Herpesvirus quiescence in neuronal cells. V: forskolin-responsiveness of the herpes simplex virus type 1 alpha0 promoter and contribution of the putative cAMP response element.
Herpesvirus quiescence in neuronal cells. V: forskolin-responsiveness of the herpes simplex virus type 1 alpha0 promoter and contribution of the putative cAMP response element.
复制标题
疱疹病毒在神经元细胞中静止。
DOI：
10.1080/13550280390218797
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发表时间：
2003
影响因子：
3.2
通讯作者：
Miller,CraigS
中科院分区：
文献类型：
--
作者：
Danaher,RobertJ;Jacob,RobertJ;Miller,CraigS
单纯疱疹病毒(HSV)-1 f0启动子包含一个假定的cAMP应答元件（CRE），位于起始转录的m68至m60位置。在本报告中，作者使用(1)神经生长因子分化(ND)-PC12细胞荧光素酶报告基因检测和(2)病毒诱导的静态感染(QIF)-PC12细胞激活检测了该元件的功能。通过限制性内切酶Tsp45I的酶切和绿豆核酸酶处理，可以完全消除假定的0 CRE。通过DNA测序验证突变区，并将其插入0-荧光素酶报告质粒（pRD f 0-luc）创建（pRD f 0 j CRE- luc）和菌株17 + HSV-1基因组（f0 j CRE）。插入f0启动子的两个拷贝通过Southern blot分析得到验证。转染0- luc pRD和0 j CRE-LUC pRD质粒的ND-PC12细胞对福斯克林（50 w M）的反应类似，在处理后3天测量，与模拟处理的培养物相比，荧光素酶活性增加了约250%。用HSV-1株17 +和f0 j CRE建立的QIF-PC12培养物在感染后17天用福斯克林（50 w M）处理，在处理后第8天分别在9/24（37.5%）和13/24（54.2%）的诱导培养物中检测到病毒。相比之下，野生型和突变型病毒处理后第8天，模拟处理培养物中分别有0/23和1/24（4.2%）检测到病毒。这些发现表明f0启动子对福斯克林具有响应性，f0启动子的CRE并不赋予ND-PC12细胞对福斯克林的响应性，并且该元件不是从QIF-PC12细胞中再激活HSV-1所必需的。
The herpes simplex virus (HSV)-1 f 0 promoter contains a putative cAMP response element (CRE) located at positions m 68 to m 60 with respect to the initiation of transcription. In this report, the authors examined the functionality of this element using (1) luciferase reporter gene assays in nerve growth factor-differentiated (ND)-PC12 cells and (2) virus-induced activation from quiescently infected (QIF)-PC12 cells. The putative f 0 CRE was completely eliminated by digestion with the restriction enzyme Tsp45I followed by mung bean nuclease treatment. The mutated region was verified by DNA sequencing and was inserted into the f 0-luciferase reporter plasmid (pRD f 0-LUC) creating (pRD f 0 j CRE-LUC), and into the HSV-1 genome of strain 17 + ( f 0 j CRE). Insertion into both copies of the f 0 promoter was verified by Southern blot analysis. ND-PC12 cells transfected with pRD f 0-LUC and pRD f 0 j CRE-LUC plasmids responded similarly to forskolin (50 w M), with approximately 250% increases in luciferase activity compared to mock-treated cultures as measured 3 days following treatment. When QIF-PC12 cultures established with HSV-1 strain 17 + and f 0 j CRE were treated with forskolin (50 w M) 17 days post infection, virus was detected in 9/24 (37.5%) and 13/24 (54.2%) of induced cultures by day 8 post treatment, respectively. In contrast, virus was detected in 0/23 and 1/24 (4.2%) of mock-treated cultures by day 8 post treatment for wild-type and mutant viruses, respectively. These findings indicate that the f 0 promoter is forskolin responsive, the purported CRE of the f 0 promoter does not confer forskolin responsiveness in ND-PC12 cells, and this element is not required for reactivation of HSV-1 from QIF-PC12 cells.