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摘要：
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摘要：目的 探讨丙型肝炎病毒 NS5A反式调节蛋白9（hepatitis C virus NS5Atransactivated protein 9，NS5ATP9）在乙型肝炎病毒（hepatitis B virus，HBV）共价闭
合环状DNA（covalently closed circular DNA，cccDNA）形成与转录中的作用机制。
方法 利用1.3拷贝HBV表达质粒转染Huh7和HepG2细胞、整合有4拷贝HBV基因组的
HepG2.2.15细胞、在诱导型四环素启动子控制下表达HBV的HepAD38细胞构建NS5ATP9
过表达或干扰的HBV细胞模型，收集样品和细胞上清液，提取RNA、HBV核心DNA
（coreDNA）、cccDNA和蛋白，利用酶联免疫吸附试验、实时荧光定量聚合酶链反应
（polymerase chain reaction，PCR）、Southern blot和Western blot技术检测HBV总RNA、
前基因组RNA（pregenomic RNA，pgRNA）、乙型肝炎病毒s抗原（hepatitis B virus s
antigene，HBsAg）、乙型肝炎病毒e抗原（hepatitis B virus e antigene，HBeAg）、松弛
环状DNA（relax circular DNA，rcDNA）以及cccDNA水平。在HepG2细胞中转染乙型肝
炎病毒x蛋白（hepatitis B virus x protein，HBx），通过免疫荧光成像及免疫共沉淀方法
检测NS5ATP9与HBx的结合情况。双荧光素酶报告基因实验检测NS5ATP9对HBx启动子
活性的影响。利用Huh7细胞转染HBV1.3及HBV稳定表达细胞株HepG2.2.15和HepAD38
转染NS5ATP9过表达/干扰质粒，通过Western blot技术检测DDB1和SMC6的蛋白水平。
结果 在HBV病毒活跃的细胞中，NS5ATP9 mRNA水平 [HepG2.2.15细胞：1.891 ± 0.567
比1.00 ± 0.034，t = 2.87，P = 0.0351；HepAD38 tet+细胞：1.978 ± 0.399比1.00 ± 0.034，
t = 4.131，P = 0.0091；HepAD38 tet-细胞：2.642 ± 0.672比1.00 ± 0.034，t = 4.127，
P = 0.0091] 和蛋白水平均显著增加。过表达NS5ATP9后可显著增加HBeAg [（5.402 ±
0.327）S/COV比（2.68 ± 0.552）S/COV，t = 7.35，P = 0.0018]、HBsAg [（2.846 ±
0.185）S/COV比（1.512 ± 0.221）S/COV，t = 8.02，P = 0.0013]、HBV pgRNA及rcDNA
的表达水平，而干扰NS5ATP9后此增加作用消失 [HBeAg：（2.029 ± 0.09）S/COV比
（3.733 ± 0.445）S/COV，t = 6.501，P = 0.0029；HBsAg：（1.501 ± 0.105）S/COV比
（1.878 ± 0.174）S/COV，t = 3.216，P = 0.0324）]。机制研究显示，NS5ATP9和HBx
蛋白主要位于细胞核核仁内，并具有共定位信号，且NS5ATP9可显著提高HBx启动
子（1071.06 ± 79.44比488.47 ± 40.12，t = 13.09，P = 0.00012）的转录活性。另外，过
表达NS5ATP9可显著降低DDB1和SMC6的蛋白水平，而沉默NS5ATP9则可显著提高
DDB1和SMC6的蛋白水平。结论 HBV上调NS5ATP9的表达，形成HBV-NS5ATP9-HBV
cccDNA-HBV的正反馈环路，NS5ATP9通过与HBx相互作用上调肝细胞中HBV cccDNA
的形成与转录，进而促进慢性乙型肝炎的发生发展。
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Abstract: Objective To investigate the role of hepatitis C virus NS5A-transactivated protein
9 (NS5ATP9) on the formation and transcription of hepatitis B virus (HBV) covalently closed
circular DNA (cccDNA). Methods HBV cell models overexpressing or interfering with
NS5ATP9 were respectively constructed by using Huh7 and HepG2 cells with 1.3 copies
of the HBV expression plasmid, HepG2.2.15 cells, and HepAD38 cells. Samples and cell
supernatants were collected and RNA, coreDNA, cccDNA and protein were extracted. The
levels of total HBV RNA, pregenomic RNA (pgRNA), hepatitis B virus s antigene (HBsAg),
hepatitis B virus e antigene (HBeAg), relax circular DNA (rcDNA) and cccDNA were detected
by enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain
reaction (qRT-PCR), Southern blot and Western blot. HBx was transfected into HepG2 cells
and the interaction of NS5ATP9 and HBx was detected by immunofluorescence imaging and
Co-immunoprecipation. The effect of NS5ATP9 on the activity of HBx promoter was detected
by dual luciferase reporter gene assay. The protein levels of DDB1 and SMC6 were detected
by Western blot after transfecting Huh7 cells with HBV 1.3, HepG2.2.15 and HepAD38
with NS5ATP9 over expression plasmid/siRNA. Results In cells with active HBV virus,
both mRNA levels [HepG2.2.15 cells: 1.891 ± 0.567 vs. 1.00 ± 0.034; t = 2.87, P = 0.0351;
HepAD38 tet+ cells: 1.978 ± 0.399 vs. 1.00 ± 0.034; t = 4.131, P = 0.0091; HepAD38 tetcells: 2.642 ± 0.672 vs. 1.00 ± 0.034; t = 4.127, P = 0.0091] and protein levels of NS5ATP9
significantly increased. Over expression of NS5ATP9 significantly increased the levels of
HBeAg [(5.402 ± 0.327) S/COV vs. (2.68 ± 0.552) S/COV; t = 7.35, P = 0.0018], HBsAg
[(2.846 ± 0.185) S/COV vs. (1.512 ± 0.221) S/COV; t = 8.02, P = 0.0013], HBV pgRNA and
rcDNA expression. However, the increasing was abolished after silencing NS5ATP9 [HBeAg:
(2.029 ± 0.09) S/COV vs. (3.733 ± 0.445) S/COV; t = 6.501, P = 0.0029; HBsAg:
(1.501 ± 0.105) S/COV vs. (1.878 ± 0.174) S/COV; t = 3.216, P = 0.0324]. Mechanistic
studies revealed that NS5ATP9 and HBx protein are mainly located in the cell nucleolus
and have co-localization signals, with NS5ATP9 significantly enhancing the transcriptional
activity of the HBx promoter (1071.06 ± 79.44 vs. 488.47 ± 40.12; t = 13.09, P = 0.00012).
Additionally, overexpression of NS5ATP9 significantly decreased the protein levels of DDB1
and SMC6, whereas silencing NS5ATP9 significantly increased the protein levels of DDB1
and SMC6. Conclusion HBV increases the expression of NS5ATP9, which forms a positive
feedback loop of HBV-NS5ATP9-HBV cccDNA-HBV. NS5ATP9 promotes the formation and
transcription of HBV cccDNA by interacting with HBx, thus promoting the occurrence and
development of chronic hepatitis B.
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