针对小GTP酶Rab蛋白的小干扰RNA筛选将Rab5B鉴定为乙型肝炎病毒

StephenW

Small Interfering RNA Screening for the Small GTPase Rab Proteins Identifies Rab5B as a Major Regulator of Hepatitis B Virus Production
Jun Inoue, Masashi Ninomiya, Teruyuki Umetsu, Takuya Nakamura, Takayuki Kogure, Eiji Kakazu, Tomoaki Iwata, Satoshi Takai, Akitoshi Sano, Mitsunori Fukuda, Koichi Watashi, Masanori Isogawa, Yasuhito Tanaka, Tooru Shimosegawa, Mark A. McNiven, Atsushi Masamune
J.-H. James Ou, Editor
DOI: 10.1128/JVI.00621-19

  
ABSTRACT

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 HNF4α, 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.

StephenW

针对小GTP酶Rab蛋白的小干扰RNA筛选将Rab5B鉴定为乙型肝炎病毒生产的主要调节因子
Jun Inoue，Masashi Ninomiya，Teruyuki Umetsu，Takuy​​a Nakamura，Takayuki Kogure，Eiji Kakazu，Tomoaki Iwata，Satoshi Takai，Akitoshi Sano，Mitsunori Fukuda，Koichi Watashi，Masanori Isogawa，Yasuhito Tanaka，Tooru Shimosegawa，Mark A. McNiven，Atsushi Masamune
J.-H. James Ou，编辑
DOI：10.1128 / JVI.00621-19

  
抽象

病毒被认为在感染细胞中使用囊泡运输，但乙型肝炎病毒（HBV）的组装/释放途径的细节仍然未知。为了鉴定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，其被认为是使用HepG2.2.15细胞在囊泡运输中作为分子开关。在62种Rab蛋白中，Rab5B的抑制最显着地增加了培养上清液中的HBV DNA。令人惊讶的是，在转染Rab5B siRNA后5天，上清液中的HBV DNA增加超过30倍，反映了感染性HBV颗粒的增加。 Northern印迹显示，含有大型乙型肝炎表面蛋白（LHBs）的编码包膜蛋白的2.4 / 2.1-kb mRNA的转录增加。对肝细胞核因子（HNF）的分析表明，已知增强2.4-kb mRNA转录的HNF4α的转录受Rab5B调节。此外，揭示了在Rab5B消耗后LHBs在内质网（ER）中积累但在多泡体（MVB）中没有，其被认为是用于HBV包膜形成的细胞器。因此，认为Rab5B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，与Rab5B耗竭细胞的ER中的HBc共定位，表明HBV包膜不仅发生在MVB中，而且发生在ER中。总之，Rab5B是HBV产生的关键调节因子，可能是抗病毒治疗的目标。

重要性HBV感染是一个全球性的健康问题，但人们对HBV如何利用细胞机制进行生命周期的机制知之甚少。特别是，尚不清楚病毒成分和病毒粒子是如何在细胞器之间转运的。据报道，HBV萌芽部位是ER或MVB，但尚未明确确定。在该研究中，使用表达HBV的细胞基于siRNA的Rab蛋白筛选显示Rab5B（Rab5同种型之一）在HBV生命周期的后期步骤中具有重要作用。虽然已知Rab5可用于早期内体，但该研究表明Rab5B在ER和MVB之间的LHB转运中起作用。此外，它影响LHB的转录。这是关于细胞器中HBV包膜蛋白转运机制的首次报道，该结果为HBV感染的治疗控制提供了重要的见解。