Inhibitory Activity of a Scorpion Defensin BmKDfsin3 against Hepatitis C Virus

Hepatitis C virus (HCV) infection is a major worldwide health problem which can cause chronic hepatitis, liver fibrosis and hepatocellular carcinoma (HCC). There is still no vaccine to prevent HCV infection. Currently, the clinical treatment of HCV infection mainly relies on the use of direct-acting antivirals (DAAs) which are expensive and have side effects. Here, BmKDfsin3, a scorpion defensin from the venom of Mesobuthus martensii Karsch, is found to dose-dependently inhibit HCV infection at noncytotoxic concentrations and affect viral attachment and post-entry in HCV life cycle. Further experimental results show that BmKDfsin3 not only suppresses p38 mitogen-activated protein kinase (MAPK) activation of HCV-infected Huh7.5.1 cells, but also inhibits p38 activation of Huh7.5.1 cells stimulated by tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) or lipopolysaccharide (LPS). BmKDfsin3 is also revealed to enter into cells. Using an upstream MyD88 dimerization inhibitor ST2345 or kinase IRAK-1/4 inhibitor I, the inhibition of p38 activation represses HCV replication in vitro. Taken together, a scorpion defensin BmKDfsin3 inhibits HCV replication, related to regulated p38 MAPK activation.


Introduction
Defensin is a class of cationic peptides rich in disulfide bonds and widely distributed in fungi, plants and animals, and is also an important part of the defense system [1]. Scorpion defensins have a variety of biological activities, including antiviral activity [2,3], antibacterial activity [4,5], immunomodulatory function [6,7], antitumor action [8,9], and ion channel modulation [10,11]. A scorpion defensin BmKDfsin4 derived from the venom of the scorpion Mesobuthus martensii Karsch was reported to inhibit hepatitis B virus (HBV) replication by our group [12]. Although such report demonstrated that the scorpion defensin can repress viral production, the specific mechanism of this effect during viral infection is not well understood.   Virus in Huh 7.5.1 cells were observed by immunofluorescence microscope.
HCV, green. 4',6-diamidino-2-phenylindole (DAPI), blue. Scale bar, 100 µm. (E) The statistics of the fluorescence ratio of extracellular virus particles as described in (D). (F) Cytotoxicity of BmKDfsin3 to Huh7.5.1 cells by the MTT assay. BmKDfsin3 was dissolved in the medium and the medium without BmKDfsin3 was used as a negative control in all experiments. The internal control of subfigure (C) was glyceraldehyde-phosphate dehydrogenase (GAPDH). ***, p < 0.001. Data represented the mean ± standard deviation (SD) of at least three independent experiments.

BmKDfsin3 Affects the Viral Attachment and Post-Entry Stages in HCV Infection Cycle
We have proven that the scorpion defensin BmKDfsin3 concentration-dependently inhibits HCV replication under noncytotoxic concentrations in Huh7.5.1 cells. To determine the action stage of the peptide BmKDfsin3 during the HCV life cycle, we conducted an experiment with different modes of peptide treatment, shown in the schematic diagram of Figure 2A. The experimental results indicated that BmKDfin3 had almost no effect on the free virion ( Figure 2B) and viral entry/fusion ( Figure 2D) stages but had 42% inhibition on the viral attachment stage ( Figure 2C). Importantly, BmKDfsin3 added during the post-entry stage had significant restriction (64%) on HCV replication in Huh7.5.1 cells ( Figure 2E). These data suggest that BmKDfsin3 suppresses HCV replication by acting on the viral attachment and post-entry phases, and the latter is more effective than the former.

BmKDfsin3 Affects the Viral Attachment and Post-Entry Stages in HCV Infection Cycle
We have proven that the scorpion defensin BmKDfsin3 concentration-dependently inhibits HCV replication under noncytotoxic concentrations in Huh7.5.1 cells. To determine the action stage of the peptide BmKDfsin3 during the HCV life cycle, we conducted an experiment with different modes of peptide treatment, shown in the schematic diagram of Figure 2A. The experimental results indicated that BmKDfin3 had almost no effect on the free virion ( Figure 2B) and viral entry/fusion ( Figure 2D) stages but had 42% inhibition on the viral attachment stage ( Figure 2C). Importantly, BmKDfsin3 added during the post-entry stage had significant restriction (64%) on HCV replication in Huh7.5.1 cells ( Figure 2E). These data suggest that BmKDfsin3 suppresses HCV replication by acting on the viral attachment and post-entry phases, and the latter is more effective than the former.  . All experiments were detected by qPCR. BmKDfsin3 was dissolved in the medium and the medium without BmKDfsin3 was used as a negative control. The internal controls of subfigures (B-E) were GAPDH. ns, no significance. *, p < 0.05. **, p < 0.01. Data represented the mean ± SD of at least three independent experiments.

