A Scorpion Defensin BmKDfsin4 Inhibits Hepatitis B Virus Replication in Vitro

Hepatitis B virus (HBV) infection is a major worldwide health problem which can cause acute and chronic hepatitis and can significantly increase the risk of liver cirrhosis and primary hepatocellular carcinoma (HCC). Nowadays, clinical therapies of HBV infection still mainly rely on nucleotide analogs and interferons, the usage of which is limited by drug-resistant mutation or side effects. Defensins had been reported to effectively inhibit the proliferation of bacteria, fungi, parasites and viruses. Here, we screened the anti-HBV activity of 25 scorpion-derived peptides most recently characterized by our group. Through evaluating anti-HBV activity and cytotoxicity, we found that BmKDfsin4, a scorpion defensin with antibacterial and Kv1.3-blocking activities, has a comparable high inhibitory rate of both HBeAg and HBsAg in HepG2.2.15 culture medium and low cytotoxicity to HepG2.2.15. Then, our experimental results further showed that BmKDfsin4 can dose-dependently decrease the production of HBV DNA and HBV viral proteins in both culture medium and cell lysate. Interestingly, BmKDfsin4 exerted high serum stability. Together, this study indicates that the scorpion defensin BmKDfsin4 also has inhibitory activity against HBV replication along with its antibacterial and potassium ion channel Kv1.3-blocking activities, which shows that BmKDfsin4 is a uniquely multifunctional defensin molecule. Our work also provides a good molecule material which will be used to investigate the link or relationship of its antiviral, antibacterial and ion channel–modulating activities in the future.


Introduction
Hepatitis B virus (HBV) is the prototypic member of hepadnaviridae and the pathogenic cause of acute and chronic type B hepatitis in humans. Chronic HBV infection, which now affects health of more than 400 million people around the world, has been demonstrated to result in a 100-fold elevation in the risk of developing hepatocellular carcinoma (HCC) [1]. Although effective vaccines have been extensively applied for years, the increase in the amount of infected individuals and the difficulties of treatment after infection are still perplexing to physicians and researchers. Interferon-α and nucleotide analogues are available for alleviating a patient's condition, but their therapeutic application is seriously limited by side effects and resistant mutated virus strains. Furthermore, combination therapies based on these drugs exerted unsatisfactory response efficiency [2,3]. Therefore, studies aimed at developing new, efficient anti-HBV agents are very urgently desired. Scorpions evolved particular venom systems during their hundreds of millions of years of evolution. For the sake of predation and defending predators, multiple types of neurotoxins have been generated, which can block or regulate the activities of a variety of ion channels [4][5][6][7]. In addition, anti-microbial peptides (AMPs) are also major components of scorpion venom peptides. These small peptides have exerted functional diversity, such as modulating innate immunity, regulating oncogenesis and directly inhibiting various bacteria, viruses, fungi and parasites [8][9][10]. In recent years, several studies about scorpion-derived antiviral peptides have been reported. For example, a scorpion venom-derived synthetic peptide, Mucroporin-M1, was reported to immediately interfere the virion of measles, SARS-CoV and influenza H5N1 viruses [11], and inhibit HBV replication in vitro and in vivo by activating the mitogen-activated protein kinase (MAPK) pathway and down-regulating HNF4α [10]. Mutations of another scorpion-derived peptide, Ctry2459, showed an extremely enhanced bioavailability and anti-HCV activity compared with the wild-type peptide [12]. In addition, another two scorpion venom peptides were also reported due to their inhibitory activity against herpes simplex virus type I [13]. Thus, scorpion venom is a resource of novel antiviral peptides.
In this study, we present a scorpion defensin BmKDfsin4, derived from the scorpion Mesobuthus martensii Karsch, which effectively inhibited HBV replication in vitro. BmKDfsin4 stood out from 25 scorpion peptides because of its high inhibition rate of HBeAg and HBsAg in the supernatant of HepG2.2.15 cells and relatively low cytotoxicity. Then, a further investigation of cytotoxicity and anti-HBV activity of BmKDfsin4 had been conducted which concluded that the inhibitory activity of BmKDfsin4 against HBV replication is concentration-dependent and its selective index (ratio of CC 50 to IC 50 ) is relatively high compared with its cytotoxicity. Furthermore, BmKDfsin4 also exerted considerable serum stability. BmKDfsin4 is the first reported scorpion defensin, which is a novel anti-HBV agent with potential for future modification and application.

