Hepatitis B Virus Virions Produced Under Nucleos(t)ide Analogue Treatment Are Mainly Not Infectious Because of Irreversible DNA Chain Termination
Hepatitis B Virus Virions Produced Under Nucleos(t)ide Analogue Treatment Are Mainly Not Infectious Because of Irreversible DNA Chain Termination
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核苷类似物治疗产生的乙型肝炎病毒病毒粒子由于不可逆的 DNA 链终止而基本上不具有传染性
DOI：
10.1002/hep.30844
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发表时间：
2020-02-01
期刊：
影响因子：
13.5
通讯作者：
Lu, Fengmin
中科院分区：
文献类型：
--
作者：
Liu, Yongzhen;Liu, Hui;Lu, Fengmin
核糖核酸类似物(Nas)已被广泛用于治疗慢性乙型肝炎(CHB)。由于病毒DNA聚合酶缺乏校对功能(3‘外切酶活性)，理论上，掺入的Nas会不可逆转地终止病毒DNA的合成。本研究探讨新生乙肝病毒(HBVDNA)DNA的性质及其在NA处理下产生的子代病毒粒子的传染性。通过对HepG2-NTCP细胞和原代人肝细胞(PHHs)的感染来确定乙肝病毒的感染性。采用定量聚合酶链式反应、Northern杂交、Southern杂交、蔗糖梯度离心法和体外内源性DNA聚合酶检测等方法研究病毒粒子中HBVDNA的生化特性。NA处理产生的子代病毒粒子对HepG2-NTCP细胞或PHH主要不具感染性。生化分析表明，在NA处理下，核衣壳或病毒粒子中的HBVDNA主要是短链负链DNA，具有不可逆转的终止作用。在接受NA治疗的患者的系列血清中，首先观察到松弛的环状DNA消失，然后与前C/C区、S基因和X基因对应的HBVDNA水平成比例下降，这支持了这一发现。结论：在NA处理下产生的病毒粒子主要是复制缺陷，因为病毒基因组被截断，DNA链的延长被不可逆转地终止。临床上，我们的结果表明，在NA治疗下，慢性乙肝患者的病毒载量随着qPCR方法检测到的基因组不同区域的不同而不同。我们的发现还表明，NA不仅通过减少病毒载量，还通过产生非传染性病毒粒子来预防围产期和性传播的乙肝病毒以及移植肝脏的感染。核苷(T)类似物(Nas)已被广泛用于治疗慢性乙型肝炎(CHB)。由于病毒DNA聚合酶缺乏校对功能(3‘外切酶活性)，理论上，掺入的Nas会不可逆转地终止病毒DNA的合成。本研究探讨新生乙肝病毒(HBVDNA)DNA的性质及其在NA处理下产生的子代病毒粒子的传染性。通过对HepG2-NTCP细胞和原代人肝细胞(PHHs)的感染来确定乙肝病毒的感染性。采用定量聚合酶链式反应、Northern杂交、Southern杂交、蔗糖梯度离心法和体外内源性DNA聚合酶检测等方法研究病毒粒子中HBVDNA的生化特性。NA处理产生的子代病毒粒子对HepG2-NTCP细胞或PHH主要不具感染性。生化分析表明，在NA处理下，核衣壳或病毒粒子中的HBVDNA主要是短链负链DNA，具有不可逆转的终止作用。在接受NA治疗的患者的系列血清中，首先观察到松弛的环状DNA消失，然后与前C/C区、S基因和X基因对应的HBVDNA水平成比例下降，这支持了这一发现。结论：在NA处理下产生的病毒粒子主要是复制缺陷，因为病毒基因组被截断，DNA链的延长被不可逆转地终止。临床上，我们的结果表明，在NA治疗下，慢性乙肝患者的病毒载量随着qPCR方法检测到的基因组不同区域的不同而不同。我们的发现还表明，NA不仅通过减少病毒载量，还通过产生非传染性病毒粒子来预防围产期和性传播的乙肝病毒以及移植肝脏的感染。
Nucleos(t)ide analogues (NAs) have been widely used for the treatment of chronic hepatitis B (CHB). Because viral DNA polymerase lacks proofreading function (3 ' exonuclease activity), theoretically, the incorporated NAs would irreversibly terminate viral DNA synthesis. This study explored the natures of nascent hepatitis B virus (HBV) DNA and infectivity of progeny virions produced under NA treatment. HBV infectivity was determined by infection of HepG2-NTCP cells and primary human hepatocytes (PHHs). Biochemical properties of HBV DNA in the progeny virions were investigated by qPCR, northern blotting, or Southern blotting hybridization, sucrose gradient centrifugation, and in vitro endogenous DNA polymerase assay. Progeny HBV virions produced under NA treatment were mainly not infectious to HepG2-NTCP cells or PHHs. Biochemical analysis revealed that under NA treatment, HBV DNA in nucleaocapsids or virions were predominantly short minus-strand DNA with irreversible termination. This finding was supported by the observation of first disappearance of relaxed circular DNA and then the proportional decline of HBV-DNA levels corresponding to the regions of PreC/C, S, and X genes in serial sera of patients receiving NA treatment. Conclusion: HBV virions produced under NA treatment are predominantly replication deficient because the viral genomes are truncated and elongation of DNA chains is irreversibly terminated. Clinically, our results suggest that the viral loads of CHB patients under NA therapy vary with the different regions of genome being detected by qPCR assays. Our findings also imply that NA prevention of perinatal and sexual HBV transmission as well as infection of transplanted livers works not only by reducing viral loads, but also by producing noninfectious virions.Nucleos(t)ide analogues (NAs) have been widely used for the treatment of chronic hepatitis B (CHB). Because viral DNA polymerase lacks proofreading function (3' exonuclease activity), theoretically, the incorporated NAs would irreversibly terminate viral DNA synthesis. This study explored the natures of nascent hepatitis B virus (HBV) DNA and infectivity of progeny virions produced under NA treatment. HBV infectivity was determined by infection of HepG2-NTCP cells and primary human hepatocytes (PHHs). Biochemical properties of HBV DNA in the progeny virions were investigated by qPCR, northern blotting, or Southern blotting hybridization, sucrose gradient centrifugation, and in vitro endogenous DNA polymerase assay. Progeny HBV virions produced under NA treatment were mainly not infectious to HepG2-NTCP cells or PHHs. Biochemical analysis revealed that under NA treatment, HBV DNA in nucleaocapsids or virions were predominantly short minus-strand DNA with irreversible termination. This finding was supported by the observation of first disappearance of relaxed circular DNA and then the proportional decline of HBV-DNA levels corresponding to the regions of PreC/C, S, and X genes in serial sera of patients receiving NA treatment. Conclusion: HBV virions produced under NA treatment are predominantly replication deficient because the viral genomes are truncated and elongation of DNA chains is irreversibly terminated. Clinically, our results suggest that the viral loads of CHB patients under NA therapy vary with the different regions of genome being detected by qPCR assays. Our findings also imply that NA prevention of perinatal and sexual HBV transmission as well as infection of transplanted livers works not only by reducing viral loads, but also by producing noninfectious virions.