Division of Infectious Diseases and Public Global Health, University of California, San Diegogrid.266100.3, La Jolla, California, USA.
J Virol. 2022 Feb 23;96(4):e0165521. doi: 10.1128/JVI.01655-21. Epub 2021 Dec 1.
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 nucleotides (nt) 1905 to 1909 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 located within nt 1606 to 1809 correspond to the coding region of HBx mRNA and 3' untranslated region (UTR) of other viral mRNAs. Using the mutants of additional m6A sites in nt 1606 to 1809 and a depletion strategy of m6A methyltransferases (METTL3/14) and reader proteins (YTHDFs), we show that m6A modification at nt 1616, located in the HBx coding region, regulates HBx protein expression. The HBx RNA and protein expression levels 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 expression levels were not affected by the m6A modification at nt 1616. 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. N6-methyladenosien (m6A) modifications recently have been implicated in the HBV life cycle. Previously, we observed that m6A modification occurs in the adenosine at nt 1907 of the HBV genome, and this modification regulates the viral life cycle. Here, we identified an additional m6A site located in nt 1616 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 mA in the regulation of HBx protein expression.
乙型肝炎病毒 (HBV) 编码一种调节蛋白，称为 HBx，过去对其进行了深入研究，结果表明它在病毒转录和复制中发挥关键作用。此外，大量文献涉及信号转导和可能的机制，这些机制与感染相关的肝癌发生有关。我们之前报道过，HBV 转录本在 epsilon 结构元件中核苷酸 (nt) 1905 到 1909 处的单个一致 DRACH 基序处被 N6-甲基腺苷 (m6A) 修饰，这种 m6A 修饰会影响 HBV 的生命周期。在这项研究中，我们提供了证据表明，位于 nt 1606 到 1809 处的其他 m6A(DRACH)基序的变体对应于 HBx mRNA 的编码区和其他病毒 mRNA 的 3'非翻译区 (UTR)。我们使用 nt 1606 到 1809 处的额外 m6A 位点的突变体和 m6A 甲基转移酶 (METTL3/14) 和读码器蛋白 (YTHDF) 的消耗策略，表明位于 HBx 编码区的 nt 1616 处的 m6A 修饰调节 HBx 蛋白表达。HBx RNA 和蛋白表达水平通过沉默 m6A 读码器 YTHDF2 和甲基转移酶以及 HBx 编码区 m6A 位点的突变显著增加。然而，其他病毒蛋白表达水平不受 nt 1616 处 m6A 修饰的影响。因此，HBx 开放阅读框 (ORF) 中的 m6A 修饰下调 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，这与它在建立慢性肝炎中的可能作用一致。N6-甲基腺苷 (m6A) 修饰最近已被牵连到 HBV 的生命周期中。之前，我们观察到 m6A 修饰发生在 HBV 基因组的 nt 1907 处的腺苷上，这种修饰调节病毒的生命周期。在这里，我们鉴定了 HBV 基因组中 nt 1616 处的另一个 m6A 位点。这种修饰会对 HBx RNA 和蛋白表达产生负面影响。在没有 m6A 甲基转移酶 (METTL3/14) 和读码器蛋白 (YTHDF2) 的情况下，HBx RNA 和蛋白表达增加。使用缺乏 HBx 编码区 m6A 的 HBV 突变体，我们展示了 mA 在调节 HBx 蛋白表达中的独特位置效应。
