Betaine Inhibits Hepatitis B Virus with an Advantage of Decreasing

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Betaine inhibits hepatitis B virus with an advantage of decreasing resistance to lamivudine and interferon alpha Mengmeng Zhang, Xiaoying Wu, Furao Lai, Xiaoyuan Zhang, Hui Wu, and Tian Min J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01180 • Publication Date (Web): 04 May 2016 Downloaded from http://pubs.acs.org on May 8, 2016

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Journal of Agricultural and Food Chemistry

Betaine Inhibits Hepatitis B Virus with an Advantage of

1 2

Decreasing Resistance to Lamivudine and Interferon Alpha

3

Mengmeng Zhang†, Xiaoying Wu§, Furao Lai†, Xiaoyuan Zhang*#, Hui Wu*†, Tian Min†

4 5 6

Affiliation

7

† College of Food Science and Engineering, South China University of Technology,

8

Guangzhou, Guangdong 510640, China

9

§ School of Bioscience and Bioengineering, South China University of Technology,

10


11

# Industrial Technology Research Institute, South China University of Technology,

12


13 14 15

Co-corresponding authors:

16

*Xiaoyuan Zhang, Industrial Technology Research Institute, South China

17

University of Technology, Wushan Road 381, Guangzhou, Guangdong, China.

18

Tel: (+86) 20-22236722; E-mail: [email protected]

19

*Hui Wu, Department of Food Quality and Safety, South China University of

20 21

Technology, Wushan Road 381, Guangzhou, Guangdong, China. Tel: (+86) 20-87112853; E-mail: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT: Betaine (BET) is a native compound known for its ability to protect the

23

liver from toxicants. However, few studies have examined the effects of BET on the

24

most common cause of liver disease, hepatitis B virus (HBV). In this study, the

25

anti-HBV activity of BET was assessed in vitro and in vivo using ELISA, QPCR and

26

Southern blotting. The resistance of HBV to lamivudine and interferon alpha is

27

challenging in the clinical treatment of HBV. The effect of BET on resistance was

28

also investigated. The results showed that the secretion of HBsAg, HbeAg, and HBV

29

DNA in HepG2.2.15 cells was significantly decreased by BET via suppressing GRP78

30

expression. In duck HBV (DHBV)-infected ducklings, 1.0 or 2.0 g/kg of BET

31

significantly reduced serum DHBV DNA, and DHBV DNA did not rebound after the

32

five-day withdrawal period. BET suppressed HBV DNA rebound produced by the

33

resistance of HBV to lamivudine and decreased the resistance mutation (rtM204V/I)

34

of HBV DNA. Supplementation of BET may improve the anti-HBV effect of

35

interferon-α (IFN-α) by increasing the expression of antiviral dsRNA-dependent

36

protein kinase induced by the JAK-STAT signaling pathway. These results may

37

provide be useful information for the clinical application of BET and solution of HBV

38

drug resistance in anti-HBV therapy.

39

KEYWORDS: betaine, hepatitis B virus, drug resistance, lamivudine, interferon

40

alpha

41 42 43 2


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44

Introduction

45

Hepatitis B virus (HBV) infection is correlated with a significantly increased risk

46

of liver failure, liver fibrosis, and cirrhosis, which predisposes individuals to

47

hepatocellular carcinoma 1. Over 350 million people are chronically infected with this

48

virus worldwide 2. Interferon and nucleoside analogues are commonly used drugs to

49

treat HBV. However, the low response of patients to interferon and resistance of HBV

50

to nucleoside analogues make the clinical treatment of HBV challenging. Lamivudine

51

(3TC) was the first nucleoside analogue used to treat patients with chronic hepatitis B

52

(CHB). Although 3TC is potent against HBV, long-term use in patients induces HBV

53

drug-resistant

54

tyrosine-methionine-aspartate-aspartate (YMDD) motif of HBV DNA polymerase 3.