Screening of Anti-HBV Agents from Scorpion-Derived Peptides
Fifteen putative peptides from the venomous cDNA libraries of four scorpion species (Mesobuthus martensii, Heterometrus petersii, Chaerilus tricostatus and Chaerilus tryznai) and 10 derivative peptides were identified as candidate antimicrobial agents and were prepared by the prokaryotic expression technique [14,15] or chemical synthesis [10,12,13] (Table S1). The anti-HBV activity of these peptides at 10 µM was then determined by measuring the concentrations of HBeAg ( Figure 1a) and HBsAg (Figure 1b) in cell culture medium of HepG2.2.15 cells by ELISA assays. BmKDfsin4 exerted a higher inhibitory activity against both HBeAg and HBsAg compared with any other scorpion-derived peptide. The inhibitory effect was 77.46% and 82.46%, respectively. The cytotoxicity of these peptides at 50 µM on HepG2.2.15 cells was measured by an MTT assay (Figure 1c). After being treated with BmKDfsin4 at 50 µM for 48 h, the cell viability of HepG2.2.15 cells was 83.18%, which indicates a relatively low cytotoxicity.

Cytotoxicity and Hemolysis of BmKDfsin4
The feasibility of the further development of BmKDfsin4 as a candidate anti-HBV agent was determined by measuring its cytotoxicity. After being incubated with a serial dilution of BmKDfsin4 for 48 h, the cell viability of HepG2.2.15 (Figure 2a), HepG2 ( Figure 2b) and L-02 ( Figure 2c) cells was measured using MTT assays. The 50% cytotoxicity concentrations (CC 50 ) of BmKDfsin4 to HepG2.2.15, HepG2 and L-02 were 167.82, 154.24 and 103.77 µM, respectively. At the concentration of 10 µM, the viability of the BmKDfsin4-treated cells was greater than 90% in all three kinds of cell lines. The hemolysis assay also showed that the viability of erythrocytes was more than 90% when the concentration of BmKDfsin4 was lower than 10 µM (Figure 2d). These data indicated that 10 µM or less BmKDfsin4 was minimally cytotoxic and suitable for further anti-HBV studies. ability against HBV by ELISA assays of HBeAg and HBsAg in the culture medium of HepG2.2.15 cells. The average inhibitory rate of BmKDfsin4 against HBeAg and HBsAg at 10 μM was 77.46% and 82.46%, respectively; (c) Cytotoxicity of these 25 peptides at 50 μM was measured by an MTT assay. The cell viability of HepG2.2.15 cells after being treated with 50 μM BmKDfsin4 for 48 h was 83.18%.*** denotes p < 0.001. Values represent the mean ± SEM of five independent samples.

Cytotoxicity and Hemolysis of BmKDfsin4
The feasibility of the further development of BmKDfsin4 as a candidate anti-HBV agent was determined by measuring its cytotoxicity. After being incubated with a serial dilution of BmKDfsin4 for 48 h, the cell viability of HepG2.2.15 (Figure 2a), HepG2 ( Figure 2b) and L-02 ( Figure 2c) cells was measured using MTT assays. The 50% cytotoxicity concentrations (CC50) of BmKDfsin4 to HepG2.2.15, HepG2 and L-02 were 167.82, 154.24 and 103.77 μM, respectively. At the concentration of 10 μM, the viability of the BmKDfsin4-treated cells was greater than 90% in all three kinds of cell lines. The hemolysis assay also showed that the viability of erythrocytes was more than 90% when the concentration of BmKDfsin4 was lower than 10 μM (Figure 2d). These data indicated that 10 μM or less BmKDfsin4 was minimally cytotoxic and suitable for further anti-HBV studies.

