Desai P, Ramakrishnan R, Lin Z W, Osak B, Glorioso J C, Levine M
Department of Human Genetics, University of Michigan, Ann Arbor 48109-0618.
J Virol. 1993 Oct;67(10):6125-35. doi: 10.1128/JVI.67.10.6125-6135.1993.
As has been demonstrated for herpes simplex virus type 2, we show in this report that the herpes simplex virus type 1 ribonucleotide reductase large subunit (RR1) gene is trans activated in transient transfection assays by VP16 and ICP0 but not by ICP4. Deletion analysis demonstrated that responsiveness to induction to VP16 resides in an octamer/TAATGARAT sequence of the RR1 promoter and that the TATA box alone is sufficient to provide induction by ICP0. The induction of the RR1 gene by ICP0 but not by ICP4 suggested that it might be possible to identify the cis-acting element(s) responsive to ICP4 in an ICP4-inducible promoter. To this end, a series of chimeric promoters containing various portions of the regulatory sequences of the RR1 promoter and thymidine kinase (TK) promoter were constructed. The TK promoter is trans activated by both ICP0 and ICP4 in transient transfection assays and by ICP4 in infection. The data show that replacing the RR1 TATA region with the TK TATA region permits ICP4 inducibility even if the rest of the RR1 promoter elements remain intact. To test whether the RR1 gene is induced by ICP0 during infection, four mutant viruses were constructed. (i) TAATGARAT+ has the wild-type RR1 promoter driving chloramphenicol acetyltransferase (CAT) and the RR2 promoter driving the lacZ gene. The RR2 gene codes for the small subunit of the ribonucleotide reductase and is expressed as a beta gene. (ii) TAATGARAT- has a triple-base change in the octamer/TAATGARAT element which renders it unresponsive to VP16 trans activation, eliminating that portion of the activation of the RR1 gene. (iii) TAATGARAT- delta alpha 0 has a deletion of the alpha 0 gene. (iv) TAATGARAT- delta alpha 4 has a deletion of the alpha 4 gene. Infections were carried out in Vero cells at a multiplicity of infection of 10 per cell; cells were assayed for CAT and beta-galactosidase (beta-Gal) activities and for virus yields. The first two infections gave strong CAT and beta-Gal activities and high yields of progeny virus. Infection with the third virus showed no CAT activity but did produce high levels of beta-Gal activity and virus progeny. The fourth infection resulted in strong CAT activity but no beta-Gal activity or progeny virus. The data demonstrated that the RR1 promoter was activated in the absence of ICP4 but not in the absence of ICP0 in these infections.(ABSTRACT TRUNCATED AT 400 WORDS)
正如对2型单纯疱疹病毒所证明的那样，我们在本报告中表明，在瞬时转染试验中，1型单纯疱疹病毒核糖核苷酸还原酶大亚基（RR1）基因可被VP16和ICP0反式激活，但不能被ICP4激活。缺失分析表明，对VP16诱导的反应性存在于RR1启动子的一个八聚体/TAATGARAT序列中，而单独的TATA框足以提供由ICP0诱导的作用。RR1基因由ICP0而非ICP4诱导，这表明有可能在一个ICP4诱导型启动子中鉴定出对ICP4有反应的顺式作用元件。为此，构建了一系列嵌合启动子，其包含RR1启动子和胸苷激酶（TK）启动子调控序列的不同部分。在瞬时转染试验中，TK启动子可被ICP0和ICP4反式激活，在感染时可被ICP4激活。数据表明，用TK的TATA区域替换RR1的TATA区域可使ICP4具有诱导性，即使RR1启动子的其余元件保持完整。为了测试在感染期间RR1基因是否由ICP0诱导，构建了四种突变病毒。（i）TAATGARAT+具有驱动氯霉素乙酰转移酶（CAT）的野生型RR1启动子和驱动lacZ基因的RR2启动子。RR2基因编码核糖核苷酸还原酶的小亚基，并作为β基因表达。（ii）TAATGARAT-在八聚体/TAATGARAT元件中有一个三碱基变化，使其对VP16反式激活无反应，消除了RR1基因激活的那部分。（iii）TAATGARAT-Δα0缺失α0基因。（iv）TAATGARAT-Δα4缺失α4基因。以每细胞10个感染复数在Vero细胞中进行感染；对细胞进行CAT和β-半乳糖苷酶（β-Gal）活性以及病毒产量的检测。前两次感染产生了很强的CAT和β-Gal活性以及高产的子代病毒。用第三种病毒感染未显示出CAT活性，但确实产生了高水平的β-Gal活性和病毒子代。第四次感染导致很强的CAT活性，但没有β-Gal活性或子代病毒。数据表明，在这些感染中，RR1启动子在没有ICP4时被激活，但在没有ICP0时未被激活。（摘要截短于400字）