55

Interferon alpha (IFN-α) is also commonly used to treat CHB patients. However, a

56

successful response to IFN-α therapy occurs in only 25-50% of CHB patients 4.

57

Although the underlying reason for the low response to IFN-α remains unclear, the

58

low methylation of signal transducer and activator of transcription 1 (STAT1) in

59

JAK-STAT signaling pathway may be an important factor 5, 6.

mutations,

such

as

the

rtM204V/I

mutation

in

a

60

Currently, the use of a combination of drugs is the main strategy for combatting

61

drug resistance. However, the combinations of different nucleoside analogues, or

62

nucleoside analogue and interferon, have failed to increase the success rate in

63

compared to monotherapy 7. Alternative strategies and drugs are urgently needed. In

64

recent years, many native compounds with anti-HBV activity were reported, such as

65

ursolic acid, mulberrofuran G, and pu-erh tea extracts2, 8, 9. These native compounds 3



66

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are major potential drug sources for overcoming HBV drug resistance 10, 11.

67

Betaine (BET) is a native compound distributed widely in animals, plants, and

68

microorganisms 12. Particularly, BET is abundantly present in common foods, such as

69

cereals, spinach, and beets. Wheat is a major source of BET in the human diet

70

BET is known as an osmoregulant that protects plants under environmental stresses

71

such as drought, high salinity, or low temperature

72

methionine-homocysteine cycle as a methyl donor. Thus, BET is widely used in

73

human and animal nutrition 16. Furthermore, BET is known for its ability to maintain

74

liver, cardiovascular, and kidney health

75

reported that BET can be used to prevent or treat liver diseases, such as alcohol liver

76

disease

77

toxins

78

traditional oriental medicine to treat hepatic disorders in Southeast Asia

79

methylation in the CpG islands is one of the epigenetic mechanisms regulating gene

80

expression

81

NAFLD and Wilson’s disease, are affected by aberrant DNA methylation

82

methylation depends on the availability of methyl groups from S-adenosylmethionine

83

(SAM), the product of the methionine-homocysteine cycle

84

reported that the protective effects of BET in the liver depend on the regulation of

85

DNA methylation via participating in methylation as a methyl donor

86

the most common cause of liver disease. However, few studies have examined the

87

effect of BET on HBV. Studies of BET for treating HBV will increase the

13, 14

.

15

. BET also participates in the

17

. Particularly, many recent studies have

18

, nonalcoholic fatty liver disease (NAFLD)

19

, and liver injury induced by

20-25

. In fact, water extracts of BET from Lycium chinensis have been used in 26

. DNA

27

. Increasing evidence indicates that many liver diseases, including

4


28, 29

. DNA

30

. Some studies have

27, 30, 31

.

HBV is

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88

understanding of the role of BET in the liver injury induced by viruses.

89

HBV infection is species-specific, and only humans and orangutans can be

90

infected under natural conditions. Thus, few animal models are available for

91

experimental studies of HBV. Therefore, HBV-like animal virus is used as substitution

92

for HBV in the laboratory. Duck hepatitis B virus (DHBV) is very similar to HBV,

93

and DHBV-infected ducks are commonly used as animal models in HBV research

94

32-34

.

95

In the present study, the anti-HBV activity of BET was assessed in a cell model

96

and duckling model. The possible mechanism was also explored. Next, the effect of

97

BET on HBV resistance to 3TC and the rtM204V/I mutation of HBV DNA was

98

evaluated. Moreover, the effect of BET on the anti-HBV activity of IFN-α and the

99

expression of antiviral protein, dsRNA-dependent protein kinase (PKR), induced by

100

the JAK-STAT signaling pathway was further explored.

101

Materials and Methods

102

Materials and Chemicals

103

BET, 3TC, and IFN-α with purity over 98% were purchased from Sigma (St.

