Rader K A, Ackland-Berglund C E, Miller J K, Pepose J S, Leib D A
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St Louis, Missouri 63110.
J Gen Virol. 1993 Sep;74 ( Pt 9):1859-69. doi: 10.1099/0022-1317-74-9-1859.
Transient expression assays in PC12 cells showed that the cAMP response element (CRE) and the TATA box of the herpes simplex virus type 1 latency-associated transcripts (LATs) promoter are essential for basal expression. Recombinant viruses were generated containing site-specific mutations in these motifs. The abilities of these recombinants to replicate, express LATs and reactivate from latency were compared with wild-type and marker-rescued viruses in a murine ocular model. The acute replication of these TATA and CRE mutant viruses was at a level equivalent to their respective marker-rescued viruses. The reactivation of virus was unaffected by mutation in the TATA box as compared with wild-type or marker-rescued viruses. In situ hybridization of TATA box mutant virus-infected ganglia, however, showed threefold fewer LAT-positive neurons than wild-type virus-infected ganglia, with consistently weaker hybridization signals. Thus, this TATA box is required for normal expression of the LATs but not for efficient reactivation. The LATs CRE mutant reactivated with slightly but reproducibly reduced frequency and delayed kinetics relative to marker-rescued virus. By in situ hybridization, however, the percentage and intensity of LATs-positive neurons were found to be comparable for the CRE mutant- and wild-type virus-infected ganglia, suggesting that the CRE is dispensable for abundant LATs expression but that a reactivation function of the LATs may depend upon the presence of the CRE. Finally, using a modified assay for examining the timing of reactivation, we showed that the induction of viral reactivation by addition of exogenous cAMP can occur independently of the LATs.
在PC12细胞中的瞬时表达分析表明，单纯疱疹病毒1型潜伏相关转录本（LATs）启动子的环磷酸腺苷反应元件（CRE）和TATA盒对于基础表达至关重要。构建了在这些基序中含有位点特异性突变的重组病毒。在小鼠眼部模型中，将这些重组体与野生型和标记拯救病毒在复制、表达LATs以及从潜伏状态重新激活的能力方面进行了比较。这些TATA盒和CRE突变病毒的急性复制水平与其各自的标记拯救病毒相当。与野生型或标记拯救病毒相比，TATA盒突变对病毒的重新激活没有影响。然而，TATA盒突变病毒感染的神经节的原位杂交显示，LAT阳性神经元比野生型病毒感染的神经节少三倍，杂交信号始终较弱。因此，这个TATA盒是LATs正常表达所必需的，但不是有效重新激活所必需的。相对于标记拯救病毒，LATs CRE突变体重新激活的频率略有降低但可重复，动力学延迟。然而，通过原位杂交发现，CRE突变体和野生型病毒感染的神经节中LAT阳性神经元的百分比和强度相当，这表明CRE对于丰富的LATs表达是可有可无的，但LATs的重新激活功能可能取决于CRE的存在。最后，使用一种改良的检测方法来检查重新激活的时间，我们发现添加外源性环磷酸腺苷诱导病毒重新激活可以独立于LATs发生。
