Hepatocyte Endoplasmic Reticulum Stress Inhibits Hepatitis B Virus Secretion and Delays Intracellular Hepatitis B Virus Clearance After Entecavir Treatment

Background: The aim of this study was to explore the effects of endoplasmic reticulum (ER) stress on hepatitis B virus (HBV) replication and the antiviral effect of entecavir (ETV). Methods: Thapsigargin (TG) and stearic acid (SA) were used to induce ER stress in HepG2.2.15 cells and HepAD38 cells that contained an integrated HBV genome, while ETV was used to inhibit HBV replication. The expression levels of glucose-regulated protein 78 (GRP78) and phosphorylated eukaryotic translation initiation factor 2 subunit alpha (p-eIF2α) were measured by western blotting. Intracellular HBV DNA was determined by qPCR; HBsAg by western blotting; HBV RNA by real-time RT-qPCR; HBsAg and HBeAg in supernatants by enzyme-linked immunosorbent assay (ELISA); and HBV DNA in supernatants by qPCR. Results: TG and SA induced ER stress in HepG2.2.15 cells and HepAD38 cells from 12 to 48 h post treatment. However, 4-phenylbutyric acid (PBA) partly alleviated the TG-induced ER stress. Moreover, TG inhibited HBsAg, HBeAg, and HBV DNA secretion from 12 to 48 h, while different concentrations of SA inhibited HBsAg and HBV DNA secretion at 48 h. TG promoted intracellular HBV DNA and HBsAg accumulation and the transcription of the HBV 3.5-kb mRNA and S mRNA. PBA treatment restored the secretion of HBsAg and HBV DNA. Finally, ER stress accelerated extracellular HBV DNA clearance but delayed intracellular HBV DNA clearance after ETV treatment. Conclusions: Hepatocyte ER stress promoted intracellular HBV DNA and HBsAg accumulation by inhibiting their secretion. Our study also suggested that hepatocyte ER stress delayed intracellular HBV DNA clearance after ETV treatment.


INTRODUCTION
Chronic hepatitis B virus (HBV) infection constitutes a global health concern. More than 240 million people are chronically infected with HBV, which puts them at great risk of developing end-stage liver diseases such as liver failure, cirrhosis, and liver cancer (1). The progression of patients with chronic HBV infection depends on a complex interaction between the host and the virus (2).
The HBV genome contains four partially overlapping open reading frames, named C, S, P, and X, from which 3.5-, 2.4-, 2.1-, and 0.7 kb-long RNAs, respectively, are transcribed. Translation of the 2.4-and 0.7-kb fragments produces the large envelope protein and the HBx protein, respectively. The 2.1-kb mRNA yields both the middle and small envelope proteins. The 3.5kb long RNA, also called pregenomic RNA, is the only HBV transcript required for genome replication (1,3).
The endoplasmic reticulum (ER) is an important cellular organelle and plays a major role in protein synthesis, folding, modification, and transport, as well as in lipid synthesis and the maintenance of calcium homeostasis. When ER homeostasis is disturbed under physical or pathological stimuli, such as the disruption of calcium homeostasis, the accumulation of unfolded or misfolded proteins, glucose starvation, and hypoxia, the unfolded protein response (UPR) is activated (4). ER stress can initiate the UPR via the binding of glucose-regulated protein 78 (GRP78) to unfolded or misfolded proteins and by activating three different sensors: protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6).
ER stress is a common pathological phenomenon in patients with liver disease, including viral hepatitis, fatty liver disease, alcoholic liver disease, drug-induced hepatitis, and ischemic liver damage, among others (5,6). Viruses, fatty acids, alcohol, and drugs can all induce ER stress in hepatocytes, which can damage these cells and lead to metabolic disorders.
HBV and hepatocytes have a complex and close interaction. The activation of HBV enhancers largely depends on cellular factors expressed in hepatocytes (7,8). Transcription factors in hepatocytes such as CCAAT/enhancer-binding protein (C/EBP), cyclic-adenosine monophosphate-responsive element-binding protein (CREB), and several other nuclear factors have been found to play an important role in HBV enhancer activation (8,9). In the HBV life cycle, secretory proteins, such as HBsAg and HBeAg, are folded and assembled in the ER of hepatocytes (10). However, how hepatocyte ER stress affects HBV replication, and the antiviral effect of nucleos(t)ide analogs, remains unknown. In this study, we employed thapsigargin (TG) and stearic acid (SA) as ER stress inducers in HBV-transfected HepG2.2.15 cells and HepAD38 cells to investigate the impact of ER stress on HBV replication and the antiviral efficacy of entecavir (ETV). h, whereas ETV (10 µM) and TG (1 µM) were simultaneously administered for the same length of time. SA (0.017 g) was dissolved in 3 ml of 0.1 mM NaOH in a water bath at 100 • C. Stock solutions were prepared by adding 3 ml of 40% fatty acid-free bovine serum albumin to each tube and incubating for 30 min at 55 • C. The final SA concentration was 10 mM. All control conditions included the corresponding vehicles at the appropriate concentrations.

