Background
Hepatitis B virus (HBV) infections annually cause 1 million of deaths worldwide[
1,
2]. Antiviral therapy is an important way to improve the prognoses of these victims. Hepatitis B surface antigen (HBsAg) loss or seroconversion is thought as a perfect endpoint of current antiviral therapy. However, hepatitis B e antigen (HBeAg) seroconversion (HBeAg serological response) and undetectable HBV DNA (virological response) are common goals to be pursued in clinical practice since on-treatment HBsAg loss or seroconversion is difficult to realize, but it would automatically occurs in patients from years to decades after HBeAg seroconversion. HBeAg is a well-known immune-toleragen[
3]. Its persistence is an independent risk factor for hepatocellular carcinoma and is associated with a lower survival rate among cirrhotic patients[
4,
5]. In contrast, HBeAg seroconversion is thought to be important in establishing a benign prognosis[
6,
7]. Compared with virological response only, virological response plus HBeAg serological response have a low relapse relate while off treatment of current antiviral therapy[
7]. Though those patients with HBeAg seroconversion (HBeAg-negative chronic hepatitis B) due to infection of HBeAg-defective variants also have poor prognoses, the significance of HBeAg to HBV infection is not minimized since these variants rarely cause a
de novo chronic infection[
8], implying that HBeAg loss may be helpful for termination of chronic HBV infection. Therefore, early antiviral intervention in HBeAg-positive chronic hepatitis B may benefit all patients. In addition, early therapeutic intervention is helpful to reduce the risks for long-term complications while on-treatment[
9,
10]. However, current antiviral options including recombinant interferon and nucleoside/nucleoside analogs cannot rapidly and economically realize the dual goals of the antiviral therapy. For example, nucleoside analog entecavir (ETV) blocks HBV replication rapidly, but induce HBeAg seroconversion unpredictably. For these reasons, ETV combined with some direct HBeAg secretion-inhibitory measures seems a strategy to improve the current antiviral therapy of chronic hepatitis B.
HBeAg is encoded by the C open reading frame of the viral genome. This frame also encodes viral core protein (also called hepatitis B core antigen, HBcAg, 21 kDa). Compared with HBcAg, the initial peptide of HBeAg has an extra precore region consisting of a 19-amino acid signal peptide that directs the nascent peptide into the secretory pathway. After the signal peptide is removed in the lumen of the endoplasmic reticulum, the HBeAg precursor is generated and transported to the
trans-Golgi network. The HBeAg precursor (pre-HBe, 22 kDa) is further cleaved by proprotein convertase furin in the arginine-rich domains of the C-terminus to generate the mature HBeAg (17 ~ 20 kDa)[
11,
12]. Furin belongs to the subtilisin/kexin-like serine protease family. It is responsible for the majority of proprotein processing, and thus not only plays critical roles in normal cell growth and differentiation, but is also involved in many disease states, such as Alzheimer’s disease, tumoriogenesis, and infections[
13]. Our previous studies have shown that persistent HBV infection prefers to occur in patients carrying highly active genotypes of furin and furin inhibitors, decanoyl-RVKR-chloromethylketone (CMK) and hexa-D-arginine (D6R), reduce HBeAg secretion without interfering with cellular protein secretion in HepG2.2.15 cells[
14,
15]. In addition, HBeAg reduction resulted from furin inhibition leads to increase in cell surface expression of immune-promoting pre-HBe[
15], which is different from HBeAg reduction or loss caused by infection of HBeAg-defective variants, suggesting that furin inhibition may have less risk to let the infection develop into poorly prognostic HBeAg-negative chronic hepatitis B. Though it is not a traditional antiviral strategy, the direct inhibition of HBeAg secretion mediated by the immune-regulation effects may be helpful for the treatment of chronic HBV infection. Thus, the suppression of furin may be a promising candidate way to improve HBeAg serological response to ETV by inhibiting HBeAg secretion directly.
CMK and D6R are small synthetic furin inhibitors that are suitable for the clinical purpose. CMK is a referential furin inhibitor in many laboratories. It is more effective than D6R in reduction of HBeAg secretion[
15]. However, CMK and a mutation (T147A) adjacent to a putative furin recognition site (
151RRGR
154) in HBcAg of HBeAg-defective variant (carrying the precore stop mutation, G1896A) have been found to enhance HBV replication[
15,
16]. In the current study, we wonder whether the suppression of furin by other measures enhances HBV replication and the combination with ETV inhibits HBV replication and HBeAg secretion simultaneously. As results, furin suppressed by measures other than CMK did not enhance HBV replication. CMK was found to enhance HBV replication by inhibiting the trypsin-like (TL) activity of proteasomes. The viral replication-enhancing effect of CMK was abrogated by ETV. ETV combined with CMK could simultaneously reduce HBV replication and HBeAg secretion. These findings highlight a novel approach to improve the antiviral therapy for chronic HBV infections.