BmKDfsin3 Inhibits p38 Activation
Many previous studies have reported that the infection of host cells with some viruses including HCV [17] can activate p38. Here, we found that HCV infection increased p38 phosphorylation in Huh7.5.1 cells. Interestingly, the activated p38 was significantly inhibited by the treatment with BmKDfsin3 ( Figure 3A). To further confirm this effect, we added different concentrations of BmKDfsin3 to HCV-infected Huh7.5.1 cells and then detected changes of the phosphorylated p38 using western blotting. The results showed that p38 phosphorylation was inhibited during HCV infection in a concentration-dependent manner after the addition of BmKDfsin3 ( Figure 3B,C). However, considering that HCV infection can activate p38, it is still unknown whether the inhibitory effect of BmKDfsin3 on p38 activation results from its anti-HCV activity or its effect on the p38 cascade. To analyze the effect of BmKDfsin3 on the p38 cascade, we used three p38 activation stimulation factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and lipopolysaccharide (LPS) [29] to incubate Huh7.5.1 cells for 2 h, and then treated the cells with BmKDfsin3. We analyzed the phosphorylation of p38 by western blotting. Results showed that BmKDfsin3 significantly inhibited the phosphorylation of p38 induced by TNF-α, IL-1β and LPS ( Figure 3D,E). Additionally, we also chemically synthesized an His tag-fused BmKDfsin3 (His-BmKDfsin3) and incubated it with Huh7.5.1 cells for 0 h, 1 h and 12 h, respectively. Then, the entry of His-BmKDfsin3 in Huh7.5.1 cells was analyzed using a confocal microscope. The results showed that His-BmKDfsin3 could enter Huh7.5.1 cells ( Figure 3F). Taken together, these data suggest that BmKDfsin3 inhibits p38 activation during HCV infection, which is possibly related to regulation of the p38 MAPK signal pathway. Huh7.5.1 cells. Huh7.5.1 cells were infected with J399EM at an MOI of 0.1 and treated with BmKDfsin3 as described in (A). All experiments were detected by qPCR. BmKDfsin3 was dissolved in the medium and the medium without BmKDfsin3 was used as a negative control. The internal controls of subfigures (B-E) were GAPDH. ns, no significance. *, p < 0.05. **, p < 0.01. Data represented the mean ± SD of at least three independent experiments.

BmKDfsin3 Inhibits p38 Activation
Many previous studies have reported that the infection of host cells with some viruses including HCV [17] can activate p38. Here, we found that HCV infection increased p38 phosphorylation in Huh7.5.1 cells. Interestingly, the activated p38 was significantly inhibited by the treatment with BmKDfsin3 ( Figure 3A). To further confirm this effect, we added different concentrations of BmKDfsin3 to HCV-infected Huh7.5.1 cells and then detected changes of the phosphorylated p38 using western blotting. The results showed that p38 phosphorylation was inhibited during HCV infection in a concentration-dependent manner after the addition of BmKDfsin3 ( Figure 3B and C). However, considering that HCV infection can activate p38, it is still unknown whether the inhibitory effect of BmKDfsin3 on p38 activation results from its anti-HCV activity or its effect on the p38 cascade. To analyze the effect of BmKDfsin3 on the p38 cascade, we used three p38 activation stimulation factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and lipopolysaccharide (LPS) [29] to incubate Huh7.5.1 cells for 2 h, and then treated the cells with BmKDfsin3. We analyzed the phosphorylation of p38 by western blotting. Results showed that BmKDfsin3 significantly inhibited the phosphorylation of p38 induced by TNF-α, IL-1β and LPS ( Figure 3D and E). Additionally, we also chemically synthesized an His tag-fused BmKDfsin3 (His-BmKDfsin3) and incubated it with Huh7.5.1 cells for 0 h, 1 h and 12 h, respectively. Then, the entry of His-BmKDfsin3 in Huh7.5.1 cells was analyzed using a confocal microscope. The results showed that His-BmKDfsin3 could enter Huh7.5.1 cells ( Figure 3F). Taken together, these data suggest that BmKDfsin3 inhibits p38 activation during HCV infection, which is possibly related to regulation of the p38 MAPK signal pathway.

