Huang Q S, Valyi-Nagy T, Kesari S, Fraser N W
Wistar Institute, Philadelphia, PA 19104, USA.
Gene Ther. 1997 Aug;4(8):797-807. doi: 10.1038/sj.gt.3300476.
beta-Galactosidase enzyme expression can be detected in only a small percentage of trigeminal ganglia (TG) neurons acutely and latently infected with herpes simplex virus (HSV), in which the lacZ reporter gene was placed down-stream of the latency associated transcript (LAT) promoter at the LAT locus. However, DNA quantification suggests that a larger percentage of cells is infected than is expressing beta-galactosidase enzyme. To investigate the mechanism involved in regulation of genes expressed from the LAT promoter in trigeminal ganglia, in situ hybridization and histochemical staining assays were employed to determine on a cell-by-cell basis beta-gal gene expression both at the RNA and protein level. Using a LAT promoter-driven beta-gal construct in HSV-1 strain HFEM, it was found that there were 89-fold more cells positive for beta-gal transcript than cells positive for beta-gal enzyme in acutely infected trigeminal ganglia and a 10-fold difference in latently infected trigeminal ganglia. Thus, there is a discordance between beta-gal mRNA and beta-gal enzyme levels in HFEM/LAT-lacZ infected cells during acute and latent infection, and the beta-gal reporter gene activity does not faithfully compare the LAT promoter activity between acute and latently infected tissue. In contrast, in situ hybridization and histochemical staining assays were performed in mice acutely infected with a virus in which 140 bp of the LAT promoter sequences flanking the TATA element were replaced by 1.8 kbp of the neurofilament promoter (HSV-1 HFEM/NF-lacZ). This construct showed a correlation between beta-gal mRNA and enzyme expression in trigeminal ganglia in acute and latent infections. These findings suggest that sequences at the 5' end of the beta-gal transcript influence translation of the beta-gal message.
β-半乳糖苷酶的表达仅在一小部分急性和潜伏感染单纯疱疹病毒（HSV）的三叉神经节（TG）神经元中可检测到，在这些神经元中，lacZ报告基因被置于潜伏相关转录物（LAT）基因座的LAT启动子下游。然而，DNA定量分析表明，被感染的细胞比例高于表达β-半乳糖苷酶的细胞比例。为了研究三叉神经节中LAT启动子调控基因表达的机制，采用原位杂交和组织化学染色分析，在单细胞水平上确定β-半乳糖苷酶基因在RNA和蛋白质水平的表达。使用HSV-1 HFEM株中LAT启动子驱动的β-半乳糖苷酶构建体，发现在急性感染的三叉神经节中，β-半乳糖苷酶转录物阳性细胞比β-半乳糖苷酶阳性细胞多89倍，在潜伏感染的三叉神经节中两者相差10倍。因此，在急性和潜伏感染期间，HFEM/LAT-lacZ感染细胞中β-半乳糖苷酶mRNA水平与β-半乳糖苷酶水平不一致，并且β-半乳糖苷酶报告基因活性不能如实反映急性和潜伏感染组织之间的LAT启动子活性。相比之下，在急性感染一种病毒的小鼠中进行原位杂交和组织化学染色分析，该病毒中TATA元件侧翼的140 bp LAT启动子序列被1.8 kbp神经丝启动子取代（HSV-1 HFEM/NF-lacZ）。该构建体在急性和潜伏感染的三叉神经节中显示出β-半乳糖苷酶mRNA与酶表达之间的相关性。这些发现表明，β-半乳糖苷酶转录物5'端的序列影响β-半乳糖苷酶信息的翻译。