Discussion
The current antiviral therapy of chronic HBV infection pursues the dual goals, virological and HBeAg serological responses; however, the later seems difficult to realize. Our previous study has shown that furin activity correlated with the outcome of HBV infection and furin inhibitors can significantly reduce HBeAg secretion without interference with the secretion of cellular secretory proteins[
14,
15]. Unfortunately, CMK, the more effective inhibitor, enhances HBV replication[
15,
16]. In this study, the suppressions of furin using D6R and the expression of furin inhibitory prosegment were not found to enhance HBV replication and HBV replication enhanced by CMK was found to correlate with its redundant inhibitory effect on the TL activity of proteasomes, suggesting that furin inhibition itself does not promote HBV replication. Furthermore, ETV combined with CMK was found to reduce HBV replication and HBeAg secretion simultaneously, which are concordant with the virological and serological responses pursued with a vengeance in antiviral practice.
HBV replication enhancements of CMK and the mutation (T147A) are accompanied by intracellular HBcAg accumulation[
16], and HBcAg is structural protein with its intracellular level usually correlating with viral replication level[
18,
19]. The findings support that HBcAg accumulation accounts for the HBV replication enhancement of CMK and the mutation. Because HBcAg and pre-HBe have identical furin-sensitive arginine-rich domains, the above findings also suggest that furin may proteolyze HBcAg. Therefore, it is reasonable to concern the unfavorable HBV replication-enhancing effect of furin inhibition. Fortunately, the suppression of furin itself was not found to enhance HBV replication in this study, which was evidenced by the facts: (i) D6R incubation and furin inhibitory prosegment expression inhibited HBeAg secretion, but did not enhance HBV replication; (ii) CMK enhanced HBV replication, but which was via a redundant inhibitory effect on the TL activity of proteasomes.
As furin is involved in the maturation of membrane fusion proteins and pro-toxins of bacteria and viruses, furin inhibition using inhibitors is viewed as a potential therapeutic strategy for anthrax, influenza A and Ebola virus infection[
13,
29,
30].The significance of HBeAg to persistent HBV infection[
8], the correlations of furin activity with the outcomes of HBV infection[
14], and the lack of influences on cell secretory function[
15], and HBV replication in this study suggest that the suppression of furin is also a promising novel strategy for the antiviral therapy of HBV infection in the future. Due to the lack of natural inhibitors, furin inhibitors used at present all are man-made compound. Although many researchers make effort to develop new inhibitors[
24,
29,
30], no medicinal inhibitors are available at present. CMK and D6R are small synthetic inhibitors suitable for clinical purpose. CMK has used as a reference furin inhibitor in cell-based tests and inhibits HBeAg secretion in HepG2.2.15 cells[
15,
16]; however, it enhances HBV replication, which is in conflict with the goal of antiviral therapy. Compared with CMK, D6R is more sensitive in cell-free test system or targeting furin on the cell surface, but less effective in inhibiting HBeAg secretion in cell-based test system[
15,
24,
31,
32], perhaps due to its poor permeability[
24]. For these reasons, the development of new furin inhibitors is imperative in the future. Our findings, including those in this study, are helpful to determine the target and orientation of the development process, paying attention to the specificity, especially not to affect the TL activity of proteasomes, and the permeability to reach the
trans-Golgi network.
Since the successful development of new inhibitors of furin has a long way to go, to make use of current inhibitors is a reasonable option. In this study, ETV was found to abrogate the HBV replication enhancement of CMK, and ETV combined with CMK inhibited HBV replication and HBeAg secretion simultaneously in HepG2.2.15 cells. Although no synergistic effect was observed, these results imply that it would be possible to realize the virological and serological responses of the antiviral therapy rapidly and economically. However, much more studies should be carried out to clarify the effectiveness and safety of the combination strategy
in vivo. Theoretically, ETV abrogates HBV replication enhancement but HBcAg accumulation of CMK. The remained HBcAg accumulation may have a chance to induce the aggravation of the inflammation and cell injury by increasing the presentation of HBcAg epitopes. It is less likely, however, to lead to fatal liver dysfunction since the generation of these epitopes depends on cellular proteasomes that have been inhibited by CMK. Of course, new effective inhibitors without effects on HBcAg accumulation would be the first choice of the combination therapy in the future. Besides HBeAg, CMK and furin knockdown with small interfering RNA lead to the reduction of HBsAg[
33]. Our previous study further demonstrates that CMK only at higher concentration (100 μmol/L) suppresses the biosynthesis of HBsAg[
15]. In this study, HBsAg-reducing effect of CMK was not found, perhaps due to the lower concentration (20 μmol/L) employed. Nonetheless, it is important to pursue the inhibitory effect on HBsAg in novel inhibitor development or therapy regimen establishment in the future since HBsAg seroconversion is the perfect end-point of antiviral therapy for chronic HBV infection.
