Background

PreS1-binding protein (PreS1BP), recognized as a nucleolar protein and tumor suppressor, influences the replication of various viruses, including vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1). Its role in hepatitis B virus (HBV) replication and the underlying mechanisms, however, remain elusive.

Methods

We investigated PreS1BP expression levels in an HBV-replicating cell and animal model and analyzed the impact of its overexpression on viral replication metrics. HBV DNA, covalently closed circular DNA (cccDNA), hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), and HBV RNA levels were assessed in HBV-expressing stable cell lines under varying PreS1BP conditions. Furthermore, co-immunoprecipitation and ubiquitination assays were used to detect PreS1BP- hepatitis B virus X protein (HBx) interactions and HBx stability modulated by PreS1BP.

Results

Our study revealed a marked decrease in PreS1BP expression in the presence of active HBV replication. Functional assays showed that PreS1BP overexpression significantly inhibited HBV replication and transcription, evidenced by the reduction in HBV DNA, cccDNA, HBsAg, HBcAg, and HBV RNA levels. At the molecular level, PreS1BP facilitated the degradation of HBx in a dose-dependent fashion, whereas siRNA-mediated knockdown of PreS1BP led to an increase in HBx levels. Subsequent investigations uncovered that PreS1BP accelerated HBx protein degradation via K63-linked ubiquitination in a ubiquitin-proteasome system-dependent manner. Co-immunoprecipitation assays further established that PreS1BP enhances the recruitment of the proteasome 20S subunit alpha 3 (PSMA3) for interaction with HBx, thereby fostering its degradation.

Conclusions

These findings unveil a previously unidentified mechanism wherein PreS1BP mediates HBx protein degradation through the ubiquitin-proteasome system, consequentially inhibiting HBV replication. This insight positions PreS1BP as a promising therapeutic target for future HBV interventions. Further studies are warranted to explore the clinical applicability of modulating PreS1BP in HBV therapy.

中文翻译：

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PreS1BP 通过促进 HBx 蛋白降解介导抑制乙型肝炎病毒复制

背景

PreS1 结合蛋白 (PreS1BP) 被认为是核仁蛋白和肿瘤抑制因子，影响多种病毒的复制，包括水泡性口炎病毒 (VSV) 和 1 型单纯疱疹病毒 (HSV-1)。然而，它在乙型肝炎病毒（HBV）复制中的作用及其潜在机制仍然难以捉摸。

方法

我们研究了 HBV 复制细胞和动物模型中的 PreS1BP 表达水平，并分析了其过度表达对病毒复制指标的影响。在不同 PreS1BP 条件下，评估表达 HBV 的稳定细胞系中的 HBV DNA、共价闭合环状 DNA (cccDNA)、乙型肝炎表面抗原 (HBsAg)、乙型肝炎核心抗原 (HBcAg) 和 HBV RNA 水平。此外，使用免疫共沉淀和泛素化测定来检测 PreS1BP-乙型肝炎病毒 X 蛋白 (HBx) 相互作用以及 PreS1BP 调节的 HBx 稳定性。

结果

我们的研究表明，在 HBV 复制活跃的情况下，PreS1BP 表达显着下降。功能测定表明，PreS1BP 过表达显着抑制 HBV 复制和转录，证据是 HBV DNA、cccDNA、HBsAg、HBcAg 和 HBV RNA 水平降低。在分子水平上，PreS1BP 以剂量依赖性方式促进 HBx 的降解，而 siRNA 介导的 PreS1BP 敲低则导致 HBx 水平增加。随后的研究发现，PreS1BP 通过 K63 连接的泛素化以泛素-蛋白酶体系统依赖性方式加速 HBx 蛋白降解。免疫共沉淀分析进一步证实，PreS1BP 会增强蛋白酶体 20S 亚基 α 3 (PSMA3) 的募集，从而与 HBx 相互作用，从而促进其降解。

结论

这些发现揭示了一种先前未知的机制，其中 PreS1BP 通过泛素蛋白酶体系统介导 HBx 蛋白降解，从而抑制 HBV 复制。这一见解使 PreS1BP 成为未来 HBV 干预的一个有前景的治疗靶点。需要进一步的研究来探索调节 PreS1BP 在 HBV 治疗中的临床适用性。