CTDG in non-activated Jurkat cells. Homo sapiens

Cyclin T1-dependent genes in non-activated Jurkat cells. HIV-1 is dependent upon cellular co-factors to mediate its replication cycle in CD4+ T cells and macrophages, the two major cell types infected by the virus in vivo. One critical co-factor is Cyclin T1, a subunit of a general RNA polymerase II elongation factor known as P-TEFb. Cyclin T1 is targeted directly by the viral Tat protein to activate proviral transcription. Cyclin T1 is up-regulated when resting CD4+ T cells are activated and during macrophage differentiation or activation, conditions that are also necessary for high levels of HIV-1 replication. Because Cyclin T1 is a subunit of a transcription factor, the up-regulation of Cyclin T1 in these cells results in the induction of cellular genes, some of which might be HIV-1 co-factors. Using shRNA depletions of Cyclin T1 and transcriptional profiling, we identified 54 cellular mRNAs that are Cyclin T1-dependent for their induction in activated CD4+ T cells and during macrophage differentiation and activation. The promoters for these Cyclin T1-dependent genes (CTDGs) are over-represented in two transcription factor binding sites, SREBP1 and ARP1. Notably, 10 of these CTDGs have been reported to be involved in HIV-1 replication, a significant over-representation of such genes when compared to randomly generated lists of 54 genes (p value < 0.00021). SiRNA depletions of two CTDGs identified here, CDK11 and Casein kinase1gamma1, suggest that these genes are also involved in HIV-1 replication. It is therefore likely that the 54 CTDGs identified here include novel HIV-1 co-factors. The presence of CTDGs in the protein space that was available for HIV-1 to sample during its evolution and acquisition of Tat function may provide an explanation for why CTDGs are enriched in viral co-factors. Keywords: cyclin T1 knockdown Overall design: Using shRNA knockdown of cyclin T1, cyclin T1-dependent genes were identified in non-activated Jurkat cells.

非激活态Jurkat细胞中依赖周期蛋白T1（Cyclin T1）的基因。HIV-1（人类免疫缺陷病毒1型）依赖细胞辅助因子来完成其在CD4+ T细胞和巨噬细胞中的复制周期——这两类是体内该病毒主要的感染靶细胞。其中关键的辅助因子之一为周期蛋白T1（Cyclin T1），它是通用RNA聚合酶II（RNA polymerase II）延伸因子P-TEFb的一个亚基。病毒Tat蛋白可直接靶向周期蛋白T1，以激活前病毒转录。
当静息CD4+ T细胞被激活，以及巨噬细胞发生分化或激活时，周期蛋白T1的表达会上调；而这些状态同样是HIV-1实现高水平复制所必需的条件。由于周期蛋白T1是转录因子的亚基，这类细胞中周期蛋白T1的上调会诱导细胞基因的表达，其中部分基因可能即为HIV-1的辅助因子。
本研究通过短发夹RNA（short hairpin RNA, shRNA）敲低周期蛋白T1并结合转录组分析，鉴定出54种细胞mRNA，它们的诱导表达依赖于周期蛋白T1，且该过程发生于激活的CD4+ T细胞以及巨噬细胞分化与激活阶段。这些依赖周期蛋白T1的基因（Cyclin T1-dependent genes, CTDGs）的启动子区域，在两种转录因子结合位点SREBP1和ARP1上呈现富集现象。
值得注意的是，其中已有10种CTDGs被报道参与HIV-1的复制过程；相较于随机选取的54基因列表，这类基因的富集程度具有统计学显著性（p值<0.00021）。针对本研究鉴定出的两种CTDGs——CDK11与酪蛋白激酶1γ1（Casein kinase1gamma1）进行小干扰RNA（small interfering RNA, siRNA）敲低实验，结果显示这两类基因同样参与HIV-1的复制过程。
因此，本研究鉴定出的54种CTDGs很可能包含新型HIV-1辅助因子。在HIV-1进化并获得Tat功能的过程中，其可靶向的蛋白质组中存在CTDGs，这或许可以解释为何CTDGs在病毒辅助因子中呈现富集现象。
关键词：周期蛋白T1敲低 实验设计：通过短发夹RNA（shRNA）敲低周期蛋白T1，在非激活态Jurkat细胞中鉴定出依赖周期蛋白T1的基因。