Basic Research Laboratory National Cancer Institute, Frederick, MD 21702, USA.
Department of Molecular Microbiology and Immunology, St. Louis University, St. Louis, MO 63104, USA.
Molecules. 2020 Sep 27;25(19):4434. doi: 10.3390/molecules25194434.
Initiation of protein-primed (-) strand DNA synthesis in hepatitis B virus (HBV) requires interaction of the viral reverse transcriptase with epsilon (ε), a -acting regulatory signal located at the 5' terminus of pre-genomic RNA (pgRNA), and several host-encoded chaperone proteins. Binding of the viral polymerase (P protein) to ε is necessary for pgRNA encapsidation and synthesis of a short primer covalently attached to its terminal domain. Although we identified small molecules that recognize HBV ε RNA, these failed to inhibit protein-primed DNA synthesis. However, since initiation of HBV (-) strand DNA synthesis occurs within a complex of viral and host components (e.g., Hsp90, DDX3 and APOBEC3G), we considered an alternative therapeutic strategy of allosteric inhibition by disrupting the initiation complex or modifying its topology. To this end, we show here that 3,7-dihydroxytropolones (3,7-dHTs) can inhibit HBV protein-primed DNA synthesis. Since DNA polymerase activity of a ribonuclease (RNase H)-deficient HBV reverse transcriptase that otherwise retains DNA polymerase function is also abrogated, this eliminates direct involvement of RNase (ribonuclease) H activity of HBV reverse transcriptase and supports the notion that the HBV initiation complex might be therapeutically targeted. Modeling studies also provide a rationale for preferential activity of 3,7-dHTs over structurally related α-hydroxytropolones (α-HTs).
乙型肝炎病毒 (HBV) 蛋白启动子 (-) 链 DNA 合成的起始需要病毒逆转录酶与 ε 的相互作用，ε 是位于前基因组 RNA (pgRNA) 5' 末端的 - 作用调节信号，以及几种宿主编码的伴侣蛋白。病毒聚合酶 (P 蛋白) 与 ε 的结合对于 pgRNA 的包裹和与其末端结构域共价连接的短引物的合成是必需的。虽然我们已经鉴定出识别 HBV ε RNA 的小分子，但这些小分子未能抑制蛋白启动子 DNA 的合成。然而，由于 HBV (-) 链 DNA 合成的起始发生在病毒和宿主成分的复合物中（例如，Hsp90、DDX3 和 APOBEC3G），我们考虑了一种通过破坏起始复合物或改变其拓扑结构来进行变构抑制的替代治疗策略。为此，我们在此表明，3,7-二羟基色酮 (3,7-dHTs) 可以抑制 HBV 蛋白启动子 DNA 的合成。由于缺乏核糖核酸酶 (RNase) H 活性的 HBV 逆转录酶保留了 DNA 聚合酶功能，否则其 DNA 聚合酶活性也被废除，这排除了 HBV 逆转录酶的 RNase (核糖核酸酶) H 活性的直接参与，并支持了这样一种观点，即 HBV 起始复合物可能是治疗的靶点。建模研究也为 3,7-dHTs 相对于结构上相关的 α-羟基色酮 (α-HTs) 的优先活性提供了依据。
