Transcriptional Regulation and Expression of Human APOBEC3G

项目描述（由申请人提供）：本项目将研究人类免疫缺陷病毒1型（HIV-1）限制性因子载脂蛋白B mRNA编辑酶催化多肽样3G （APOBEC3G）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APOBEC3G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。如果能在体内做到这一点，它将抑制HIV-1在感染患者体内的复制，减少发病机制。APOBEC3G是一种胞质胞苷脱氨酶e1, 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细胞系缺乏Vif的情况下，它限制HIV-1的传染性3。为了防止APOBEC3G的限制，病毒蛋白Vif在产生细胞中诱导其泛素化和随后的降解。在hiv - 1 ?vif， APOBEC3G被整合到逆转录病毒颗粒中。在感染靶细胞后，APOBEC3G在逆转录过程中通过脱氨病毒ssDNA中的胞苷残基触发G- bbbba突变4。抑制HIV-1？vif与APOBEC3G表达相关。细胞限制HIV- 1？vif被称为非允许型，而那些支持HIV-1复制的？vif被称为允许型，缺乏APOBEC3G的表达。控制APOBEC3G细胞类型特异性表达的因素尚不清楚。SA1将表征APOBEC3G表达的调控机制。由APOBEC3G特异性启动子片段驱动的荧光素酶表达将用于评估APOBEC3G在允许和非允许T细胞系中的表达。启动子片段将被解剖，以确定导致转录激活或抑制的新元件和转录因子。SA2将鉴定控制APOBEC3G差异表达的转录因子。转录调节因子的基因表达水平将通过微阵列分析在非允许和允许T细胞系之间进行比较。候选药物将通过过表达和shRNA介导的敲低分析来评估其对APOBEC3G表达的影响。此外，将筛选一个逆转录病毒巨噬细胞来源的cDNA表达文库，以确定一个细胞因子，使APOBEC3G在允许细胞中表达。

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.