104

Louis, MO, USA). HepG2.2.15 cells were obtained from the Type Culture Collection

105

of the Chinese Academy of Sciences (Shanghai, China). Dulbecco’s modified Eagle’s

106

medium (DMEM), fetal bovine serum (FBS), G418, penicillin, and streptomycin were

107

purchased from Gibco Life Technologies (Grand Island, NY, USA). All other

108

chemical reagents used in this study were of analytical grade.

109

Cell Culture 5



110

The human hepatoblastoma cell line HepG2.2.15, which includes the stably

111

transfected HBV genome 35, was used in this study. HepG2.2.15 cells were cultivated

112

in DMEM supplemented with 10% FBS, 100 IU/mL of penicillin, 100 mg/mL of

113

streptomycin, and 380 µg/mL of G418 in a humidified atmosphere with 5% CO2 at

114

37℃.

115

CCK-8 Assay

116

The effect of BET on the viability of HepG2.2.15 cells was determined using the

117

CCK-8 (Dojindo, Kumamoto, Japan) method. The cells were seeded at a density of

118

5000 cells/well in a 96-well plate. After 24 h, the cells were treated with different

119

concentrations of BET (12.5-400 mM) for 8 days. The control wells contained an

120

equivalent amount of medium. All treated groups included three replicates. Next,

121

CCK-8 was added to each well, and the absorbance was evaluated in a microplate

122

reader (BioTek, Winooski, VT, USA) at 450 nm after 40 min. The percentage of cell

123

viability was calculated as follows:

124

125

Percentage of cell viability (%) =

Mean OD450 of test group × 100% Mean OD450 of control group

Treatment of HepG2.2.15 cells with BET

126

HepG2.2.15 cells were inoculated into culture flasks (25 cm2, NUNC, Roskilde,

127

Denmark) at a density of 1×105/mL. After 24 h, the cells were treated with 2.5, 5, 10,

128

20 or 40 mM BET for 8 days. The cultural medium was used as a negative control and

129

3TC (40 µM) was used as a positive control. All groups included three replicates. The

130

supernatants were collected every 2 days to detect HBV surface antigen (HBsAg),

6


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131

HBV e antigen (HBeAg), and HBV DNA, and the cells were lysed for RNA analysis

132

after treatment.

133

Detection of HBsAg and HBeAg

134

Detection of HBsAg and HBeAg in the culture medium was measured by using a

135

commercially available kit (Kehua, Shanghai, China). The medium samples were

136

centrifuged at 2000×g for 10 min and diluted to appropriate concentrations before

137

measurement. The measurement was carried out according to the manufacturer’s

138

instructions. Absorbance at 450 and 630 nm was measured using the microplate reader.

139

Inhibition rates were calculated as follows:

140

141

 Mean（ OD450 - OD630) of tested group  Inhibition(%) = 1  × 100%  Mean (OD450 - OD630) of control group  Measurement of HBV DNA

142

HBV DNA was extracted from the culture medium as described previously with

143

some modifications 36. To correct for the loss of HBV DNA during the extraction, 1

144

µg pGL3 Basic Vector (Promega, Madison, WI, USA) was added to 1 mL supernatant

145

from HepG2.2.15 cells before the extraction. Next, the supernatant was centrifuged at

146

2000 × g for 10 min, and polyethylene glycol (Mr, 8000) was added at a

147

concentration of 10% (wt/vol) followed by overnight precipitation at 4℃. The virions

148

were pelleted (30 min, 10,000 ×g), and the pellet was resuspended in lysis buffer (10

149

mM Tris-Cl, 5 mM EDTA, 150 mM NaCl, 1% SDS) at room temperature for 15 min.

150

Proteinase K was added at 500 µg/mL and the suspension incubated for 2 h at 56℃.

151

The digest was extracted with phenol/chloroform, 1:1 (vol/vol), or chloroform, and

7



152

the DNA was precipitated with 2.5 vol of ethanol. The DNA pellet was dissolved in

153

TE buffer.