Conclusions
Furin-inhibiting measures other than CMK did not enhance HBV replication and CMK enhanced HBV replication by affecting the TL activity of proteasomes, suggesting that furin inhibition itself does not lead to HBV replication. ETV could completely abrogate the HBV replication enhancement of CMK. Moreover, ETV combined with CMK reduced HBV replication and HBeAg secretion simultaneously in HepG2.2.15 cells, which implies that nucleotide/nucleoside analogs combined with some furin inhibitors may be an easy way to realize the dual goals of antiviral therapy. Nonetheless, more studies on the effectiveness and safety of the combination strategy in vivo are warranted in the future.
Methods
Plasmid construct
Furin inhibitory prosegment-expressing vector (pfurin-PS) was constructed using plasmid pIRES2-EGFP (Clontech, Palo Alto, CA). The sequence of the inhibitory prosegment was designed from those coding 109 amino acids of the N-terminus of furin (gene ID: 5045). The sequence of the construct had been confirmed using DNA sequencing.
Cell culture, transfection, and protease inhibitor treatments
HepG2.2.15 cells were regularly grown in Dulbecco’s modified Eagle’s medium, supplemented with 10% (vol/vol) fetal calf serum and 380 μg/mL of geneticin if necessary. Transient transfection was performed using FuGENE HD transfection reagent (Roche Applied Science, Indianapolis, IN). Cells were treated with 10 ~ 50 μmol/L CMK (EMD Biosciences, La Jolla, CA, USA) or 100 μmol/L D6R (EMD Biosciences) with or without 30 nmol/L ETV (Sigma-Aldrich Corporation, St. Louis, MO, USA) for 48 hours in a growth arrest medium containing 0.5% (vol/vol) fetal calf serum after confluent growth. The cells (107) were harvested to evaluate HBV replication and viral antigen expression. To perform virion release and cell viability assays, cells were further cultivated using fresh medium for 12 hours. To evaluate the turnover rate of HBcAg, cells were treated with or without cycloheximide, a protein synthesis inhibitor (Sigma-Aldrich Corporation, St. Louis, MO, USA), and harvested in 12 hour intervals to a maximum of 48 hours.
Detections of core-associated HBV DNA
The isolation of supernatant and intracellular core particles was performed as reported[
34]. Sampling was balanced based on the protein level in cell lysate. Supernatant core-associated HBV DNA was quantitatively analyzed using commercial real-time fluorescent polymerase chain reaction (PCR) kits (Daan Gene Inc., Guangzhou, China). The intracellular core-associated HBV DNA was detected used Southern blot analysis. The isolated DNA was separated and transferred onto nylon membranes (Roche Applied Science, Indianapolis, IN, USA). After hybridized with digoxigenin-labeled DNA probes, all membranes were incubated with horseradish peroxidase-labeled anti-digoxigenin antibody (Roche Applied Science), and developed with an enhanced chemiluminescence reagent (Invitrogen Corporation, Shanghai, China).
Detections of intracellular viral antigens, furin inhibitory prosegment and proteasome subunits
For the detections of proteasome subunits or intercellular HBeAg, pre-HBe and HBcAg, total cellular protein or cytosolic and non-cytosolic cellular proteins were extracted as reported[
20]. The total or sorted cellular proteins were separated and transferred onto polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA, USA) using standard techniques. Immunoblot analysis was performed using polyclonal antibodies to HBcAg (DAKO, Carpinteria, CA, USA), furin (LS-C23720; LifeSpan BioSciences Inc., Seattle, WA, USA) or the proteasome subunits (ab22673; Abcam, Cambridge, UK).
Protease digestion assay
The trypsin (Sigma-Aldrich Corporation) digestion of recombinant HBcAg, a fragment of 156 amino acids with a 6 × histidine (his)-tag in the C-terminus (Millipore Corporation), was performed as described elsewhere[
22]. To study the putative proteolysis of recombinant HBc by the cytosolic proteases of cells, cytosolic extract was prepared from 10
6 HepG2 cells by incubating for 3 minutes on ice with 1 mL of a digitonin buffer (50 mmol/L Tris [pH 8], 150 mmol/L NaCl, and 22.5 μg/mL digitonin), and centrifuging at 1500 × g for 2 minutes at 4°C.
Proteasome activity assay
The TL, chymotrypsin-like, and caspase-like activities of the proteasomes were measured using commercial cell-based kits (Proteasome-Glo
TM, Promega, Madison, WI, USA) as described previously[
35]. Luminescence was recorded using a luminometer (Promega).
Statistical analysis
The differences in virion release, HBeAg and HBsAg secretions and proteasome activities were analyzed using the Student’s t-test. A P < 0.05 was considered statistically significant. All statistical analyses were conducted using SPSS software (version 11; SPSS, Inc., Chicago, IL, USA).
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Competing interests
The authors declare that they have no known competing interests.
Authors’ contributions
Conception of the idea and design of the experiments are due in part to XMP. The manuscript was written and drafted by XMP, HYY and NQZ. Western Blot, Southern Blot and proteasome activity analysis were conducted by HYY, NQZ, DML and LG. All authors read and approved the final manuscript.