Transcriptional Regulation and Expression of Human APOBEC3G

描述（由申请人提供）：该项目将研究编码人类免疫缺陷病毒1型（HIV-1）限制因子载脂蛋白B mRNA编辑酶催化多肽样3G（APOBEC 3G）的基因的转录调控。了解该基因的转录调控可能提供一种方法来增加APOBEC 3G在HIV-1靶细胞中的表达水平，从而抵消病毒辅助蛋白Vif的拮抗作用。如果这可以在体内完成，它将抑制感染患者的HIV-1复制，减少发病率。 APOBEC 3G是一种胞质胞苷脱氨酶1，2，作为宿主先天免疫应答的一部分。在原代人T细胞、单核细胞、巨噬细胞和淋巴细胞衍生的细胞系中不存在Vif的情况下，它限制HIV-1的感染性3。为了防止APOBEC 3G的限制，病毒蛋白Vif诱导其在生产细胞中的泛素化和随后的降解1。在HIV-1中？vif，将APOBEC 3G掺入逆转录病毒颗粒中。在感染靶细胞后，APOBEC 3G通过在逆转录过程中将病毒ssDNA中的胞苷残基脱氨基而触发G->A突变4。抑制HIV-1？vif与APOBEC 3G表达相关。限制HIV- 1的细胞？vif被称为非允许的，而那些支持HIV-1的复制？vif被称为允许的，缺乏APOBEC 3G表达。控制APOBEC 3G细胞类型特异性表达的因素尚不清楚。SA 1将表征APOBEC 3G表达的调控机制。由APOBEC 3G特异性启动子片段驱动的荧光素酶表达将用于评估APOBEC 3G在允许和非允许T细胞系中的表达。将解剖启动子片段以鉴定导致转录激活或抑制的新元件和转录因子。SA 2将鉴定控制APOBEC 3G差异表达的转录因子。将通过微阵列分析在非允许性和允许性T细胞系之间比较转录调节因子的基因表达水平。将通过过表达和shRNA介导的敲低分析评价候选物对APOBEC 3G表达的影响。此外，将筛选逆转录病毒巨噬细胞衍生的cDNA表达文库，以鉴定能够在允许细胞中表达APOBEC 3G的细胞因子。 公共卫生相关性：艾滋病仍然是当今主要的公共卫生问题之一。目前的抗逆转录病毒药物已经成功地降低了死亡率，延长了患者的寿命，但价格昂贵，容易产生副作用，并导致耐药病毒。我们建议探索一种替代策略，以减少患者体内的病毒复制，其中在人体细胞中产生的酶可以有效地阻断病毒复制，并在感染细胞中以更高的水平表达。

DESCRIPTION (provided by applicant): The project will investigate the transcriptional regulation of the gene that encodes the human immunodeficiency virus type 1 (HIV-1) restriction factor Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G (APOBEC3G). An understanding of the transcriptional regulation of this gene may provide a means to increase APOBEC3G expression levels in HIV-1 target cells, thereby counteracting the antagonistic effect of the viral accessory protein Vif. If this could be done in vivo it would suppress HIV-1 replication in infected patients, reducing pathogenesis. APOBEC3G is a cytosolic cytidine deaminase1, 2 that serves as part of the host innate immune response. It restricts the infectivity of HIV-1 in the absence of Vif in primary human T cells, monocytes, macrophages and lymphocyte derived cell lines3. To prevent restriction by APOBEC3G, the viral protein Vif induces its ubiquitination and subsequent degradation in the producer cell1. In HIV-1?vif, APOBEC3G is incorporated into retroviral particles. Upon infection of the target cell, APOBEC3G triggers G-->A mutations by deaminating cytidine residues in the viral ssDNA during reverse transcription4. Inhibition of HIV-1?vif correlates with APOBEC3G expression. Cells that restrict HIV- 1?vif are termed non-permissive, whereas those that support the replication of HIV-1?vif are called permissive and lack APOBEC3G expression. The factors that govern cell-type specific expression of APOBEC3G are unknown. SA1 will characterize the regulatory mechanisms of APOBEC3G expression. Luciferase expression driven by an APOBEC3G specific promoter fragment will be used to assess APOBEC3G expression in permissive and non-permissive T cell lines. The promoter fragment will be dissected to identify novel elements and transcription factors that result in transcriptional activation or repression. SA2 will identify the transcription factors that govern the differential expression of APOBEC3G. Gene expression levels of transcriptional regulators will be compared between non-permissive and permissive T cell lines by microarray analyses. Candidates will be evaluated for their effect on APOBEC3G expression by overexpression and shRNA mediated knock-down analyses. Additionally, a retroviral macrophage-derived cDNA expression library will be screened to identify a cellular factor that enables APOBEC3G expression in permissive cells. PUBLIC HEALTH RELEVANCE: AIDS remains one of the major modern-day public health problems. Current anti-retroviral medications have successfully reduced mortality and extended the lifespan of patients, but are expensive, prone to side effects, and result in drug-resistant viruses. We propose to explore an alternative strategy to reduce virus replication in patients in which an enzyme, produced in human cells that potently blocks virus replication, is expressed at higher levels in infected cells.