154

HBV DNA was measured by quantitative PCR (QPCR) using a 7500 Real-Time

155

PCR System (Applied Biosystems, Foster City, CA, USA) with Brilliant II Green

156

QPCR Master Mix (Stratagene, Palo Alto, CA, USA). All quantification data were

157

normalized to the pGL3 Basic vector. The primer sequences for q-HBV and q-pGL3

158

are shown in Table 1. PCR conditions were 95℃ for 10 min; 40 cycles of 95℃ for

159

10 s, 60℃ for 20 s, and 72℃ for 15 s; then 25℃ for 10 min.

160

RNA Isolation and QPCR Analysis

161

Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA)

162

according to the manufacturer’s protocol, and the RNA was used for cDNA synthesis

163

using M-MLV RTase (Promega). cDNA encoding the GRP78 gene was quantified by

164

QPCR. The kit and PCR conditions were the same as described above. β- Actin was

165

used as an internal reference. The primers for GRP78 and β-actin are shown in Table

166

1.

167

Experimental of Ducklings with BET

168

DHBV-positive (from congenital infection) ducklings (Sheldrakes of Longyan) at

169

1 day of age were maintained under normal daylight and fed with standard

170

commercial diet and water in accordance with the procedures outlined in the Guide

171

for the Care and Use of Laboratory Animals prepared by the National Academy of

172

Sciences and published by the National Institutes of Health.

8


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BET (0.25, 1.0 or 2.0 g/kg) was administered to the ducklings orally. Water was

174

used as a negative control and 3TC (20 mg/kg) was used as a positive control. All

175

groups included eight ducklings and drugs were administered for 21 days. On days 0

176

(T0), 10 (T10), and 21 (T21) of treatment and day 5 (P5) of post-treatment follow-up,

177

blood samples were taken by jugular puncture and prepared for analysis of DHBV

178

DNA. After the withdrawal period, ducklings were killed by decapitation.

179

A previously described method was used with modifications to measure DHBV

180

DNA 33. First, 50 µL duckling serum was spotted directly onto nitrocellulose filters.

181

After denaturation (0.5 M NaOH, 1.5 M NaCl), neutralization (0.5 M Tris-HCl [pH

182

8.0] with 1.5 M NaCl followed by 2× SSC), and fixation (80℃ for 2 h), filters were

183

hybridized with a full-length 32P-labeled DHBV genomic DNA probe (FuRui, Beijing,

184

China). Filters were autoradiographed and quantitative measurement was conducted

185

by examination of OD (490 nm) values.

186

HBV Resistance to 3TC in Long-term Treatment

187

HepG2.2.15 cells were treated with BET (10 mM), 3TC (40 µM), or a combination

188

of these drugs. All groups included three replicates. Fresh culture medium containing

189

the drugs was changed every 2 days. The cells were passaged every 6 days and the

190

medium samples were collected to further analysis. The treatment was continued for

191

60 days.

192

Constructions of YMDD, YVDD and YIDD Plasmids

193

A 600-bp sequence of HBV DNA containing the YMDD region

194

(TATATGGATGAT) was amplified by PCR using Prime STARTM HS DNA 9



195

Polymerase (TaKaRa, Shiga, Japan) and cloned into the pGL3 Basic Vector between

196

the XhoI and HindIII sites using primers s-YMDD (Table 1). This plasmid was named

197

YMDD. Mutants of YMDD, including YVDD (TATGTGGATGAT) and YIDD

198

(TATATTGATGAT) were generated from this template and cloned in a similar

199

manner, using two-step PCR with the primers m-YVDD and m-YIDD (Table 1).

200

Plasmid construction is shown in Figure 1.

