Role of DDX5 in Hepatitis B virus transcription and hepatocarcinogenesis

摘要 该提案研究了RNA解旋酶DDX 5/p68在调节免疫信号传导途径中的作用， 基因表达，因为它与B型肝炎病毒（HBV）感染和HBV相关的肝细胞癌有关 （HCC）。我们早期的研究表明，DDX 5是HBV生物合成的宿主限制因子， 机制尚不清楚，DDX 5在HBV相关的HCC中下调，预后不良， 对用于治疗晚期HCC的多激酶抑制剂（mTKI）的反应，以及DDX 5控制 STAT 1翻译。我们对表现出DDX 5敲低的人肝癌细胞系的RNAseq分析， Wnt和非经典NF-κB信号传导的激活，作为DDX 5的下游靶点。 进一步了解DDX 5在病毒生物合成和肝癌方面的作用机制 为了研究DDX 5的发病机制，我们进行了蛋白质组学（LC-MS/MS）研究以鉴定DDX 5的细胞相互作用伴侣。 令人惊讶的是，我们的研究确定了干扰素γ诱导蛋白16（IFI 16），一种核DNA传感器， 病毒在细胞核中复制的限制因子，作为与病毒相互作用的最突出的细胞因子， DDX5最近的一项研究表明，IFI 16结合HBV微型染色体，导致HBV抑制 复制的然而，HBV下调IFI 16表达作为逃避先天免疫监视的策略 慢性HBV感染者。值得注意的是，DDX 5在这种相互作用中的作用目前尚不清楚。我们 基于天然核提取物的色谱分析，然后进行无标记定量MS分析的结果显示， DDX 5和IFI 16与辅助和核心PRC 2亚基HDAC 1，2和DNMT 1共洗脱。这是一致 使用CORUM数据库预测，DDX 5/IFI 16与MeCP 1复合物相互作用，包括 组蛋白去乙酰化酶（HDAC）和DNA甲基转移酶（DNMT），以及染色质沉默Polycomb 抑制复合物2（PRC 2）。重要的是，用重组核酸酶benzonase处理核裂解物 DDX 5与IFI 16的结合减少，表明RNA参与了该复合物的形成。 然而，参与DDX 5/IFI 16复合物形成的RNA底物目前尚不清楚。 基于这些初步观察，我们假设DDX 5/IFI 16复合物抑制病毒， 转录，以及对HCC进展重要的基因的细胞转录。为了解决这个假设 我们提出了两个独立的具体目标，侧重于这一机制的关键方面： 目的1：确定表观遗传DDX 5/IFI 16沉默复合物的抗病毒作用。 目的2：探讨DDX 5/IFI 16复合物在肝癌相关基因表达中的作用。 影响：该项目的成功完成将确定一个基本机制作为治疗靶点， 沉默HBV微型染色体的转录，逆转细胞基因表达变化 与HBV相关肝癌预后不良有关。

Abstract This proposal investigates the role of the RNA helicase DDX5/p68 in regulating immune signaling pathways and gene expression, as it relates to hepatitis B virus (HBV) infection and HBV-related hepatocellular carcinoma (HCC). Our earlier studies demonstrated that DDX5 is a host restriction factor in HBV biosynthesis by a mechanism not yet understood, that DDX5 is downregulated in HBV-related HCC with poor prognosis and in response to multi-kinase inhibitors (mTKIs) used for the treatment of advanced HCC, and that DDX5 controls STAT1 translation. Our RNAseq analyses of human liver cancer cell lines exhibiting DDX5 knockdown, identified activation of Wnt and non-canonical NF-κB signaling, as downstream targets of DDX5. To gain further insight into the mechanism of DDX5 action in terms of virus biosynthesis and liver cancer pathogenesis, we performed a proteomics (LC-MS/MS) study to identify cellular interacting partners of DDX5. Surprisingly, our studies identified the Interferon gamma induced protein 16 (IFI16), a nuclear DNA sensor and restriction factor for viruses that replicate in the nucleus, as the most prominent cellular factor interacting with DDX5. A recent study has demonstrated that IFI16 binds the HBV minichromosome leading to inhibition of HBV replication. However, HBV downregulates IFI16 expression as a strategy to escape innate immune surveillance in chronically HBV infected patients. Significantly, the role of DDX5 in this interaction is currently unknown. Our results based on chromatography of native nuclear extracts followed by label-free quantitative MS profiling show that DDX5 and IFI16 co-eluted with auxiliary and core PRC2 subunits, HDAC1, 2 and DNMT1. This is consistent with the prediction using the CORUM database, that DDX5/IFI16 interact with the MeCP1 complex, comprised of histone deacetylases (HDACs) & DNA methyl transferases (DNMTs), and the chromatin silencing Polycomb Repressive Complex 2 (PRC2). Importantly, treatment of nuclear lysates with recombinant nuclease benzonase reduced association of DDX5 with IFI16, suggesting involvement of an RNA in the formation of this complex. However, the RNA substrate involved in formation of the DDX5/IFI16 complex is currently unknown. Based on these preliminary observations, we hypothesize that the DDX5/IFI16 complex represses viral transcription, as well as cellular transcription of genes important for HCC progression. To address this hypothesis we propose two independent specific aims focusing on key aspects of this mechanism: Aim1: To determine the antiviral effect of the epigenetic DDX5/IFI16 silencing complex. Aim2: To determine the role of the DDX5/IFI16 complex in the expression of liver cancer genes. Impact: Successful completion of this project will identify an essential mechanism as a therapeutic target for silencing transcription from the HBV minichromosome, and reversing cellular gene expression changes associated with poor prognosis HBV-related liver cancer.