HBV核心内的内源性胞苷脱氨酶对HBV基因组的不对称修饰提示

StephenW

J Virol. 2018 Feb 28. pii: JVI.02190-17. doi: 10.1128/JVI.02190-17. [Epub ahead of print]
Asymmetric modification of HBV genomes by an endogenous cytidine deaminase inside HBV cores informs a model of reverse transcription.Nair S1, Zlotnick A2.
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AbstractCytidine deaminases inhibit replication of broad range of DNA viruses by deaminating cytidines on single stranded DNA to generate uracil. While several lines of evidence have revealed HBV genome editing by deamination, it is still unclear which nucleic acid intermediate of HBV is modified. Hepatitis B virus has a relaxed circular double-stranded DNA (rcDNA) genome that is reverse transcribed within virus cores from a RNA template. The HBV genome also persists as covalently closed circular DNA (cccDNA) in the nucleus of an infected cell. In the present study, we find that in HBV-producing HepAD38 and Hep2.2.15 cell lines, endogenous cytidine deaminases edited 10-25% of HBV rcDNA genomes, asymmetrically with almost all mutations on the 5' half of the minus strand. This region corresponds to the last half of the minus strand to be protected by plus strand synthesis. Within this half of the genome, the number of mutations peaks in the middle. Over-expressed APOBEC3A and APOBEC3G could be packaged in HBV capsids but did not change the amount or distribution of mutations. We found no deamination on pgRNA indicating that an intact genome is encapsidated and deaminated during or after reverse transcription. The deamination pattern suggests a model of rcDNA synthesis where pgRNA and then newly synthesized minus-sense single stranded DNA are protected from deaminase by interaction with the virus capsid; during plus strand synthesis, when enough dsDNA has been synthesized to displace the remaining minus strand from the capsid surface that single stranded DNA becomes deaminase-sensitive.IMPORTANCE Host-induced mutation of the HBV genome, as by APOBEC proteins, may be a path to clearing the virus. We examined Cytidine to Thymidine mutations in the genomes of HBV particles grown in the presence or absence of overexpressed APOBEC proteins. We found that genomes were subjected to deamination activity during reverse transcription, which takes place within the virus capsid. These observations provide a direct insight into the mechanics of reverse transcription, suggesting that newly synthesized dsDNA displaces ssDNA from the capsid walls making the ssDNA accessible to deaminase activity.


PMID:29491156DOI:10.1128/JVI.02190-17

StephenW

J Virol。 2018年2月28日。pii：JVI.02190-17。 doi：10.1128 / JVI.02190-17。 [电子版提前打印]
HBV核心内的内源性胞苷脱氨酶对HBV基因组的不对称修饰提示了逆转录模型。
奈尔S1，Zlotnick A2。
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胞苷脱氨酶通过将单链DNA上的胞苷脱氨基以产生尿嘧啶来抑制各种DNA病毒的复制。尽管几条证据显示脱氨基作用对HBV基因组进行了编辑，但仍不清楚HBV的哪个核酸中间体被修饰。乙型肝炎病毒具有松弛的环状双链DNA（rcDNA）基因组，其在RNA模板的病毒核心内反转录。 HBV基因组在感染细胞的细胞核中也以共价闭合环状DNA（cccDNA）的形式存在。在本研究中，我们发现在HBV产生的HepAD38和Hep2.2.15细胞系中，内源性胞苷脱氨酶编辑HBV rcDNA基因组的10-25％，几乎与负链的5'半部分上的所有突变不对称。该区域对应于待被正链合成保护的负链的后一半。在这一半的基因组中，突变的数量在中间出现峰值。过度表达的APOBEC3A和APOBEC3G可以包装在HBV衣壳中，但不改变突变的量或分布。我们发现pgRNA上没有脱氨基，表明完整的基因组在反转录过程中或之后被包膜和脱氨。脱氨模式提出了一种rcDNA合成模型，其中pgRNA然后新合成的负义单链DNA通过与病毒衣壳相互作用而被保护免于脱氨酶;在正链合成期间，当足够的dsDNA被合成以置换来自衣壳表面的剩余负链时，单链DNA变为脱氨酶敏感性。意向由于APOBEC蛋白质，宿主诱导的HBV基因组突变可能是清除病毒。我们检查了在存在或不存在过表达的APOBEC蛋白的情况下生长的HBV颗粒基因组中的胞嘧啶核苷至胸苷的突变。我们发现，基因组在逆转录过程中发生脱氨基活性，发生在病毒衣壳内。这些观察提供了对逆转录力学的直接了解，表明新合成的dsDNA从衣壳壁置换ssDNA，从而使得ssDNA可脱除酶活性。

结论：
    29491156
DOI：
    10.1128 / JVI.02190-17