201

Detection of rtM204V/I Mutation

202

To discriminate mutants from wild-type viruses, Taqman-MGB probes were

203

designed, including YVDD probe and YIDD probe (Table 1). The YMDD, YVDD,

204

and YIDD plasmids were used to determine probe specificity. The copy number of

205

total HBV DNA was measured as described above. The copy numbers of mutants

206

(YVDD, YIDD) were measured by QPCR using Taqman-MGB probes. A Premix Ex

207

TaqTM (TaKaRa) kit was used. PCR conditions were 95℃ for 1 min; 40 cycles of 95℃

208

for 5 s,

209

Plasmids containing the YMDD, YVDD and YIDD sequences were used as standards.

210

The percentage of mutants in total HBV DNA was calculated as follows:

211

Percentage of rtM204V/I mutantion (%) =

212

Treatment of HepG2.2.15 cells with BET and IFN-α

60℃ for 40 s; then 25℃ for 10 min. The primer t-DNA was used (Table 1).

the copy number of mutants × 100% the copy number of total HBV DNA

213

HepG2.2.15 cells were treated with BET (0.5 mM), IFN-α (100 IU/mL), or their

214

combination for 8 days. Treatment with culture medium was used as a control. All

215

groups included three replicates. The medium samples were collected to measure

216

HBsAg, HbeAg, and HBV DNA. The total RNA in the cells was isolated to measure 10


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217

the mRNA level of PKR. The kit and PCR conditions used were same as those used

218

for GRP78. β-Actin was used as an internal reference. The primer used to amplify

219

PKR is shown in Table 1.

220

Statistical Analysis

221

Data is expressed as the mean± standard deviation (SD) of three replicates.

222

One-way ANOVA and Duncan multiple-range test were used to detect significant

223

differences between groups with SPSS 21.0 (SPSS, Inc., Chicago, IL, USA). p < 0.05

224

was considered statistically significant.

225

Results and Discussion

226

Cytotoxicity of BET on HepG2.2.15 cells

227

HBV production in HepG2.2.15 cells is thought to be significantly affected by 37, 38

228

the cytotoxicity of the drugs

. To exclude this influence, the cell viability of BET

229

on HepG2.2.15 cells was tested in the CCK8 assay. Over the 8-day BET treatment

230

period, cell viability was close to 100% with BET below 100 mM. Cytotoxicity was

231

observed when the treatment dose was 200 mM (Figure 2). This indicated that BET

232

below 100 mM did not have the cytotoxicity. This result was used to determine the

233

dose range of BET for subsequent experiments.

234

Anti-HBV Activity of BET in HepG2.2.15 cells

235

Treatment of HepG2.2.15 cells with 20 mM BET for 2 days resulted in 38.6%

236

reduction of HBsAg secretion. After treatment for 8 days, the maximum inhibition

237

rate of BET on HBsAg was 53.4% and the minimum inhibition rate was 29.2%

238

(Figure 3A). The inhibitory effect of BET on HBeAg secretion was slightly less than 11



Page 12 of 33

239

that for HBsAg. After 2 days, the maximum inhibition rate of BET on HBeAg

240

approximated 5%. After the treatment for 8 days, the maximum inhibition rate of BET

241

on HBeAg reached 36.7% and the minimum inhibition rate was 17.1% (Figure 3B).

242

The above results showed that BET inhibited the secretion of HBV particles and the

243

effect exhibited a dose-dependent manner. To further confirm the anti-HBV activity of

244

BET in HepG2.2.15 cells, the levels of extracellular HBV DNA were evaluated after

245

8

246

similarly to the reduction in HBsAg and HBeAg,

247

significantly after treatment of BET.

days

of

BET

treatmnent.

As

shown HBV

in

Figure

3C,

DNA decreased

248

In addition, the inhibitory effect of 3TC on HBsAg and HBeAg was less potent

249

than that on HBV DNA. However, BET inhibited HBsAg and HBeAg as well as HBV

250

DNA. This may be because HBV RNA transcription and protein production are

251

separated from viral genome replication, resulting from the presence of a long-lived

252

population of covalently closed circular DNA in the host cell nucleus 39, whereas the

253

target of 3TC is DNA polymerase. Thus, 3TC effectively inhibited HBV DNA

254

replication and slightly affected HBV protein production. This also indicates that the

255

mechanism of BET against HBV was different from that of 3TC.

