乙型肝炎病毒 X (HBx) 蛋白表达受其 mRNA 的 N6-甲基腺苷修饰的

StephenW

乙型肝炎病毒 X (HBx) 蛋白表达受其 mRNA 的 N6-甲基腺苷修饰的严格调控
Geon-Woo Kim 1 , Aleem Siddiqui 1
隶属关系
联系

    1
    加利福尼亚大学圣地亚哥分校传染病和全球公共卫生部，拉霍亚，加利福尼亚 92093，美国。

    PMID：34851655 DOI：10.1128/JVI.01655-21

抽象的

乙型肝炎病毒 (HBV) 编码一种称为 HBx 的调节蛋白，过去对其进行了深入研究，并显示其在病毒转录和复制中起关键作用。此外，在与信号转导和导致与感染相关的肝癌发生的可能机制相关的文献中存在大量工作。我们之前曾报道过，HBV 转录本在 epsilon 结构元件中 1905-1909 核苷酸 (nt) 的单一共有 DRACH 基序上被 N6-甲基腺苷 (m6A) 修饰，并且这种 m6A 修饰会影响 HBV 生命周期。在这项研究中，我们提供了 m6A (DRACH) 基序的其他变体位于 1606 至 1809 nt 内的证据，对应于 HBx mRNA 的编码区和其他病毒 mRNA 的 3' 非翻译区 (UTR)。使用 1606 至 1809 nt 中额外 m6A 位点的突变体以及 m6A 甲基转移酶 (METTL3/14) 和阅读器蛋白 (YTHDF) 的消耗策略，我们表明位于 HBx 编码区的 1616 nt 处的 m6A 修饰调节 HBx 蛋白表达. m6A 阅读器 YTHDF2 和甲基转移酶的沉默以及 HBx 编码区中 m6A 位点的突变显着增加了 HBx RNA 和蛋白质表达。然而，其他病毒蛋白表达不受 1616 nt 处 m6A 修饰的影响。因此，HBx 开放阅读框 (ORF) 中的 m6A 修饰会下调 HBx 蛋白表达，这在 HBV 转染、转基因小鼠和人类肝细胞的自然感染过程中很常见。这些研究确定了 m6A 修饰在 HBx 蛋白表达的微妙调节中的功能作用，与其在建立慢性肝炎中的可能作用一致。重要性 N6-甲基腺苷 (m6A) 修饰最近与 HBV 生命周期有关。以前，我们观察到 m6A 修饰发生在 HBV 基因组 1907 nt 的腺苷中，这种修饰调节病毒生命周期。在这里，我们确定了一个额外的 m6A 位点，位于 HBV 基因组的 1616 nt。这种修饰会对 HBx RNA 和蛋白质表达产生负面影响。在没有 m6A 甲基转移酶 (METTL3/14) 和阅读器蛋白 (YTHDF2) 的情况下，HBx RNA 和蛋白质表达增加。使用在 HBx 编码区缺乏 m6A 的 HBV 突变体，我们展示了 m6A 在调节 HBx 蛋白表达中的独特位置效应。

StephenW

Hepatitis B virus X (HBx) Protein Expression is Tightly Regulated by N6-methyladenosine Modification of its mRNA
Geon-Woo Kim  1 , Aleem Siddiqui  1
Affiliations
Affiliation

    1
    Division of Infectious Diseases and Public Global Health, University of California, San Diego, La Jolla, CA 92093, USA.

    PMID: 34851655 DOI: 10.1128/JVI.01655-21

Abstract

Hepatitis B virus (HBV) encodes a regulatory protein termed HBx, that has been intensely studied in the past and shown to play a key role(s) in viral transcription and replication. In addition, a huge body of work exists in the literature related to signal transduction and possible mechanism(s) leading to hepatocarcinogenesis associated with infection. We have previously reported that HBV transcripts are modified by N6-methyladenosine (m6A) at the single consensus DRACH motif at 1905-1909 nucleotide (nt) in the epsilon structural element and this m6A modification affects the HBV life cycle. In this study, we present evidence that additional variants of m6A (DRACH) motifs are located within 1606 to 1809 nt correspond on the coding region of HBx mRNA and 3' untranslated region (UTR) of other viral mRNAs. Using the mutants of additional m6A site in 1606 to 1809 nt and a depletion strategy of m6A methyltransferases (METTL3/14) and reader proteins (YTHDFs), we show that m6A modification at 1616 nt, located in HBx coding region, regulates HBx protein expression. The HBx RNA and protein expressions were notably increased by the silencing of m6A reader YTHDF2 and methyltransferases as well as the mutation of m6A sites in the HBx coding region. However, other viral protein expressions were not affected by the m6A modification at 1616 nt. Thus, m6A modifications in the HBx open reading frame (ORF), downregulate HBx protein expression, commonly seen during HBV transfections, transgenic mice, and natural infections of human hepatocytes. These studies identify the functional role of m6A modification in the subtle regulation of HBx protein expression consistent with its possible role in establishing chronic hepatitis. Importance N6-methyladenosien (m6A) modifications have been recently implicated in the HBV life cycle. Previously, we observed that m6A modification occurs in the adenosine at 1907 nt of HBV genome and this modification regulates the viral life cycle. Here, we identified an additional m6A site located in 1616 nt of the HBV genome. This modification negatively affects HBx RNA and protein expression. In the absence of m6A methyltransferases (METTL3/14) and reader protein (YTHDF2), the HBx RNA and protein expression were increased. Using HBV mutants that lack m6A in the HBx coding region, we present the unique positional effects of m6A in the regulation of HBx protein expression.