Inhibition of p38 Activation Suppresses HCV Replication In Vitro
Next, we ask whether the p38 signaling pathway played an important role in HCV replication. MyD88 and IRAK1/4 are two important upstream cascade proteins of the p38 signaling pathway, as shown in Figure 4A. ST2345 is a polypeptide with 23 amino acid residues ( Figure 4B) that interferes with MyD88 toll/IL-1R (TIR) domain dimerization [30]. ST2345 peptide was chemically synthesized by GL Biochem Ltd. (GL Biochem (Shanghai) Ltd. Limited Liability Company, Shanghai, China). High-performance liquid chromatography (HPLC) analysis indicated that the elution time of the peptide ST2345 was in 17.07 min at 230 nm and the purity of ST2345 was more than 95% ( Figure 4C). Mass spectrometry analysis indicated that the mass of the synthetic ST2345 peptide matches its  Figure D was analyzed with Image J software (E). TNF-α, IL-1β, and LPS were dissolved in phosphate buffered saline (PBS) and PBS was used as a negative control. **, p < 0.01. ***, p < 0.001. Data represented the mean ± SD of at least three independent experiments. (F) The entry of His-BmKDfsin3 to Huh7.5.1 cells. Cells were treated with His-BmKDfsin3 (5 µM) for 0 h, 1 h and 12 h, respectively, and then stained with anti-His antibody and DAPI. Cells were observed using a confocal microscopy. His-BmKDfsin3, green. DAPI, blue. Scale bar, 10 µm. Cells were treated with His-BmKDfsin3 (5 µM) for 0 h as a control. The subfigure (F) was representative of at least ten independent pictures.

Inhibition of p38 Activation Suppresses HCV Replication In Vitro
Next, we ask whether the p38 signaling pathway played an important role in HCV replication. MyD88 and IRAK1/4 are two important upstream cascade proteins of the p38 signaling pathway, as shown in Figure 4A. ST2345 is a polypeptide with 23 amino acid residues ( Figure 4B) that interferes with MyD88 toll/IL-1R (TIR) domain dimerization [30]. ST2345 peptide was chemically synthesized by GL Biochem Ltd. (GL Biochem (Shanghai) Ltd. Limited Liability Company, Shanghai, China). High-performance liquid chromatography (HPLC) analysis indicated that the elution time of the peptide ST2345 was in 17.07 min at 230 nm and the purity of ST2345 was more than 95% ( Figure 4C). Mass spectrometry analysis indicated that the mass of the synthetic ST2345 peptide matches its theoretical molecular weight (2985.60 Da) ( Figure 4C). IRAK-1/4 inhibitor I is a small molecule that suppresses the phosphorylations of IRAK1 and IRAK4 with IC 50 of 0.3 µM and 0.2 µM, respectively [31].
Both ST2345 and IRAK-1/4 inhibitor I can inhibit the activation of p38 by regulating the p38 signaling pathway. Therefore, they were used to investigate anti-HCV activity, respectively. The experimental results showed that the peptide ST2345 significantly inhibited HCV replication in a dose-dependent manner ( Figure 4D). Similarly, IRAK-1/4 inhibitor I was observed to have a concentration-dependent antiviral activity against HCV infection in Huh7.5.1 cells ( Figure 4E). These results indicate that inhibition of p38 activation suppresses HCV replication and the p38 signaling pathway may play an important role in viral infection.  Figure 4C). IRAK-1/4 inhibitor I is a small molecule that suppresses the phosphorylations of IRAK1 and IRAK4 with IC50 of 0.3 μM and 0.2 μM, respectively [31]. Both ST2345 and IRAK-1/4 inhibitor I can inhibit the activation of p38 by regulating the p38 signaling pathway. Therefore, they were used to investigate anti-HCV activity, respectively. The experimental results showed that the peptide ST2345 significantly inhibited HCV replication in a dose-dependent manner ( Figure 4D). Similarly, IRAK-1/4 inhibitor I was observed to have a concentration-dependent antiviral activity against HCV infection in Huh7.5.1 cells ( Figure 4E). These results indicate that inhibition of p38 activation suppresses HCV replication and the p38 signaling pathway may play an important role in viral infection. The expression level of the HCV core protein was determined by western blotting. The ST2345 peptide was dissolved in medium and the medium without ST2345 was used as a negative control. The IRAK-1/4 inhibitor I was dissolved in dimethyl sulfoxide (DMSO) and the DMSO without IRAK-1/4 inhibitor I was used as a negative control.