256

Suppression of BET on GRP78 Expression

257

The endoplasmic reticulum (ER) is an important compartment involved in the

258

modification and folding of membrane and secretary proteins. The accumulation of

259

surface proteins of HBV, particularly HBsAg, induces the unfolded protein response

260

and subsequent ER stress

40

. Glucose-regulated protein 78 (GRP78) is a chaperone 12


Page 13 of 33


261

protein induced by ER stress and catalyzes the correct folding of proteins. GRP78 was

262

found to function during HBV viral morphogenesis where it interacts with the large

263

surface protein of HBV and regulates its posttranscriptional topological reorientation

264

41

265

the HepG2.2.15 cells. A previous report indicated that the suppression of GRP78

266

protein expression leads to blocks viral particle assembly and secretion 11. Therefore,

267

the inhibition of HBV by BET may occur through a similar mechanism.

. As shown in Figure 4A, BET significantly decreased the mRNA level of GRP78 in

268

Moreover, GRP78 is a product of ER stress, and suppression of GRP78 indicates

269

that BET can alleviate ER stress induced by HBV. As described above, HBsAg

270

accumulation induces ER stress. ER stress can promote the expression of HBsAg and

271

replication of HBV through a feedback mechanism

272

inhibitory effect of BET on HBsAg was more potent than that on HBeAg. Therefore,

273

the anti-HBV activity of BET was mediated via the regulation of ER stress (Figure

274

4B). The mechanism of BET was similar to those of some constituents of medicinal

275

plants with anti-HBV activities 42, 43. However, how these constituents alleviate ER

276

stress remains unclear.

33

. This explains why the

277

A previous study showed that BET decreases ER stress by reducing homocysteine

278

in alcoholic mice livers 44. Homocysteine is known to induce the ER stress response in

279

hepatocytes. High levels of homocysteine have been detected in patients with HBV

280

infection

281

methyltransferase in the methionine-homocysteine cycle

282

BET on ER stress may be related to the methyl donor function.

45

. BET reduces homocysteine by affecting betaine-homocysteine 44, 46

13


. Therefore, the effect of


Page 14 of 33

283

In addition, 3TC directly inhibits the replication of HBV DNA. However, 3TC

284

can also decrease the expression of GRP78 (Figure 4A). This indicates that the

285

decrease in HBV by another antiviral mechanism also alleviates ER stress and that

286

BET has another anti-HBV mechanism. The effects of BET in increasing the

287

endogenous antioxidant system or inhibiting inflammation in cells

288

contribute to the anti-HBV activity. The specific mechanism requires further

289

investigation.

290

Inhibition of BET on DHBV Replication

47, 48

, may also

291

The anti-HBV activity of BET was further evaluated in a DHBV-infected duckling

292

model. The results are shown in Table 2. Duck serum DHBV DNA replication levels

293

were markedly decreased in the groups treated with 3TC or high-dose (2.0 g/kg) or

294

medium-dose (1.0 g/kg) BET compared with the control group. Interestingly, the

295

DHBV DNA replication level in the 3TC group was dramatically increased after the

296

5-day withdrawal period. The relatively rapid rebound in ducks treated with 3TC has

297

been reported previously

298

groups, although the inhibition of BET on DHBV DNA was less potent than in 3TC.

299

This may be because BET inhibited DHBV replication indirectly, whereas 3TC

300

directly targeted viral replication. The inhibition of 3TC on DHBV depends on

301

existing 3TC. The regulatory effects of BET on ER stress or other stress responses in

302

cells showed a long-term effect after treatment with BET. Therefore, DHBV DNA

303

rebounded more rapidly after 3TC treatment than after BET treatment. This indicates

304

that BET has a longer-term effect on HBV than 3TC in the clinic.