Extracellular Anti-HBV Activity of BmKDfsin4 in Vitro
To further investigate the effect of BmKDfsin4 on HBV replication in vitro, HepG2.2.15 cells were treated with a serial dilution of BmKDfsin4 for 48 h. HBeAg and HBsAg levels in HepG2.2.15 cell supernatant after being treated with BmKDfsin4 were measured by ELISA assays. As shown in Figure 3a,b, the cellular supernatant HBeAg and HBsAg levels were significantly reduced by BmKDfsin4 in a dose-dependent manner. The 50% inhibitory concentrations (IC 50 ) were 3.95 and 2.28 µM, yielding selective indexes (ratio of CC 50 to IC 50 ) as 42.78 and 74.11, respectively (Table 1). HBV progeny DNA in the supernatant of HepG2.2.15 cells was quantitated using real-time PCR. Real-time PCR data indicated that HBV progeny DNA in the culture media was also inhibited by BmKDfsin4 in a concentration-dependent manner (IC 50 = 1.26 µM) ( Figure 3c) and the selective index was 133.20 (Table 1). Thus, BmKDfsin4 can reduce levels of both HBV antigens and HBV progeny DNA in HepG2.2.15 cell culture supernatant in a concentration-dependent manner.
To further investigate the effect of BmKDfsin4 on HBV replication in vitro, HepG2.2.15 cells were treated with a serial dilution of BmKDfsin4 for 48 h. HBeAg and HBsAg levels in HepG2.2.15 cell supernatant after being treated with BmKDfsin4 were measured by ELISA assays. As shown in Figure 3a,b, the cellular supernatant HBeAg and HBsAg levels were significantly reduced by BmKDfsin4 in a dose-dependent manner. The 50% inhibitory concentrations (IC50) were 3.95 and 2.28 μM, yielding selective indexes (ratio of CC50 to IC50) as 42.78 and 74.11, respectively (Table 1). HBV progeny DNA in the supernatant of HepG2.2.15 cells was quantitated using real-time PCR. Real-time PCR data indicated that HBV progeny DNA in the culture media was also inhibited by BmKDfsin4 in a concentration-dependent manner (IC50 = 1.26 μM) (Figure 3c) and the selective index was 133.20 (Table 1). Thus, BmKDfsin4 can reduce levels of both HBV antigens and HBV progeny DNA in HepG2.2.15 cell culture supernatant in a concentration-dependent manner.

Intracellular Anti-HBV Activity of BmKDfsin4 in Vitro
The levels of intracellular HBV viral proteins were measured by Western blot analysis. Similar to the effects in HepG2.2.15 cell culture supernatant, HepG2.2.15 intracellular HBsAg (Figure 4a was almost undetectable. BmKDfsin4 seems to have extremely strong inhibitory activity against HBx protein (Figure 4c). HepG2.2.15 cells treated by BmKDfsin4 at 5 µM had only a slight band of HBx protein, while the HBx protein of HepG2.2.15 cells was totally undetectable after the treatment of 10 µM BmKDfsin4. The inhibitory activity of BmKDfsin4 against HBV core (Figure 4b) and HBV RT (Figure 4d) seems lower compared with HBsAg and HBx protein, but it still exerted a significantly concentration-dependent inhibition. These results suggest that BmKDfsin4 has an inhibitory activity against intracellular HBV viral proteins at minimally cytotoxic concentrations and these inhibitory effects were dose-dependent.

Intracellular Anti-HBV Activity of BmKDfsin4 in Vitro
The levels of intracellular HBV viral proteins were measured by Western blot analysis. Similar to the effects in HepG2.2.15 cell culture supernatant, HepG2.2.15 intracellular HBsAg (Figure 4a) was also inhibited by BmKDfsin4 in a dose-dependent manner. When treated by BmKDfsin4 at 10 μM, HBsAg was almost undetectable. BmKDfsin4 seems to have extremely strong inhibitory activity against HBx protein (Figure 4c). HepG2.2.15 cells treated by BmKDfsin4 at 5 μM had only a slight band of HBx protein, while the HBx protein of HepG2.2.15 cells was totally undetectable after the treatment of 10 μM BmKDfsin4. The inhibitory activity of BmKDfsin4 against HBV core (Figure 4b) and HBV RT (Figure 4d) seems lower compared with HBsAg and HBx protein, but it still exerted a significantly concentration-dependent inhibition. These results suggest that BmKDfsin4 has an inhibitory activity against intracellular HBV viral proteins at minimally cytotoxic concentrations and these inhibitory effects were dose-dependent.

Serum Stability of BmKDfsin4
The serum stability of BmKDfsin4 was tested by inhibitory activity against HBeAg and HBsAg in the supernatant of HepG2.2.15 at 10 μM after incubation in 10% FBS for less than 24 h. After being incubated with 10% serum for 8 h or less, the inhibitory rate of BmKDfsin4 against HBeAg and HBsAg was similar to the rate of the serum-untreated BmKDfsin4. There was a slight decrease of inhibitory ratio after being treated with 10% serum for 12 and 24 h, but the inhibitory ratio of BmKDfsin4 against HBeAg was still 65.01% and 48.85%, respectively (Figure 5a). Meanwhile, the inhibitory ratio of BmKDfsin4 against HBsAg was 63.69% and 54.00%, respectively (Figure 5b). These results suggest that BmKDfsin4 has a high serum stability.

