Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan.
PLoS Pathog. 2010 Oct 28;6(10):e1001162. doi: 10.1371/journal.ppat.1001162.
It remains unclear what determines the subcellular localization of hepatitis B virus (HBV) core protein (HBc) and particles. To address this fundamental issue, we have identified four distinct HBc localization signals in the arginine rich domain (ARD) of HBc, using immunofluorescence confocal microscopy and fractionation/Western blot analysis. ARD consists of four tight clustering arginine-rich subdomains. ARD-I and ARD-III are associated with two co-dependent nuclear localization signals (NLS), while ARD-II and ARD-IV behave like two independent nuclear export signals (NES). This conclusion is based on five independent lines of experimental evidence: i) Using an HBV replication system in hepatoma cells, we demonstrated in a double-blind manner that only the HBc of mutant ARD-II+IV, among a total of 15 ARD mutants, can predominantly localize to the nucleus. ii) These results were confirmed using a chimera reporter system by placing mutant or wild type HBc trafficking signals in the heterologous context of SV40 large T antigen (LT). iii) By a heterokaryon or homokaryon analysis, the fusion protein of SV40 LT-HBc ARD appeared to transport from nuclei of transfected donor cells to nuclei of recipient cells, suggesting the existence of an NES in HBc ARD. This putative NES is leptomycin B resistant. iv) We demonstrated by co-immunoprecipitation that HBc ARD can physically interact with a cellular factor TAP/NXF1 (Tip-associated protein/nuclear export factor-1), which is known to be important for nuclear export of mRNA and proteins. Treatment with a TAP-specific siRNA strikingly shifted cytoplasmic HBc to nucleus, and led to a near 7-fold reduction of viral replication, and a near 10-fold reduction in HBsAg secretion. v) HBc of mutant ARD-II+IV was accumulated predominantly in the nucleus in a mouse model by hydrodynamic delivery. In addition to the revised map of NLS, our results suggest that HBc could shuttle rapidly between nucleus and cytoplasm via a novel TAP-dependent NES.
HBV 核心蛋白（HBc）和颗粒的亚细胞定位由什么决定尚不清楚。为了解决这个基本问题，我们使用免疫荧光共聚焦显微镜和分级/Western blot 分析，在 HBc 的精氨酸丰富区（ARD）中鉴定了四个不同的 HBc 定位信号。ARD 由四个紧密聚集的富含精氨酸的亚结构域组成。ARD-I 和 ARD-III 与两个相互依赖的核定位信号（NLS）相关，而 ARD-II 和 ARD-IV 表现为两个独立的核输出信号（NES）。这一结论基于五条独立的实验证据：i）使用肝癌细胞中的 HBV 复制系统，我们以双盲方式证明，在总共 15 个 ARD 突变体中，只有 ARD-II+IV 的 HBc 突变体主要定位于核内。ii）通过放置突变或野生型 HBc 运输信号在 SV40 大 T 抗原（LT）的异源背景下的嵌合报告系统，验证了这些结果。iii）通过异核体或同核体分析，SV40 LT-HBc ARD 的融合蛋白似乎从转染供体细胞的核转运到受体细胞的核，表明 HBc ARD 中存在 NES。这种假定的 NES 对莱普霉素 B 有抗性。iv）我们通过共免疫沉淀证明，HBc ARD 可以与一种细胞因子 TAP/NXF1（Tip-associated protein/nuclear export factor-1）发生物理相互作用，该因子对于 mRNA 和蛋白质的核输出很重要。用 TAP 特异性 siRNA 处理会显著将细胞质中的 HBc 转移到核内，并导致病毒复制减少近 7 倍，HBsAg 分泌减少近 10 倍。v）在通过水力传递的小鼠模型中，HBc 的突变体 ARD-II+IV 主要在核内积累。除了修订后的 NLS 图谱外，我们的结果表明 HBc 可以通过一种新型的 TAP 依赖性 NES 在核和细胞质之间快速穿梭。
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