49

. DHBV DNA did not rebound significantly in the BET

14


Page 15 of 33


305

BET Inhibited HBV Resistance to 3TC

306

To investigate the effect of BET on HBV resistance to 3TC, HepG2.2.15 cells

307

were treated with BET, 3TC, or their combination for a longer period. As shown in

308

Figure 5, by day 12, the level of HBV DNA had decreased in the 3TC group and

309

combination group. Between days 12 and 42, HBV DNA levels remained stable.

310

However, HBV DNA levels rebounded in the 3TC group after 48 days. The

311

combination group showed a lower level of HBV DNA than the 3TC group, and the

312

rebound was suppressed. This indicates that BET inhibited the development of HBV

313

drug resistance to 3TC.

314

The use of native compounds with antiviral activity in the clinic is restricted by

315

the low potency of these compounds. However, they provide novel modes of action

316

against HBV, particularly using the host-based antiviral strategies 9. These compounds

317

can able create an intracellular environment that does not support HBV. The

318

therapeutic pressure of these agents is not on viruses, but on the host. Thus, these

319

compounds can be used to overcome HBV drug resistance. As described above, the

320

mechanism of BET against HBV may involve the regulation of ER stress. Thus,

321

anti-HBV effect of BET results from the improved intracellular environment. The

322

most probable mechanism of BET inhibiting HBV resistance to 3TC is based on the

323

different targets of BET and 3TC.

324

rtM204V/I Mutation Change in HBV DNA

325

HBV resistance to 3TC is mainly associated with the rtM204V/I mutation of HBV

326

DNA. The mutation results in amino acid substitution, directly reducing the 15



327

susceptibility to 3TC. To further confirm the inhibitory effect of BET on HBV drug

328

resistance to 3TC, the percentage change of the rtM204V/I mutation in total HBV

329

DNA was measured following treatment with 3TC, BET, or their combination. The

330

results in Figure 6A and 6B show that the Taqman-MGB probes were highly specific

331

and could discriminate the mutant from wild or other mutant sequences.

332

The rtM204V/I mutation was not detected in the control group or BET group. This

333

indicates that the mutation did not occur or that the level of mutations was extremely

334

low under natural conditions or treatment with BET. However, as shown in Figure 6C,

335

the percentages of YVDD and YIDD reached 0.31% and 0.23%, respectively,

336

following treatment with 3TC on day 12. With passsing time, the percentage of

337

rtM204V/I mutation increased. On days 12 to 36, the percentages of both mutants

338

were below 10% respectively. Although the mutants existed, the anti-HBV effect of

339

3TC changed only minimally before day 48 (Figure 5). This may be related to the

340

properties of the mutants. After 48 days, the percentage of rtM204V/I mutation

341

surpassed 20% and HBV DNA rebounded. Using a combination of 3TC and BET, the

342

percentage of rtM204V/I mutation significantly decreased compared with the

343

treatment of 3TC alone after day 24. This result showed that BET could suppress the

344

drug resistance mutation of HBV DNA significantly.

345

BET Improved Anti-HBV Activity of IFN-α

346

To test the effect of BET on the anti-HBV activity of IFN-α, HepG2.2.15 cells

347

were treated with a combination of BET and IFN-α. As shown in Figure 7A, 0.5 mM

348

BET did not inhibit HBsAg and HBeAg, whereas combination treatment with BET 16


Page 16 of 33

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349

and IFN-α significantly improved the inhibition, compared to IFN-α. Consistently,

350

inhibition of HBV DNA in the combination group also increased (Figure 7B). These

351

results indicate that BET improves the anti-HBV activity of IFN-α. Additionally, PKR

352

is an antiviral protein induced by the JAK-STAT signaling pathway. PKR mRNA

353

levels following combination treatment increased significantly compared with IFN-α

354

treatment alone (Figure 7C). This indicates that BET enhances the JAK-STAT

355

signaling pathway. Therefore, the anti-HBV activity of IFN-α improved by BET

356

resulted from enhancement of the JAK-STAT signaling pathway.

