Small Interfering RNA Screening for the Small GTPase Rab Proteins Identifies Rab5B as a Major Regulator of Hepatitis B Virus Production
Small Interfering RNA Screening for the Small GTPase Rab Proteins Identifies Rab5B as a Major Regulator of Hepatitis B Virus Production
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DOI：
10.1128/jvi.00621-19
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发表时间：
2019-08-01
影响因子：
5.4
通讯作者：
Masamune, Atsushi
中科院分区：
文献类型：
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作者：
Inoue, Jun;Ninomiya, Masashi;Masamune, Atsushi
病毒被认为是利用囊泡运输感染的细胞，但组装/释放途径的细节B型肝炎病毒（HBV）仍然是未知的。为了确定HBV产生的关键调节因子，我们对Rab蛋白进行了短干扰RNA（siRNA）筛选，Rab蛋白被认为是使用HepG2.2.15细胞进行囊泡运输的分子开关。在62种Rab蛋白中，Rab 5 B的抑制最显著地增加了培养上清中的HBV DNA。令人惊讶的是，Rab 5 B siRNA转染后5天，上清液中的HBV DNA增加了30倍以上，反映了感染性HBV颗粒的增加。北方杂交显示，编码大分子B表面蛋白（LHBs）的包膜蛋白的2.4/2.1-kb mRNA的转录增加。肝细胞核因子（HNF）的分析表明，HNF 4 α的转录，这是已知的增强2.4 kb的mRNA转录，是由Rab 5 B调节。此外，研究表明，Rab 5 B耗尽后，LHB已积聚在内质网（ER）中，但未积聚在多泡体（MVB）中，多泡体被认为是用于形成HBV包膜的细胞器。因此，认为Rab 5 B是将LHB从ER转运至MVB所必需的。免疫荧光显微镜显示，HBs蛋白，包括LHBs，共定位与HBc在ER的Rab 5 B-耗尽的细胞，表明HBV复制不仅发生在MVB，但也在ER。总之，Rab 5 B是HBV产生的关键调节因子，可能是抗病毒治疗的靶点。重要HBV感染是一个全球性的健康问题，但HBV如何利用细胞机器进行其生命周期的机制知之甚少。特别是，目前还不清楚病毒组分和病毒粒子是如何在细胞器之间转运的。据报道，HBV的萌芽位点是ER或MVB，但尚未明确确定。在这项研究中，使用HBV表达细胞进行的基于siRNA的Rab蛋白筛选表明Rab 5亚型之一Rab 5 B在HBV生命周期的后期阶段具有重要作用。尽管已知Rab 5作用于早期内体，但这项研究表明Rab 5 B在ER和MVB之间的LHB转运中起作用。此外，它还影响LHB的转录。这是第一份关于HBV包膜蛋白在细胞器间转运机制的报告，其结果为HBV感染的治疗控制提供了重要的见解。
Viruses are considered to use vesicular trafficking in infected cells, but the details of assembly/release pathways of hepatitis B virus (HBV) are still unknown. To identify key regulators of HBV production, we performed short interfering RNA (siRNA) screening for Rab proteins, which are considered to act as molecular switches in vesicular trafficking using HepG2.2.15 cells. Among 62 Rab proteins, the suppression of Rab5B most significantly increased HBV DNA in the culture supernatant. Surprisingly, 5 days after the transfection of Rab5B siRNA, HBV DNA in the supernatant was increased more than 30-fold, reflecting the increase of infectious HBV particles. Northern blotting showed that transcription of 2.4/2.1-kb mRNA coding envelope proteins containing large hepatitis B surface protein (LHBs) was increased. Analysis of hepatocyte nuclear factors (HNFs) showed that transcription of HNF4 alpha, which is known to enhance 2.4-kb mRNA transcription, was regulated by Rab5B. Also, it was revealed that LHBs had accumulated in the endoplasmic reticulum (ER) after Rab5B depletion but not in the multivesicular body (MVB), which is thought to be an organelle utilized for HBV envelope formation. Therefore, it was considered that Rab5B is required for the transport of LHBs from the ER to MVB. Immunofluorescent microscopy showed that HBs proteins, including LHBs, colocalized with HBc in the ER of Rab5B-depleted cells, suggesting that HBV envelopment occurs not only in the MVB but also in the ER. In conclusion, Rab5B is a key regulator of HBV production and could be a target of antiviral therapy.IMPORTANCE HBV infection is a worldwide health problem, but the mechanisms of how HBV utilizes cellular machinery for its life cycle are poorly understood. In particular, it has been unclear how the viral components and virions are transported among the organelles. The HBV budding site has been reported to be the ER or MVB, but it has not been clearly determined. In this study, siRNA-based screening of Rab proteins using HBV-expressing cells showed that Rab5B, one of the Rab5 isoforms, has important roles in late steps of the HBV life cycle. Although Rab5 is known to work on early endosomes, this study showed that Rab5B plays a role in the transport of LHBs between the ER and MVB. Furthermore, it affects the transcription of LHBs. This is the first report on the mechanisms of HBV envelope protein transport among the organelles, and the results provide important insights into the therapeutic control of HBV infection.