Flow Cytometry
Apoptosis was determined using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit following the manufacturer's instructions. Briefly, 2 × 10 6 cells were harvested and washed twice with precooled PBS and resuspended in 500 µl of binding buffer. Then, 5 µl of Annexin V-FITC and 5 µl of PI was added to each sample followed by incubation at room temperature in the dark for 10 min. Analysis was performed by flow cytometry (Beckman Coulter Gallios, USA) according to the manufacturer's specifications.

Quantification of HBsAg and HBeAg
HepG2.2.15 and HepAD38 cells were cultured at a density of 1 × 10 4 cells per well in RPMI 1640 medium. After drug treatment, the levels of HBsAg and HBeAg in the supernatants were measured using an enzyme-linked immunosorbent assay (ELISA) (Kehua Bio-engineering Corp.) according to the manufacturer's recommendations.

Statistical Analysis
The results were expressed as means ± standard deviation for "n" independent observations. One-way ANOVA was used to determine the statistical differences between the mean values for the groups. The level of significance was set at P < 0.05.   (Supplementary Figures 1, 2). Furthermore, 1 µM of TG significantly induced GRP78 and p-eIF2α expression from 12 to 48 h post treatment in HepAD38 cells ( Figure 1C). Our results demonstrated that both TG and SA could induce ER stress and activate the UPR in HepG2.2.15 cells and HepAD38 cells.

Thapsigargin and Stearic Acid Treatment
Next, we investigated whether PBA could alleviate the ER stress induced by TG (Figure 1D). We found that at the 1-mM concentration, PBA could significantly inhibit the TG-mediated induction of GRP78 expression, demonstrating that PBA could alleviate TG-induced ER stress.
We next evaluated the effect of TG and SA on the apoptosis and viability of HepG2.2.15 cells. At the concentration of 1 µM, TG treatment did not lead to a marked increase in the rate of apoptosis from 12 to 48 h post treatment, and a similar effect was observed for SA treatment (50 and 100 µM) at 48 h (Supplementary Figure 3).  Figure 2C). Western blotting results confirmed that TG significantly inhibited the secretion of HBsAg from 12 to 48 h. However, SA treatment did not significantly affect the secretion of HBeAg, at the concentration of either 50 or 100 µM (Figure 3). At 100 µM, SA also significantly inhibited the   Figures 4-7).

The Effects of Thapsigargin on the Transcription of Hepatitis B Virus DNA and Intracellular HBsAg and Hepatitis B Virus DNA Levels
We further explored the effects of TG on the expression levels of the 3.5-kb mRNA and S mRNA as well as on the intracellular levels of HBsAg and HBV DNA. As shown in Figure 4, TG significantly upregulated the expression of the HBV 3.5-kb mRNA fragment and S mRNA and increased the intracellular levels of HBsAg and HBV DNA. Western blotting confirmed that the intracellular level of HBsAg was significantly increased by TG from 12 to 48 h post treatment.

Thapsigargin Inhibited the Secretion of HBsAg and Hepatitis B Virus DNA via the Induction of Endoplasmic Reticulum Stress
We then investigated whether the inhibitory effects of TG on HBsAg and HBV DNA secretion were exerted through the induction of ER stress. As shown in Figure 5, the secretion of HBsAg and HBV DNA was significantly restored by PBA at 36 h post treatment. Similarly, HBeAg levels in the supernatant were also partially restored. These results strongly suggested that TG inhibited HBsAg, HBeAg, and HBV DNA secretion through the induction of ER stress.

Thapsigargin Delayed Intracellular Hepatitis B Virus Clearance After Entecavir Treatment
Finally, we assessed whether the TG-induced ER stress impaired the antiviral effect of ETV. After 96 h of ETV treatment, HBV replication was significantly inhibited (Figure 6). TG treatment significantly delayed the intracellular clearance of HBV DNA, HBsAg, and HBV RNA; in contrast, TG treatment significantly accelerated the extracellular clearance of HBV DNA, HBsAg, and HBeAg.