357

Methylated STAT1 is a key signal molecule in the JAK-STAT signaling pathway

358

(Figure 7D). SAM is the methyl group donor for STAT1 methylation catalyzed by

359

protein arginine methyltransferase 1. The virus can inhibit protein arginine

360

methyltransferase 1 and lead to the low methylation of STAT1

361

effect of IFN-α was decreased and patients showed a low response to IFN-α. A

362

previous study showed that addition of SAM could restore the STAT1 methylation

363

and improve the antiviral effect of IFN-α

364

the conversion of L-homocysteine to methionine, a direct precursor of SAM. SAM

365

levels can be increased by adding BET

366

SAM. Thus, the mechanism of BET on JAK-STAT signaling pathway may be that

367

BET supplementation increased SAM levels, restoring the JAK-STAT signaling

368

pathway. However, further studies are required to confirm this.

50

. Thus, the antiviral

5, 50

. Betaine is the methyl group donor for

50

. In this study, BET had similar effect as

369

In summary, BET showed anti-HBV activity both in vitro and in vivo, possibly

370

through the regulation of ER stress by BET. Based on their different mechanisms, 17



371

BET inhibited HBV resistance to 3TC and suppressed the introduction of resistance

372

mutations into HBV DNA. Additionally, BET improved the anti-HBV activity of

373

IFN-α via enhancing the JAK-STAT signaling pathway. This enhancement may be the

374

result of the methyl donor function of BET. Although the anti-HBV activity of BET

375

may be less potent than those of drugs used in the clinic, it is advantageous to inhibit

376

the resistance of HBV to 3TC and improve the antiviral effect of IFN-α. Therefore,

377

BET shows potential as a complement drug for the treatment of HBV.

378

Funding

379

This work was financially supported by the Open Project Program of Provincial

380

Key Laboratory of Green Processing Technology and Product Safety of Natural

381

Products (201304) and National Natural Science Foundation of China (Grant No.

382

31201330).

18


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513 21



514

Figure captions:

515

Figure 1. Construction of YVDD plasmid. Two pairs of primers, including s-YMDD

516

forward primer and m-YVDD reverse primer and s-YMDD reverse primer and

517

m-YVDD forward primer, were used in the first PCR step to generate intermediate

518

PCR products. Next, the two mutated products were denatured as a template for the

519

second PCR step by using flanking primers s-YMDD. The final PCR products were

520

digested with Xho I and Hind III (TaKaRa), and then inserted into the pGL3 Basic

521

Vector. The recombinant vector was transformed into Escherichia coli DH5α cells.

522

The mutations were confirmed by sequencing. The YIDD plasmid was constructed in

523

the same manner. The primers used are listed in Table 1.

524

Figure 2. Cytotoxic effect of BET on HepG2.2.15 cells. To evaluate cytotoxicity, cells

525

were plated in 96-well plates for 24 h and treated with different concentrations

526

(12.5-400 mM) of BET for 8 days. After treatment, the cells were subjected to a

527

cytotoxicity assay used CCK-8. The experiments were performed by triplicates. The

528

data shown are the mean ± SD.

529

Figure 3. Inhibition of BET on HBV. (A) HBsAg, (B) HBeAg and (C) HBV DNA.

530

HepG2.2.15 cells were cultured in the presence of BET at various concentrations or

531

3TC at 40 µM for 8 days, and then HBsAg and HBeAg in the supernatants were

532

quantified using specific ELISA kits. HBV DNA levels were quantified by QPCR.

533

The pGL3 Basic Vector was used as an internal standard. The experiments were

534

performed by triplicates, and the data are presented as the mean ± SD. *, p

Betaine Inhibits Hepatitis B Virus with an Advantage of Decreasing

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