Frazier D P, Cox D, Godshalk E M, Schaffer P A
Division of Molecular Genetics, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
J Virol. 1996 Nov;70(11):7424-32. doi: 10.1128/JVI.70.11.7424-7432.1996.
Herpes simplex virus establishes latent infections in the nuclei of sensory neurons. These infections are characterized by the abundant expression of a series of 5' coterminal transcripts termed the latency-associated transcripts (LATs). Available evidence indicates that LAT expression is specifically regulated in latently infected neurons. Although previous studies have examined the regulation of LAT expression in neuronal and nonneuronal cells, the mechanism of regulation of LAT expression in neuronal cells in response to external factors has not been investigated. To address this question, we characterized the activity of LAT promoter fusion constructs in PC12 cells following treatment with nerve growth factor (NGF) and/or sodium butyrate (NaB), agents that affect expression of cell cycle-associated genes. Expression from the LAT promoter was induced 8- to 12-fold by either NGF or NaB alone and 40- to 60-fold when the two agents were added simultaneously. Fibroblast growth factor also induced expression from the LAT promoter but to a lesser extent than NGF. Treatment with factors such as epidermal growth factor, phorbol myristate acetate, cyclic AMP, or KCI had no significant effect on LAT promoter activity. Notably, promoter-reporter constructs containing immediate-early (ICP0 and ICP4), early (ICP8 and UL9), and late (UL10 and UL30) viral promoters were induced only two- to fourfold by NGF, suggesting that the LAT promoter may be unusual among herpes simplex virus genes in the magnitude of its response to this factor. To identify pathways leading to LAT activation in vitro, we characterized the response of the LAT promoter to NGF and/or NaB in PC12-derived cell lines containing mutations in specific signal transduction pathways. We found that activation of the LAT promoter requires Ras activation and that activation of the serine/threonine kinase, Raf, is sufficient to induce LAT expression. Together, these results indicate that the LAT promoter is regulated via the Ras/Raf signal transduction pathway in response to external factors such as NGF and NaB and that LAT expression may be regulated by NGF in latently infected neurons.
单纯疱疹病毒在感觉神经元的细胞核中建立潜伏感染。这些感染的特征是一系列5' 共末端转录本大量表达，这些转录本被称为潜伏相关转录本（LATs）。现有证据表明，LAT的表达在潜伏感染的神经元中受到特异性调节。尽管先前的研究已经探讨了神经元和非神经元细胞中LAT表达的调节，但尚未研究神经元细胞中LAT表达响应外部因素的调节机制。为了解决这个问题，我们在用神经生长因子（NGF）和/或丁酸钠（NaB）处理后的PC12细胞中，对LAT启动子融合构建体的活性进行了表征，NGF和NaB是影响细胞周期相关基因表达的试剂。单独使用NGF或NaB时，LAT启动子的表达可被诱导8至12倍，而同时添加这两种试剂时，表达可被诱导40至60倍。成纤维细胞生长因子也可诱导LAT启动子的表达，但程度低于NGF。用表皮生长因子、佛波酯、环磷酸腺苷或氯化钾等因子处理对LAT启动子活性没有显著影响。值得注意的是，包含即刻早期（ICP0和ICP4）、早期（ICP8和UL9）和晚期（UL10和UL30）病毒启动子的启动子-报告基因构建体仅被NGF诱导2至4倍，这表明LAT启动子在对该因子的反应强度方面可能在单纯疱疹病毒基因中是不同寻常的。为了在体外鉴定导致LAT激活的途径，我们在含有特定信号转导途径突变的PC12衍生细胞系中，对LAT启动子对NGF和/或NaB的反应进行了表征。我们发现LAT启动子的激活需要Ras激活，并且丝氨酸/苏氨酸激酶Raf的激活足以诱导LAT表达。总之，这些结果表明，LAT启动子通过Ras/Raf信号转导途径响应NGF和NaB等外部因素进行调节，并且LAT表达可能在潜伏感染的神经元中受到NGF的调节。