DISCUSSION
HBV assembly and HBsAg synthesis and secretion are highly dependent on ER function (10,12). Each of the HBV envelope proteins is cotranslationally inserted into the ER membrane. The middle and small proteins have single transmembrane domains, whereas the large protein has a unique dual transmembrane conformation topology in the ER. The large protein is modified by myristoylation, but this is dispensable for virion formation (13)(14)(15). Host chaperones, such as heat shock protein 70 (HSP70) and GRP78/Bip, play an important role in the correct folding of envelope proteins and the maintenance of the dual topology of the large protein in the ER (16,17). The synthesis of HBeAg is also closely associated with the ER (18,19). As HBV replication and antigen secretion in vivo are both dependent on a complex interaction between the host and the virus, such as the activation of immune cells and the regulation of cytokine levels, it is difficult to identify the direct effect of ER stress on HBV replication in vivo. In this study, we used TG and SA to induce ER stress in HepG2.2.15 cells containing an integrated HBV genome and HepAD38 cells, and the results obtained mostly reflect the direct effect of ER stress on HBV replication.
In this study, we found for the first time that hepatocyte ER stress greatly suppressed HBsAg and HBV DNA secretion and increased the intracellular levels of HBsAg and HBV DNA. How ER stress affects HBV replication remains unknown. Several studies have reported that the accumulation of mutated HBsAg in hepatocytes results in ER stress and the activation of the UPR both in vitro and in vivo (20)(21)(22). However, the results regarding the effect of hepatocyte ER stress on HBV replication have been contradictory, and several different mechanisms have been suggested to be involved. Xu et al. reported that hepatocyte ER stress induced by the retention of the large protein of the HBV can activate the S promoter; increase the synthesis of the middle and small proteins to restore the proportions of the large, middle, and small proteins in cells; and maintain the assembly of HBV particles (23). Additionally, the authors also found that the transcriptional activation of the S promoter was cell type-restricted and was mediated through the IRE1α/X-box binding protein 1 (XBP1) pathway. A different study demonstrated that, in HBV-infected hepatoma cells, the ER stress-associated degradation pathway was activated, leading to the degradation of the HBV envelope protein and the consequent inhibition of HBV replication (24). Cisplatin-induced ER stress in hepatocytes has also been found to evoke HBV reactivation via the peroxisome proliferatoractivated receptor gamma coactivator 1 alpha (PPARGC1A) signaling pathway (25). GRP78 is a master ER stress regulator, the expression of which is significantly upregulated during ER stress. Several studies have demonstrated that GRP78 expression can inhibit HBV DNA replication (26)(27)(28). We do not know the reasons for these differing results. In this study, we used TG and SA to induce ER stress, and we found that both inhibited HBV DNA and HBsAg secretion, while TG also promoted intracellular HBV DNA and HBsAg accumulation. PBA is a lowmolecular-weight chemical chaperone that effectively prevents misfolded protein aggregation and alleviates ER stress. After PBA administration, HBV DNA and HBsAg secretion were partly restored. These results demonstrated that the inhibitory effects of TG and SA on HBV DNA and HBsAg secretion were at least partly dependent on the induction of ER stress.
The ER is the largest Ca 2+ store in hepatocytes. Ca 2+ release from the ER is a common mechanism underlying the occurrence and progression of ER stress following both physical and pathological cues. TG induces ER stress by irreversibly inhibiting sarcoplasmic/ER Ca 2+ -ATPase, which blocks the reabsorption of calcium from the cytosol to the ER and leads to an increase in the cytosolic Ca 2+ concentration (29). Cytosolic Ca 2+ can stimulate the Pyk2/Src kinase signal transduction pathway, thereby activating HBV reverse transcription and DNA replication (30). However, one study reported that HBV DNA replication was inhibited following the blocking of the ER Ca 2+ -ATPase (12). How SA, a saturated fatty acid, induces ER stress remains unclear. High saturated fatty acid concentrations have been found to impair ER structure and function (31). In our study, both TG and SA inhibited the secretion of HBV DNA and HBsAg. However, these effects were partially attenuated following PBA-mediated alleviation of ER stress. These results further supported that the inhibition of HBV DNA and HBsAg secretion by TG and SA was partly mediated via the induction of ER stress. However, whether TG promoted the transcription of both the HBV 3.5-kb mRNA fragment and S mRNA via an increase in the cytosolic Ca 2+ concentration warrants further study.
Another major finding of this study was that, although ETV displayed high antiviral efficacy in the presence of TG, treatment with TG accelerated extracellular HBV DNA, HBsAg, and HBeAg clearance but delayed the clearance of intracellular HBV DNA, HBsAg, and HBV mRNA. These results suggested that hepatocyte ER stress delayed intracellular HBV clearance. The intracellular HBV DNA and HBsAg accumulation induced by TG exposure may partly explain this result. However, the mechanism underlying the impaired HBsAg and HBV DNA secretion and the enhanced transcription of the HBV 3.5-kb mRNA and S mRNA under ER stress needs further clarification. Additionally, given that ER stress is a common pathological phenomenon in patients with liver disease, the clinical significance of the delayed intracellular HBV clearance under ER stress after ETV treatment should also be explored.
In conclusion, this study is the first to report that hepatocyte ER stress promotes intracellular HBV DNA and HBsAg accumulation by inhibiting HBV DNA and HBsAg secretion. Our study also suggested that hepatocyte ER stress delays intracellular HBV DNA clearance after ETV treatment.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
SL designed the experiments and wrote the manuscript. HC, MM, QL, HH, and GZ performed the experiments. YL and FY revised the manuscript and analyzed the data. All authors read and approved the final manuscript.

FUNDING
This work was supported by funding from the Chinese National Natural Science Foundation Project (81460124 and 81860114). The funders had no role in the study design and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study.