State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100039, China.
CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Life Sci. 2019 Mar 1;220:1-7. doi: 10.1016/j.lfs.2019.01.039. Epub 2019 Jan 26.
To investigate the role and underlying mechanism of 4E-BP1 and S6K1 in regulating autophagy and hepatitis B virus (HBV) replication.
The mRNA relative expression of HBx and its DNA level were detected by real-time PCR. The relative levels of hepatitis B surface antigen (HBsAg) were measured by enzyme-linked immunosorbent assay (ELISA). HBx DNA level of HepG2 cells transfected with pcDNA3.1(+)-HBV1.3 plasmids were detected by Southern blot. Moreover, we determined autophagy through electron microscopy, confocal microscopy and Western blot.
Rapamycin promoted autophagy and the X protein synthesis concomitantly with elevation in Akt phosphorylation and Beclin1 expression. Either Beclin1 or Akt depletion suppresses the Rapa-enhanced HBV replication, whereas mTOR silencing inhibited HBV replication concurring with a decreased in both S6K1 and 4E-BP1 phosphorylation. Unexpectedly, Akt inhibitor suppressed Rapa-dependent autophagic flux and increased the level of p62/SQSTM1. While S6K1 ablation impaired autophagy and decreased X protein expression, 4E-BP1 silencing slightly influenced autophagy and increased X protein level.
The underlying mechanism of 4E-BP1 and S6K1, two main downstream effectors of mTOR, in mediating HBV replication and HBV-induced autophagy remains largely unknown. Here, we propose that Akt is required for both HBV replication and Rapa-induced autophagy, and 4E-BP1 and S6K1 play a distinct role in the virus replication and autophagic process.
研究 4E-BP1 和 S6K1 在调控自噬和乙型肝炎病毒（HBV）复制中的作用及其潜在机制。
采用实时 PCR 检测 HBx 的 mRNA 相对表达及其 DNA 水平，酶联免疫吸附试验（ELISA）检测乙型肝炎表面抗原（HBsAg）的相对水平，Southern blot 检测转染 pcDNA3.1(+)-HBV1.3 质粒的 HepG2 细胞中的 HBx DNA 水平。此外，我们通过电子显微镜、共聚焦显微镜和 Western blot 来确定自噬。
雷帕霉素促进自噬和 X 蛋白的合成，同时伴随着 Akt 磷酸化和 Beclin1 表达的增加。Beclin1 或 Akt 耗尽均可抑制 Rapa 增强的 HBV 复制，而 mTOR 沉默抑制 HBV 复制，同时 S6K1 和 4E-BP1 磷酸化均降低。出乎意料的是，Akt 抑制剂抑制了 Rapa 依赖的自噬流，并增加了 p62/SQSTM1 的水平。虽然 S6K1 缺失损害了自噬并降低了 X 蛋白的表达，但 4E-BP1 沉默对自噬影响较小，并增加了 X 蛋白水平。
mTOR 的两个主要下游效应物 4E-BP1 和 S6K1 介导 HBV 复制和 HBV 诱导的自噬的潜在机制在很大程度上仍不清楚。在这里，我们提出 Akt 是 HBV 复制和 Rapa 诱导的自噬所必需的，而 4E-BP1 和 S6K1 在病毒复制和自噬过程中发挥不同的作用。
