Luo Yue, Cheng Junjun, Hu Zhanying, Ban Haiqun, Wu Shuo, Hwang Nicky, Kulp John, Li Yuhuan, Du Yanming, Chang Jinhong, Viswanathan Usha, Guo Ju-Tao
Baruch S. Blumberg Institute, Doylestown, Pennsylvanian, USA; Institute of Hepatology, Second Xiangya Hospital, Central South University, Changsha, Hunan Province, China.
Baruch S. Blumberg Institute, Doylestown, Pennsylvanian, USA.
Antiviral Res. 2021 Jul;191:105080. doi: 10.1016/j.antiviral.2021.105080. Epub 2021 Apr 30.
Assembly of hepatitis B virus (HBV) capsids is driven by the hydrophobic interaction of core protein (Cp) at dimer-dimer interface. Binding of core protein allosteric modulators (CpAMs) to a hydrophobic "HAP" pocket formed between the inter-dimer interface strengths the dimer-dimer interaction and misdirects the assembly of Cp dimers into non-capsid Cp polymers or morphologically normal capsids devoid of viral pregenomic (pg) RNA and DNA polymerase. In this study, we performed a systematic mutagenesis analysis to identify Cp amino acid residues at Cp dimer-dimer interface that are critical for capsid assembly, pgRNA encapsidation and resistance to CpAMs. By analyzing 70 mutant Cp with a single amino acid substitution of 25 amino acid residues around the HAP pocket, our study revealed that residue W102 and Y132 are critical for capsid assembly. However, substitution of many other residues did not significantly alter the amount of capsids, but reduced the amount of encapsidated pgRNA, suggesting their critical roles in pgRNA packaging. Interestingly, several mutant Cp with a single amino acid substitution of residue P25, T33 or I105 supported high levels of DNA replication, but conferred strong resistance to multiple chemotypes of CpAMs. In addition, we also found that WT Cp, but not the assembly incompetent Cp, such as Y132A Cp, interacted with HBV DNA polymerase (Pol). This later finding implies that encapsidation of viral DNA polymerase may depend on the interaction of Pol with a capsid assembly intermediate, but not free Cp dimers. Taking together, our findings reported herein shed new light on the mechanism of HBV nucleocapsid assembly and mode of CpAM action.
乙型肝炎病毒（HBV）衣壳的组装是由核心蛋白（Cp）在二聚体 - 二聚体界面的疏水相互作用驱动的。核心蛋白变构调节剂（CpAMs）与二聚体间界面形成的疏水“HAP”口袋结合，增强了二聚体 - 二聚体相互作用，并将Cp二聚体的组装错误引导为非衣壳Cp聚合物或形态正常但缺乏病毒前基因组（pg）RNA和DNA聚合酶的衣壳。在本研究中，我们进行了系统的诱变分析，以鉴定Cp二聚体 - 二聚体界面处对衣壳组装、pgRNA衣壳化和对CpAMs抗性至关重要的Cp氨基酸残基。通过分析HAP口袋周围25个氨基酸残基单氨基酸取代的70个突变型Cp，我们的研究表明，残基W102和Y132对衣壳组装至关重要。然而，许多其他残基的取代并没有显著改变衣壳的数量，但减少了衣壳化pgRNA的数量，表明它们在pgRNA包装中起关键作用。有趣的是，几个P25、T33或I105残基单氨基酸取代的突变型Cp支持高水平的DNA复制，但对多种化学类型的CpAMs具有很强的抗性。此外，我们还发现野生型Cp，而不是组装无能力的Cp，如Y132A Cp，与HBV DNA聚合酶（Pol）相互作用。这一发现意味着病毒DNA聚合酶的衣壳化可能依赖于Pol与衣壳组装中间体的相互作用，而不是游离的Cp二聚体。综上所述，我们在此报道的研究结果为HBV核衣壳组装机制和CpAM作用模式提供了新的线索。
