP 2-2 2223) antibody was purchased from Novus Biologicals (Littleton, CO). Horseradish peroxidase–conjugated secondary antibody (71045-M and 12-348) and Flag Beads (M8823) were purchased from Sigma-Aldrich. Collagen Type 1 Rat tail (354236) was purchased from Corning (New York, NY).
Small interfering RNAs (siRNAs) (stB0010599) were purchased from RiboBio (Guangzhou, China). Cells were co-transfected with 3 siRNAs targeting UBXN7 at a concentration of 50 nmol/L according to the manufacturer’s instructions, and protein or RNA was extracted 48 hours after transfection for experiments.
RNA Extraction and Real-Time Quantitative Polymerase Chain Reaction
Total RNA was extracted from cells by using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA) and subjected to reverse transcription by using M-MLV Reverse Transcriptase Kit (Invitrogen, Thermo Fisher Scientific). Real-time quantitative PCR was performed with the SYBR Select Master Mix (Life Technologies, Carlsbad, CA) with indicated primers.
The HepG2.2.15 and HepG2 cells were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum and either the “light” form [12C6]-L-lysine/[12C614N4]-L-arginine or the “heavy” form [13C6]-L-lysine/[13C615N4]-L-arginine for more than 7 generations by using SILAC Protein Quantitation Kit according to manufacturer’s instructions. After SILAC labeling, the cell samples were collected for protein extraction, trypsin digestion, high-performance liquid chromatography fractionation, affinity enrichment, and liquid chromatography with tandem mass spectrometry analysis to identify the host protein and ubiquitination changes.
Co-Immunoprecipitation and Western Blot Analyses
Cells were co-transfected with 5 μg each of 2 differently tagged plasmids for 48 hours. Cells were lysed with IP buffer: 20 mmol/L Tris-Cl (pH 8.0), 150 mmol/L NaCl, 2 mmol/L EGTA, 1% NP-40, and protease inhibitor cocktails. The lysates were centrifuged at 12,000 rpm for 5 minutes. Equal number of supernatants were incubated with anti-Flag- or anti-Myc-beads overnight at 4°C. Beads were washed 4 times with beads washing buffer (20 mmol/L Tris-Cl [pH 7.5], 300 mmol/L NaCl) before Western blot analysis. For Western blot, protein samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on a 4%-20% gradient Tris-glycine gel and transferred to a nitrocellulose membrane. The membranes were blocked for 1 hour at room temperature with 5% skim milk in TBS-T (0.1% Tween 20), followed by incubation overnight at 4°C with primary antibodies. After washing in TBS-T 3 times, the membranes were incubated with horseradish peroxidase–conjugated secondary antibodies for 1 hour at room temperature. The immunoreactive bands were visualized with enhanced chemiluminescence system (Yeasen, China).
Northern and Southern Blot Analysis
Cells were co-transfected with 10 μg plasmids for 48 hours. Total RNA was electrophoresed in a 1% agarose formaldehyde gel and was transferred onto a nylon membrane. Intracellular HBV core particle-associated DNA was extracted, and Southern blot analysis was performed as described previously.
The blot was detected by using the DIG Northern starter kit (Roche Diagnostics, Indianapolis, IN) for Northern blot corresponding to nucleotides 1072 to 2171 of the HBV genome and for Southern blot corresponding to nucleotides 157 to 1068 of the HBV genome.
Luciferase Activity Assay
Cells were co-transfected with 200 ng firefly luciferase reporter vector, 20 ng internal control renilla luciferase control vector (pRL-TK), and either 400 ng UBXN7 expression construct or control vector for 36 hours. Luciferase activities in cell lysates were detected by using the Dual-Glo system (Promega, Madison, WI). The firefly luciferase activity was normalized by renilla luciferase activity.
Enzyme-Linked Immunosorbent Assay
Cells were co-transfected with 1 μg pHBV1.3 and 1–2 μg of either UBXN7 expression construct or the control vector for 48 hours. The levels of HBV surface antigen and HBV e antigen in the supernatant were determined by using ELISA (Kehua Bio-engineering, Shanghai, China).
Hydrodynamics-Based Transfection in Mice
BALB/c mice at 10 weeks old were purchased from the Hubei Provincial Center for Disease Control and Prevention (Wuhan, China). Briefly, 10 μg pHBV1.3 and 20 μg of either UBXN7 expression construct or the control vector were pre-mixed with normal saline in a volume equivalent to 10% body weight and injected via tail vein within 6–8 seconds. Ninety-six hours later, the liver tissue was isolated for immunohistochemical staining and Western blot analysis. Sera were collected for ELISA analysis of HBV surface antigen and HBV e antigen. All mice were housed in a pathogen-free mouse colony, and the animal experiments were performed according to the Guide for the Care and Use of Medical Laboratory Animals (Ministry of Health, People’s Republic of China, 1998).
Autophagosome Formation Assay
Cells were co-transfected with 500 ng GFP-LC3 and 1 μg of either UBXN7 (WT or ΔUAS) expression construct or the control vector for 48 hours. The cells were fixed, and the nuclei were stained with 4-6-diamidino-2-phenylindole (Promoter Biotechnology). The green fluorescent protein-LC3 fluorescence was observed under a confocal fluorescence microscope (Leica SP8, Wetzlar, Germany).
All experiments were performed at least 3 times. Statistical significance was determined by using unpaired Student t test between 2 groups. P <.05 was considered significant (∗P < .05, ∗∗P < .01, ∗∗∗P < .001). All data analyses were carried out by using the statistical analysis software GraphPad Prism, version 6.0 (GraphPad Software, San Diego, CA).
The authors thank Dr Yun-Bo Shi, a senior investigator at NIH/NICHD, for critically revising the manuscript.
Conflicts of interest The authors disclose no conflicts.
Funding Supported by the National Natural Science Foundation of China (81572447, 31871427, and 31370187). The funders had no role in the design of the study, data collection and interpretation, or writing the manuscript.