Tan Zhenning, Pionek Karolyn, Unchwaniwala Nuruddin, Maguire Megan L, Loeb Daniel D, Zlotnick Adam
Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, Indiana, USA.
McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.
J Virol. 2015 Mar;89(6):3275-84. doi: 10.1128/JVI.03545-14. Epub 2015 Jan 7.
Hepatitis B virus (HBV) capsid proteins (Cps) assemble around the pregenomic RNA (pgRNA) and viral reverse transcriptase (P). pgRNA is then reverse transcribed to double-stranded DNA (dsDNA) within the capsid. The Cp assembly domain, which forms the shell of the capsid, regulates assembly kinetics and capsid stability. The Cp, via its nucleic acid-binding C-terminal domain, also affects nucleic acid organization. We hypothesize that the structure of the capsid may also have a direct effect on nucleic acid processing. Using structure-guided design, we made a series of mutations at the interface between Cp subunits that change capsid assembly kinetics and thermodynamics in a predictable manner. Assembly in cell culture mirrored in vitro activity. However, all of these mutations led to defects in pgRNA packaging. The amount of first-strand DNA synthesized was roughly proportional to the amount of RNA packaged. However, the synthesis of second-strand DNA, which requires two template switches, was not supported by any of the substitutions. These data demonstrate that the HBV capsid is far more than an inert container, as mutations in the assembly domain, distant from packaged nucleic acid, affect reverse transcription. We suggest that capsid molecular motion plays a role in regulating genome replication.
The hepatitis B virus (HBV) capsid plays a central role in the virus life cycle and has been studied as a potential antiviral target. The capsid protein (Cp) packages the viral pregenomic RNA (pgRNA) and polymerase to form the HBV core. The role of the capsid in subsequent nucleic acid metabolism is unknown. Here, guided by the structure of the capsid with bound antiviral molecules, we designed Cp mutants that enhanced or attenuated the assembly of purified Cp in vitro. In cell culture, assembly of mutants was consistent with their in vitro biophysical properties. However, all of these mutations inhibited HBV replication. Specifically, changing the biophysical chemistry of Cp caused defects in pgRNA packaging and synthesis of the second strand of DNA. These results suggest that the HBV Cp assembly domain potentially regulates reverse transcription, extending the activities of the capsid protein beyond its presumed role as an inert compartment.
乙肝病毒（HBV）衣壳蛋白（Cps）围绕前基因组RNA（pgRNA）和病毒逆转录酶（P）组装。然后pgRNA在衣壳内逆转录为双链DNA（dsDNA）。形成衣壳外壳的Cp组装结构域调节组装动力学和衣壳稳定性。Cp通过其核酸结合C末端结构域也影响核酸组织。我们假设衣壳结构可能也对核酸加工有直接影响。利用结构导向设计，我们在Cp亚基之间的界面处进行了一系列突变，这些突变以可预测的方式改变衣壳组装动力学和热力学。细胞培养中的组装反映了体外活性。然而，所有这些突变都导致pgRNA包装缺陷。合成的第一链DNA的量大致与包装的RNA量成比例。然而，需要两次模板转换的第二链DNA的合成不受任何替代的支持。这些数据表明，HBV衣壳远不止是一个惰性容器，因为组装结构域中远离包装核酸的突变会影响逆转录。我们认为衣壳分子运动在调节基因组复制中起作用。
乙肝病毒（HBV）衣壳在病毒生命周期中起核心作用，并已作为潜在的抗病毒靶点进行研究。衣壳蛋白（Cp）包装病毒前基因组RNA（pgRNA）和聚合酶以形成HBV核心。衣壳在随后的核酸代谢中的作用尚不清楚。在这里，在结合抗病毒分子的衣壳结构的指导下，我们设计了在体外增强或减弱纯化Cp组装的Cp突变体。在细胞培养中，突变体的组装与其体外生物物理性质一致。然而，所有这些突变都抑制了HBV复制。具体而言，改变Cp的生物物理化学会导致pgRNA包装和DNA第二链合成缺陷。这些结果表明，HBV Cp组装结构域可能调节逆转录，将衣壳蛋白的活性扩展到其作为惰性隔室的假定作用之外。