Serum Stability of BmKDfsin4
The serum stability of BmKDfsin4 was tested by inhibitory activity against HBeAg and HBsAg in the supernatant of HepG2.2.15 at 10 µM after incubation in 10% FBS for less than 24 h. After being incubated with 10% serum for 8 h or less, the inhibitory rate of BmKDfsin4 against HBeAg and HBsAg was similar to the rate of the serum-untreated BmKDfsin4. There was a slight decrease of inhibitory ratio after being treated with 10% serum for 12 and 24 h, but the inhibitory ratio of BmKDfsin4 against HBeAg was still 65.01% and 48.85%, respectively (Figure 5a). Meanwhile, the inhibitory ratio of BmKDfsin4 against HBsAg was 63.69% and 54.00%, respectively (Figure 5b). These results suggest that BmKDfsin4 has a high serum stability.

Discussion
The application of interferons is limited by their side effects, and nucleotide analog treatments such as lamivudine (3TC) have the potential of inducing mutations in viral polymerase, which can prompt drug-resistant virus strains. Thus, the demand for the development of new potential anti-HBV drugs is still urgent. In recent years, many newly discovered or designed anti-HBV compounds or organic molecules have been reported. Cyclosporin A was reported to inhibit HBV and HDV at the entry stage by interfering with NTCP (sodium taurocholate co-transporting polypeptide) [16]. Acylated peptides derived from the large viral surface protein inhibited HBV infection with an extremely low IC50 [17].
Our experimental results showed that BmKDfsin4 exerted considerable inhibitory activity on HBV replication by decreasing the production of HBeAg (IC50 = 3.95 μM), HBsAg (IC50 = 2.28 μM) and HBV DNA (IC50 = 1.26 μM) in cell culture medium, and the production of intracellular HBsAg, HBV core protein, HBx protein and HBV RT and the inhibition were concentration-dependent. Compared with its high anti-HBV activity, the MTT assay indicated that BmKDfsin4 has a low cytotoxicity in HepG2.2.15 cells (CC50 = 167.82 μM), HepG2 cells (CC50 = 154.24 μM) and L-02 cells (CC50 = 103.77 μM). As for HepG2.2.15 cells, which is a stable transferred cell line of 1.3-fold HBV genome, the selective index of BmKDfsin4 was valued as high as 133.20. In addition, BmKDfsin4 also exerted extremely low hemolytic activity on human erythrocytes. The serum stability of BmKDfsin4 was measured by the inhibitory rate of HBeAg and HBsAg, after being incubated with 10% serum for a serial time points. As there was only a slight decrease in inhibitory activity against both HBeAg and HBsAg after a 24 h incubation, BmKDfsin4 was thought to have a strong serum stability.
To the best of our knowledge, all reported antiviral peptides derived from scorpion venom were short-chain polypeptides with 13-20 amino acid residues and without any disulfide bridge [10][11][12][13]31]. Having the typical sequence and predicted structure characteristics of defensin, BmKDfsin4 is the first scorpion defensin with experimental evidence of antiviral activity. It is worth mentioning that only the scorpion defensin BmKDfsin4 can inhibit HBV replication in the cultured HepG2.2.15 system among the tested 25 peptides from scorpion venoms. Different from BmKDfsin4, all the other 24 non-

