Decreased reporter gene expression during latent infection with HSV LAT promoter constructs.
Decreased reporter gene expression during latent infection with HSV LAT promoter constructs.
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HSV LAT 启动子构建体潜伏感染期间报告基因表达降低。
DOI：
10.1006/viro.1993.1632
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发表时间：
1993
期刊：
影响因子：
3.7
通讯作者：
Stevens,JG
中科院分区：
文献类型：
--
作者：
Margolis,TP;Bloom,DC;Dobson,AT;Feldman,LT;Stevens,JG
潜伏相关转录物（LAT）是HSV潜伏感染期间存在的唯一病毒转录物，其编码自HSV基因组的内部重复区的8.5kb片段。然而，很少有人知道的启动子活性，降解过程中，和元件或区域的相对贡献影响这些转录本在潜伏感染的神经元的长期表达。为了开始解决这个问题，我们研究了急性和潜伏感染期间LAT启动子的活性。用科斯/62-3感染小鼠足垫，这是一种工程化的单纯疱疹病毒，其中LAT启动子的两个拷贝用于驱动大肠杆菌Z基因的表达。接种后4天（p.i.）用酶组织化学和原位杂交方法观察到神经节细胞内有丰富的β-半乳糖苷酶（β-gal）蛋白和转录本。相比之下，到第21天（此时已建立潜伏感染），即使通过聚合酶链反应（PCR）测定，感染的神经节中也不存在β-gal转录物。这些发现表明在感染后第4天和第21天之间LAT启动子活性显著下降。为了证实这一结论，我们用第二种重要的构建体科斯/67-7感染小鼠，其中LAT启动子用于驱动神经生长因子（NGF）基因的表达。注射四天，在感染的神经节细胞中存在大量的NGF抗原和转录物，但在潜伏感染的神经节中未发现克隆的NGF基因转录的证据。我们的研究结果表明，LAT启动子活性受到严重限制，在潜伏期的神经节感染。
The latency-associated transcripts (LAT), which code from an 8.5 kb segment of the internal repeat region of the HSV genome, are the only viral transcripts that are present during HSV latent infection. However, little is known about the relative contribution of promoter activity, degradative processes, and elements or regions affecting long term expression of these transcripts in latently infected neurons. To begin to address this question we investigated LAT promoter activity during acute and latent infection. Mouse footpads were infected with KOS/62-3, an engineered herpes simplex virus in which both copies of the LAT promoter are used to drive expression of theEscherichia colilac Z gene. Four days post-inoculation (p.i.) abundant β-galactosidase (β-gal) protein and transcripts were present within ganglionic neurons as assayed by enzyme histochemistry andin situhybridization. In contrast, by Day 21 (at which time a latent infection had been established) no β-gal transcripts were present in infected ganglia, even when assayed by the polymerase chain reaction (PCR). These findings indicate a significant drop in LAT promoter activity between Day 4 and Day 21 p.i. To provide confirmatory evidence for this conclusion we infected mice with a second vital construct, KOS/67-7, in which the LAT promoter was used to drive expression of the nerve growth factor (NGF) gene. Four days p.i., abundant NGF antigen and transcripts were present in infected ganglionic neurons, but no evidence of transcription of the cloned NGF gene could be found in latently infected ganglia. Our findings suggest that LAT promoter activity is severely restricted during the latent phase of ganglionic infection.