Discussion
Many studies have previously reported that defensins have antibacterial, antiviral and ion channel-modulation activities [32][33][34]. Recently, there are many discoveries regarding antiviral The expression level of the HCV core protein was determined by western blotting. The ST2345 peptide was dissolved in medium and the medium without ST2345 was used as a negative control. The IRAK-1/4 inhibitor I was dissolved in dimethyl sulfoxide (DMSO) and the DMSO without IRAK-1/4 inhibitor I was used as a negative control.

Discussion
Many studies have previously reported that defensins have antibacterial, antiviral and ion channel-modulation activities [32][33][34]. Recently, there are many discoveries regarding antiviral defensin polypeptides from scorpions, which may be new candidates for antiviral drug molecules. Human αand β-defensins can suppress HCV replication [35]. Additionally, human α-defensin-1 inhibited protein kinase C (PKC) activation and repressed influenza virus replication [36]. Recent published data also suggest that α-defensin-1 may not only act directly on the virus in the absence of serum, but Antibiotics 2020, 9, 33 8 of 13 on the cell in the presence of serum as well [37]. Previous studies in our laboratory also found that the scorpion defensin BmKDfsin4 inhibited HBV replication [12] and blocked potassium channels [10]. Additionally, Zeng et al. designed a histidine-rich Eval418 derivative that could significantly inhibit herpes simplex virus 1 (HSV-1) replication [38]. During our study, the scorpion defensin BmKDfsin3 was found to be capable of inhibiting HCV replication in a concentration-dependent manner under non-cytotoxicity. BmKDfsin3 is a new antiviral peptide and our study provides a new molecule for anti-HCV.
HCV infection can activate the p38 MAPK [17]. p38 MAPK, a member of the mitogen-activated protein (MAP) kinase family, plays crucial roles in many biological processes in response to extracellular stimuli that mediate a variety of cellular behaviors. p38 activated by many extracellular signals executes important physiological and pathological functions in inflammation, cell proliferation, apoptosis, differentiation, aging and tumorigenesis and, especially, cellular stress and infection [39,40]. Classical p38 activation follows the activation of the MAPK signaling molecules MAP kinase kinase kinase (MAP3K), MAP kinase kinase (MAPKK), and MAP kinase (MAPK) cascades. The MKKs of p38 in this pathway are mainly MKK3 and MKK6 which activate the phosphorylations of Thr180 and Tyr182 in p38, respectively [41]. Previously, respiratory syncytial virus (RSV) was reported to induce Toll-like receptor 4 (TLR4)-mediated activation of p38 in the early stages of infection, which was important for viral infection [42]. p38 specific inhibitor can suppress RSV and influenza A virus replication, and the phosphorylated p38 induced by virus can also be inhibited [43]. Here, BmKDfsin3 was found to inhibit HCV replication. Moreover, BmKDfsin3 was revealed to repress p38 activation. Concurrently, HCV replication can also be suppressed when the p38 MAPK signal pathway is inhibited by the treatment of the MyD88 inhibitor or IRAK inhibitor. All these suggest that a scorpion defensin BmKDfsin3 inhibits HCV replication, related to regulated p38 MAPK activation.

MTT Assay
To analyze the cytotoxic effect of BmKDfsin3 on Huh7.5.1 cells, we used the MTT cytotoxicity assay, as in the previous article published in our laboratory [38]. Briefly, Huh7.5.1 cells were planted in 96-well plates (10 4 cells per well) and cultured at 37 • C for 24 h. The BmKDfsin3 was diluted with fresh medium in different concentrations and then replaced the cell culture medium, and the cells were cultured at 37 • C for 48 h. Then, the medium was removed, and the cells were incubated with the 0.25 µg/µL MTT solution in 200 µL of medium per each well at 37 • C for 4 h. The medium was then replaced with 100 µL DMSO and shaken gently for 10 min at room temperature. The absorbance was then measured at a wavelength of 570 nm using a microplate reader (BioTek, Winooski, VT, USA).

