Expression data from immature dendritic cells (iDC) expressing HIV-1 Tat alleles and mutants

HIV-1 Tat induces the expression of interferon (IFN)-inducible genes in immature dendritic cells (iDC) in the absence of IFN production. We evaluated how three alleles of Tat and some Tat mutants differ in cellular gene modulation and whether a similar gene induction pattern could be detected by treating cells with IFN’s. The three alleles and mutants, with the exception of mutants TatSF21-47 and TatSF2G48-R57A that do not localize in the nucleous, modulated to different degrees IFN-inducible genes without concomitant induction of IFN’s. The first exon TatSF21-72 and the minimal transactivator TatSF21-58, all induced genes to a significantly greater extent than full-length Tat. The 2nd exon appears to diminish the gene modulation that can be observed when the first exon alone is expressed. We investigated the effect of various Tat alleles and mutants on host cell gene expression in immature dendritic cells (iDC). The cells were infected with adenoviruses expressing the Tat constructs. The Tat alleles and mutants are under a tetracycline inducible promoter and are expressed only in cells co-infected with Ad-tTA, which expresses the tetracycline responsive transactivator. 10e6 iDC were infected with 5 plague forming units per cell of the adenoviruses and cells were collected 10 and 20 hours post-infection. RNA was isolated and mRNA expression levels were analyzed by microarrays. As controls we used cells that were left uninfected and cells that were infected with Ad-LacZ, which expresses beta-galactosidase.

HIV-1 Tat蛋白在无干扰素（interferon, IFN）产生的条件下，可诱导未成熟树突状细胞（immature dendritic cells, iDC）表达干扰素诱导基因。本研究评估了三种Tat等位基因及部分Tat突变体在调控细胞基因表达层面的差异，并探究是否可通过干扰素处理细胞来检测到与之相似的基因诱导模式。除无法定位于细胞核的TatSF2Δ1-47与TatSF2G48-R57A突变体外，其余受试的Tat等位基因及突变体均可不同程度地调控干扰素诱导基因的表达，且未伴随干扰素的诱导生成。首外显子TatSF2Δ1-72与最小反式激活因子TatSF2Δ1-58对基因的诱导效果显著强于全长Tat。第二外显子似乎会削弱仅表达首外显子时所观测到的基因调控效应。本研究进一步探究了多种Tat等位基因及突变体对未成熟树突状细胞宿主基因表达的影响。实验中，细胞被感染携带Tat表达构建体的腺病毒。该Tat等位基因及突变体受四环素诱导型启动子调控，仅在共感染表达四环素响应性反式激活因子的Ad-tTA的细胞中才会表达。以每细胞5个噬斑形成单位的病毒量感染10^6个iDC，分别于感染后10小时与20小时收集细胞。提取RNA并通过微阵列技术分析mRNA表达水平。对照组设置为未感染细胞，以及感染表达β-半乳糖苷酶的Ad-LacZ的细胞。