Discussion
The application of interferons is limited by their side effects, and nucleotide analog treatments such as lamivudine (3TC) have the potential of inducing mutations in viral polymerase, which can prompt drug-resistant virus strains. Thus, the demand for the development of new potential anti-HBV drugs is still urgent. In recent years, many newly discovered or designed anti-HBV compounds or organic molecules have been reported. Cyclosporin A was reported to inhibit HBV and HDV at the entry stage by interfering with NTCP (sodium taurocholate co-transporting polypeptide) [16]. Acylated peptides derived from the large viral surface protein inhibited HBV infection with an extremely low IC 50 [17].
Our experimental results showed that BmKDfsin4 exerted considerable inhibitory activity on HBV replication by decreasing the production of HBeAg (IC 50 = 3.95 µM), HBsAg (IC 50 = 2.28 µM) and HBV DNA (IC 50 = 1.26 µM) in cell culture medium, and the production of intracellular HBsAg, HBV core protein, HBx protein and HBV RT and the inhibition were concentration-dependent. Compared with its high anti-HBV activity, the MTT assay indicated that BmKDfsin4 has a low cytotoxicity in HepG2.  20. In addition, BmKDfsin4 also exerted extremely low hemolytic activity on human erythrocytes. The serum stability of BmKDfsin4 was measured by the inhibitory rate of HBeAg and HBsAg, after being incubated with 10% serum for a serial time points. As there was only a slight decrease in inhibitory activity against both HBeAg and HBsAg after a 24 h incubation, BmKDfsin4 was thought to have a strong serum stability.
To the best of our knowledge, all reported antiviral peptides derived from scorpion venom were short-chain polypeptides with 13-20 amino acid residues and without any disulfide bridge [10][11][12][13]31]. Having the typical sequence and predicted structure characteristics of defensin, BmKDfsin4 is the first scorpion defensin with experimental evidence of antiviral activity. It is worth mentioning that only the scorpion defensin BmKDfsin4 can inhibit HBV replication in the cultured HepG2.2.15 system among the tested 25 peptides from scorpion venoms. Different from BmKDfsin4, all the other 24 non-active peptides are short linear peptides which are not cross-linked by disulfide bridges. The scorpion defensin BmKDfsin4 should be deduced to have more strong serum stability than the other 24 short linear peptides, which was partially confirmed by the result that BmKDfsin4 has a strong serum stability in the above anti-HBV tests. Meanwhile, the phenomenon was possibly related to antiviral mechanisms. Although the antiviral mechanisms of linear peptides are various, most short linear peptides may inhibit virus infection and replication by interacting with viral envelopes or surfaces of capsids and then destroying the structure of virions. However, HepG2.2.15, the cell line used for the antiviral experiments in our study, was derived from a human hepatoblastoma (HepG2). In HepG2.2.15, HBV genome was stably incorporated into the host genome and HBV was replicated. Therefore, HepG2.2.15 could continuously produce infectious HBV virions, but it was not successfully infected by HBV particles.
In addition, most recently, our group had also reported that BmKDfsin4 can effectively and selectively inhibit the growth of Gram-positive bacteria, including some standard and antibiotic-resistant strains, and block the potassium channel with a neurotoxin-like pore-blocking mode. [14] Thus, BmKDfsin4 is the first reported multi-functional defensin with three different aspects of bio-activities. Whether there is an internal relation between its three biological functions deserves further research and exploration in the future.

Reagents
BmKDfsin4 was expressed using the prokaryotic expressing system of our laboratory. Other peptides were synthesized using the solid-phase synthesis method and amidated at the C-terminus with a purity of >95% (ChinaPeptides Co., Ltd., Shanghai, China).

MTT Assay
Cells were seeded in 96-well plates (10 4 cells per well) and cultured in DMEM containing 10% FBS in 5% CO 2 at 37˝C for 12 h. Then, peptides were added to the medium in a serial of concentrations. The concentrations ranged from 0 to 200 µM. Five repetitions were set for each concentration. After 48 h of incubation, 20 µL of MTT solution (5 mg/mL in PBS buffer; Invitrogen, Foster, CA, USA) was added to the medium in each well, and the plate was incubated in 5% CO 2 at 37˝C for 4 h. Then, the medium was replaced by 100 µL DMSO. To completely dissolve the precipitated crystal purple formazan, the plate was then gently shaken for 10 min at room temperature. The absorbance at a wavelength of 570 nm was measured using a microplate reader (BioTek, Winooski, VT, USA).

Hemolysis Assay
Erythrocytes were obtained from fresh human blood by centrifugation for 10 min at 1000ˆg. After washed by HEPES buffer for three times, erythrocytes were resuspended in normal saline and seeded in 96-well plates (10 7 cells per well). BmKDfsin4 was added in a serial of concentrations, 0.1% triton-X100 was used as positive control and normal saline was used as negative control. After incubated at 37˝C for 1 h, the supernatants were collected by centrifugation for 10 min at 1000ˆg. The absorbance of hemoglobin was measured at a wavelength of 570 nm.

Quantification of HBeAg, HBsAg and HBV DNA in the Culture Medium
HepG2.2.15 cells were seeded in 24-well plates (5ˆ10 4 cells per well) and incubated at 37˝C for 12 h. Then, the cells were treated with various concentrations of BmKDfsin4 and cultured for another 48 h. HBeAg and HBsAg levels were quantitated using an enzyme linked immunoassay (ELISA) kit. HBV DNA in the culture medium was extracted and measured using real-time PCR (QIAGEN, Valencia, CA, USA).

Serum Stability Testing
BmKDfsin4 was incubated with 10% FBS or ddH 2 O at 37˝C for 4 h, 8 h, 12 h and 24 h. Then, BmKDfsin4 was added to HepG2.2.15 cells at 10 µM and the cells were incubated at 37˝C in 5% CO 2 for 48 h. The concentrations of HBeAg and HBsAg were detected using ELISA, and the antiviral activity was then evaluated.

Statistical Analysis
Data are shown as mean˘standard deviation from at least three separate experiments. Statistical significance was tested using a two-tailed Student's t-test. IC 50 , CC 50 and HC 50 were calculated using the Statistical Product and Service Solution (SPSS) software (Version 21, IBM, Armonk, NY, USA).