Confocal Microscopy
To detect infectious HCV virions in the supernatant of HCV-infected cells treated with BmKDfsin3 or not, the collected supernatants were used to infect naïve Huh7.5.1 cells. The cells were fixed with precooled 4% paraformaldehyde, and the nuclei were stained with DAPI (ANT10072). The green fluorescence (HCV J399EM) was observed under a confocal microscope.
To test whether BmKDfsin3 can enter into cells, we added His-tagged BmKDfsin3 (5 µM) to Huh7.5.1 cells and incubated them in confocal dishes for 0 h, 1 h and 12 h, respectively. These cells were washed three times with cold PBS and fixed with precooled 4% paraformaldehyde and permeated with 0.2% Triton X-100. The cells were washed three times with PBS and then blocked with 8% body surface area (BSA) for 1 h at room temperature. Then, cells were incubated with 1% BSA diluted primary antibody and shaken overnight at 4 • C. Subsequently, the cells were incubated with the secondary antibody Alexa Fluor 488 for 1 h at room temperature in the dark, and then the nuclei were stained with DAPI and observed under a confocal microscope.

qPCR
The method of qPCR for detecting intracellular viral RNA was consistent with that of our laboratory [45]. Briefly, the detected cells were lysed using TRIzol reagent (Takara) to release the intracellular total RNA, followed by precipitation with isopropanol. The first-strand cDNA was reversed-transcribed by the RevertAid first strand cDNA synthesis kit (ThermoFisher Scientific). The cDNA was quantitated by qPCR with primers using the Bestar ® SYBR Green qPCR master mix reagent (DBI ® Bioscience). HCV primers were 5 -TCTGCGGAACCGGTGAGTA-3 (sense) and 5 -TCAGGCAGTACCACAAGGC-3 (antisense).

Western Blotting
Cells were collected and lysed 30 min on ice by lysis buffer (1% TritonX-100, 10% glycerol, 50 mM HEPES, pH 7.2, 150 mM NaCl). The cell lysate was centrifuged at 12,000 rpm for 15 min, and then the supernatant was collected and assayed with a bicinchoninic acid (BCA) protein quantification kit (ThermoFisher Scientific, Cleveland, OH, USA). Samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose (NC) membranes. The non-specific protein on the NC membrane was blocked with 5% skim milk powder and incubated on a shaker for 2 h at room temperature. The primary antibody was incubated overnight at 4 • C, and the secondary antibody was incubated for 2 h at room temperature. The results were analyzed by a chemiluminescence kit with FuJi medical X-ray film.

Peptide Oxidation and Purification
The linear BmKDfsin3, His-BmKDfsin3, and ST2345 peptides were synthesized by GL Biochem Ltd. (Shanghai, China). The methods of oxidation and purification for BmKDfsin3 and His-BmKDfsin3 were as previously described by our group [46]. Briefly, BmKDfsin3 and His-BmKDfsin3 were oxidized by dissolving them with 0.2 M Tris-HCl (pH = 8.3) for 48 h at 25 • C in a shaker (50 rpm) at a concentration of 1 mg/mL. Reduced and oxidized peptides were analyzed and purified by HPLC on a C18 column (Elite HPLC, Dalian, China, 10 × 250 mm, 5 µm, 300 A). Typical setting of HPLC was a linear gradient from 5% to 95% CH 3 CN with 0.1% trifluoroacetic acid (TFA) in 60 min with a constant flow rate of 1-4 mL/min. The injection volume of HPLC was less than 5.0 mL every time. Targeted peptides were collected with detection at a wavelength of 230 nm and freeze-dried to save. Similarly, the HPLC condition of ST2345 peptide was the same as BmKDfsin3 and His-BmKDfsin3.

Statistics Analysis
Adobe Photoshop CS6 (Adobe Systems, Inc, San Jose, CA, USA) and Graphpad Prism 5 were used for statistical analysis. Data represented the mean ± SD of at least three independent experiments and p values calculated by the t-test of Student. Statistical significance were considered at a p value less than 0.05 (*, p < 0.05. **, p < 0.01. ***, p < 0.001).

Conflicts of Interest:
The authors declare no competing financial interest.