Biogenesis and molecular characteristics of serum hepatitis B virus RNA.
Biogenesis and molecular characteristics of serum hepatitis B virus RNA.
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血清乙型肝炎病毒 RNA 的生物发生和分子特征。
DOI：
10.1371/journal.ppat.1008945
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发表时间：
2020-10
期刊：
影响因子：
6.7
通讯作者：
Guo H
中科院分区：
文献类型：
--
作者：
Shen S;Xie Z;Cai D;Yu X;Zhang H;Kim ES;Zhou B;Hou J;Zhang X;Huang Q;Sun J;Guo H
HBV是一种包膜DNA病毒，通过肝细胞中基因组前RNA （pg）中间体的逆转录复制其DNA基因组。有趣的是，HBV RNA可以在慢性乙型肝炎（CHB）患者血清中的病毒样颗粒中检测到，并已被用作治疗患者肝内cccDNA活性的生物标志物。然而，血清HBV RNA的生物发生和分子特征仍有待完全确定。在这项研究中，我们发现封装的血清HBV RNA主要由pgRNA组成，它们具有洗涤剂和核糖核酸酶抗性。通过使用引物缺陷型HBV突变体Y63D或3TC处理，在不影响pgRNA封装的情况下阻断HBV DNA复制，我们证明细胞培养上清含有大量含pgRNA的非包膜衣壳和少量含pgRNA的病毒粒子。pgrna -病毒粒子的形成需要衣壳组装和病毒包膜蛋白，这两种蛋白分别可以被衣壳组装调节剂和包膜敲除突变体抑制。此外，pgrna -病毒粒子利用多泡体途径出口，以类似于dna -病毒粒子形态发生的方式。Northern blotting、RT-PCR和3 ‘ RACE检测显示血清/上清HBV pgRNA主要剪接，缺乏3 ’端序列。此外，从使用可逆HBV启动抑制剂L-FMAU处理的细胞中收集的pgrna病毒粒子无法在HepG2-NTCP细胞中建立感染。总之，血清HBV RNA在非感染性病毒粒子中以剪接和无聚(A) pgRNA的形式分泌。我们的研究将揭示HBV生命周期中血清HBV RNA的分子生物学，并有助于开发血清HBV RNA作为慢性乙型肝炎诊断和治疗预后的新型生物标志物。尽管越来越多的证据支持细胞外HBV RNA（或血清HBV RNA）物种的存在及其作为监测慢性HBV感染的新标志物的潜力，但血清HBV RNA的来源和分子形式仍然不明确。除了感染性DNA病毒粒子外，已知HBV在其复制周期内还在细胞外产生许多不完整的病毒粒子，包括亚病毒粒子（HBsAg）、裸衣壳和空病毒粒子。在这里，我们证明了含有HBV前基因（pg） rna的病毒颗粒样颗粒存在于细胞培养液和患者血清中，它们通过多泡体（MVB）分泌途径与成熟的DNA病毒颗粒一起分泌。此外，病毒粒子衍生的HBV RNA主要是剪接和3 '截断形式，并且HBV RNA病毒粒子无法在细胞培养中启动新一轮感染。这些发现为更好地理解血清HBV RNA生物学提供了新的见解，并为血清HBV RNA作为慢性乙型肝炎患者生物标志物的进一步广泛临床应用提供了概念基础。
HBV is an enveloped DNA virus that replicates its DNA genome via reverse transcription of a pregenomic (pg) RNA intermediate in hepatocytes. Interestingly, HBV RNA can be detected in virus-like particles in chronic hepatitis B (CHB) patient serum and has been utilized as a biomarker for intrahepatic cccDNA activity in treated patients. However, the biogenesis and molecular characteristics of serum HBV RNA remain to be fully defined. In this study, we found that the encapsidated serum HBV RNA predominately consists of pgRNA, which are detergent- and ribonuclease-resistant. Through blocking HBV DNA replication without affecting pgRNA encapsidation by using the priming-defective HBV mutant Y63D or 3TC treatment, we demonstrated that the cell culture supernatant contains a large amount of pgRNA-containing nonenveloped capsids and a minor population of pgRNA-containing virions. The formation of pgRNA-virion requires both capsid assembly and viral envelope proteins, which can be inhibited by capsid assembly modulators and an envelope–knockout mutant, respectively. Furthermore, the pgRNA-virion utilizes the multivesicular body pathway for egress, in a similar way as DNA-virion morphogenesis. Northern blotting, RT-PCR, and 3’ RACE assays revealed that serum/supernatant HBV pgRNA are mainly spliced and devoid of the 3’-terminal sequences. Furthermore, pgRNA-virion collected from cells treated with a reversible HBV priming inhibitor L-FMAU was unable to establish infection in HepG2-NTCP cells. In summary, serum HBV RNA is secreted in noninfectious virion-like particle as spliced and poly(A)-free pgRNA. Our study will shed light on the molecular biology of serum HBV RNA in HBV life cycle, and aid the development of serum HBV RNA as a novel biomarker for CHB diagnosis and treatment prognosis. Although increasing evidence supports the presence of extracellular HBV RNA (or serum HBV RNA) species and their potentials to be a new marker for monitoring chronic HBV infection, the origin and molecular forms of serum HBV RNA remain ill-defined. In addition to the infectious DNA virions, HBV is known to produce a number of incomplete viral particles extracellularly, including subviral particles (HBsAg), naked capsids, and empty virions, during its replication cycle. Here, we demonstrated that HBV pregonomic (pg) RNA-containing virion-like-particles exist in cell culture fluid and patient sera, which are secreted along with mature DNA virions via the multivesicular body (MVB) secretory route. Moreover, virion-derived HBV RNAs are predominantly spliced and 3’ truncated forms, and HBV RNA virions are unable to initiate a new round of infection in cell cultures. These findings provide new insights into a better understanding of serum HBV RNA biology and a conceptual basis for further widespread clinical applications of serum HBV RNA as a biomarker in chronic hepatitis B patients.
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DOI：
10.1007/978-1-62703-484-5_13
发表时间：
2013
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Cai, Dawei;Nie, Hui;Yan, Ran;Guo, Ju-Tao;Block, Timothy M;Guo, Haitao
通讯作者：
Guo, Haitao
DOI：
10.3390/v9030056
发表时间：
2017-03-21
期刊：
Viruses
影响因子：
--
作者：
Hu J;Liu K
通讯作者：
Liu K
影响因子：
4.9
作者：
Jones, Scott A.;Murakami, Eisuke;Hu, Jianming
通讯作者：
Hu, Jianming
通过监测治疗的慢性乙型肝炎患者特征突变的出现和逆转表明乙型肝炎病毒共价闭合环状 DNA 的快速周转
DOI：
10.1002/hep.31240
发表时间：
2021-01
期刊：
Hepatology (Baltimore, Md.)
影响因子：
--
作者：
Huang Q;Zhou B;Cai D;Zong Y;Wu Y;Liu S;Mercier A;Guo H;Hou J;Colonno R;Sun J
通讯作者：
Sun J
血清 HBV RNA：慢性乙型肝炎病毒感染的新的潜在生物标志物
DOI：
10.1002/hep.30325
发表时间：
2019-04
期刊：
Hepatology (Baltimore, Md.)
影响因子：
--
作者：
Liu S;Zhou B;Valdes JD;Sun J;Guo H
通讯作者：
Guo H