Farrell M J, Margolis T P, Gomes W A, Feldman L T
Department of Microbiology and Immunology, UCLA School of Medicine 90024.
J Virol. 1994 Sep;68(9):5337-43. doi: 10.1128/JVI.68.9.5337-5343.1994.
It has been previously reported that the latency-associated transcript (LAT) promoter contains a DNA sequence at the LAT transcription start site which resembles the ICP4 consensus DNA binding site and that this site allows ICP4-mediated downregulation of the LAT promoter in transient assays (A. H. Batchelor and P. O'Hare, J. Virol. 64:3269-3279, 1990). We have confirmed these data by showing that an ICP4-expressing plasmid will downregulate lacZ expression from a plasmid containing the LAT promoter and transcription start site (pJA1) and does not downregulate lacZ expression from a plasmid in which the start site has been mutagenized (pWAG15). To determine the role of the LAT transcription start site in regulating LAT promoter activity in the context of the virus, two recombinant viruses, KOS-1 and KOS-15, were studied. KOS-1 contains an 863-bp portion of the LAT promoter, including the LAT cap site, fused to the lacZ gene and inserted into the gC locus (T.P. Margolis, F. Sedarati, A.T. Dobson, L.T. Feldman, and J.G. Stevens, Virology 189:150-160, 1992). The second virus (KOS-15) was constructed in identical fashion, using plasmid pWAG-15, which is not downregulated by ICP4. Vero cells productively infected with KOS-15 produce 10-fold more beta-galactosidase than do those infected with KOS-1. In murine dorsal root ganglia acutely infected with KOS-1, only 1.2% of dorsal root ganglion neurons that expressed viral antigen also expressed beta-galactosidase. In contrast, in KOS-15-infected mice, beta-galactosidase was detected in 18% of viral antigen-positive neurons. Similar findings were observed in trigeminal ganglia acutely infected with KOS-1 and KOS-15. Thus, the region encompassing the LAT transcription start site appears to play an important role in repression of the LAT promoter activity not only in vitro but also in acutely infected neurons in vivo. These results suggest that during productive infection with HSV-1, LAT expression is tightly regulated.
先前已有报道称，潜伏期相关转录本（LAT）启动子在LAT转录起始位点含有一段DNA序列，该序列类似于ICP4共有DNA结合位点，并且在瞬时分析中该位点允许ICP4介导的LAT启动子下调（A. H. 巴彻勒和P. 奥黑尔，《病毒学杂志》64:3269 - 3279，1990年）。我们通过以下方式证实了这些数据：表达ICP4的质粒会下调含有LAT启动子和转录起始位点的质粒（pJA1）的lacZ表达，但不会下调起始位点已发生诱变的质粒（pWAG15）的lacZ表达。为了确定LAT转录起始位点在病毒背景下调节LAT启动子活性中的作用，研究了两种重组病毒，KOS - 1和KOS - 15。KOS - 1包含LAT启动子的一个863 bp片段，包括LAT帽位点，与lacZ基因融合并插入gC基因座（T.P. 马戈利斯、F. 塞达拉蒂、A.T. 多布森、L.T. 费尔德曼和J.G. 史蒂文斯，《病毒学》189:150 - 160，1992年）。第二种病毒（KOS - 15）以相同方式构建，使用不受ICP4下调的质粒pWAG - 15。被KOS - 15有效感染的Vero细胞产生的β - 半乳糖苷酶比被KOS - 1感染的细胞多10倍。在被KOS - 1急性感染的小鼠背根神经节中，仅1.2%表达病毒抗原的背根神经节神经元也表达β - 半乳糖苷酶。相比之下，在被KOS - 15感染的小鼠中，在18%的病毒抗原阳性神经元中检测到了β - 半乳糖苷酶。在被KOS - 1和KOS - 15急性感染的三叉神经节中也观察到了类似的结果。因此，包含LAT转录起始位点的区域似乎不仅在体外而且在体内急性感染的神经元中对LAT启动子活性的抑制起着重要作用。这些结果表明，在单纯疱疹病毒1型的有效感染期间，LAT表达受到严格调控。
