Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China.
Baruch S. Blumberg Institute, Doylestown, Pennsylvania, United States of America.
PLoS Pathog. 2021 Nov 9;17(11):e1010057. doi: 10.1371/journal.ppat.1010057. eCollection 2021 Nov.
The core protein (Cp) of hepatitis B virus (HBV) assembles pregenomic RNA (pgRNA) and viral DNA polymerase to form nucleocapsids where the reverse transcriptional viral DNA replication takes place. Core protein allosteric modulators (CpAMs) inhibit HBV replication by binding to a hydrophobic "HAP" pocket at Cp dimer-dimer interfaces to misdirect the assembly of Cp dimers into aberrant or morphologically "normal" capsids devoid of pgRNA. We report herein that a panel of CpAM-resistant Cp with single amino acid substitution of residues at the dimer-dimer interface not only disrupted pgRNA packaging, but also compromised nucleocapsid envelopment, virion infectivity and covalently closed circular (ccc) DNA biosynthesis. Interestingly, these mutations also significantly reduced the secretion of HBeAg. Biochemical analysis revealed that the CpAM-resistant mutations in the context of precore protein (p25) did not affect the levels of p22 produced by signal peptidase removal of N-terminal 19 amino acid residues, but significantly reduced p17, which is produced by furin cleavage of C-terminal arginine-rich domain of p22 and secreted as HBeAg. Interestingly, p22 existed as both unphosphorylated and phosphorylated forms. While the unphosphorylated p22 is in the membranous secretary organelles and the precursor of HBeAg, p22 in the cytosol and nuclei is hyperphosphorylated at the C-terminal arginine-rich domain and interacts with Cp to disrupt capsid assembly and viral DNA replication. The results thus indicate that in addition to nucleocapsid assembly, interaction of Cp at dimer-dimer interface also plays important roles in the production and infectivity of progeny virions through modulation of nucleocapsid envelopment and uncoating. Similar interaction at reduced p17 dimer-dimer interface appears to be important for its metabolic stability and sensitivity to CpAM suppression of HBeAg secretion.
乙型肝炎病毒 (HBV) 的核心蛋白 (Cp) 组装前基因组 RNA (pgRNA) 和病毒 DNA 聚合酶形成核衣壳，在那里进行逆转录病毒 DNA 复制。核心蛋白别构调节剂 (CpAMs) 通过与 Cp 二聚体-二聚体界面上的疏水性“HAP”口袋结合来抑制 HBV 复制，从而将 Cp 二聚体错误地组装成缺乏 pgRNA 的异常或形态上“正常”的衣壳。我们在此报告，一组在二聚体-二聚体界面处具有单个氨基酸取代的 CpAM 抗性 Cp，不仅破坏了 pgRNA 包装，而且还损害了核衣壳包膜、病毒粒子感染力和共价闭合环状 (ccc) DNA 生物合成。有趣的是，这些突变也显著降低了 HBeAg 的分泌。生化分析表明，在前核心蛋白 (p25) 背景下，CpAM 抗性突变不影响信号肽切除 N 端 19 个氨基酸残基后产生的 p22 的水平，但显著降低了 p17 的水平，p17 是由 p22 的 C 端富含精氨酸的结构域经弗林蛋白酶切割产生的，并作为 HBeAg 分泌。有趣的是，p22 既存在未磷酸化形式也存在磷酸化形式。虽然未磷酸化的 p22 存在于膜性分泌细胞器中，是 HBeAg 的前体，但细胞质和细胞核中的 p22 在 C 端富含精氨酸的结构域处高度磷酸化，并与 Cp 相互作用，破坏衣壳组装和病毒 DNA 复制。结果表明，除了核衣壳组装外，Cp 在二聚体-二聚体界面的相互作用还通过调节核衣壳包膜和脱壳，在产生和感染子代病毒粒子方面发挥重要作用。在降低的 p17 二聚体-二聚体界面上的类似相互作用似乎对其代谢稳定性和对 CpAM 抑制 HBeAg 分泌的敏感性很重要。
