DeLuca N A, Schaffer P A
Mol Cell Biol. 1985 Aug;5(8):1997-2008. doi: 10.1128/mcb.5.8.1997-2008.1985.
To better define the activities on herpes simplex virus type 1 gene expression of temperature-sensitive and wild-type forms of the transcriptional regulatory protein ICP4, regulatory sequences from immediate-early, early, and late herpes simplex virus genes were fused to the gene for chloramphenicol acetyltransferase (CAT). These constructs were used in trans induction and cotransfection experiments with wild-type and temperature-sensitive mutant alleles of ICP4. The ICP4 genes used in this study were cloned from the KOS strain (wild type) and two phenotypically distinct temperature-sensitive ICP4 mutants, tsB32 and tsL14 (DeLuca et al., J. Virol. 52:767-776, 1984), both alone and in conjunction with three other immediate-early genes. The latter series of plasmids was used to assess the influence of additional immediate-early gene products on gene expression in the presence of a given ICP4 allele. The results of this study demonstrate that the phenotypes of these ICP4 mutants observed in cell culture at the nonpermissive temperature were determined in part by activities associated with the mutant ICP4 polypeptides and that these activities differed from those of wild-type ICP4. Low levels of wild-type ICP4 had a marginal but reproducible stimulatory effect on immediate-early CAT gene expression, especially the pIE4/5CAT chimera. This effect was diminished with increasing quantities of ICP4, suggesting an inhibitory role for the wild-type form of the protein. The ICP4 mutants had a strong stimulatory effect on immediate-early CAT expression, consistent with their phenotypes at 39 degrees C. The mutant forms of the ICP4 polypeptide differed in their ability to induce CAT activity from an early chimeric gene. Thus, the tsL14 form of ICP4 was effective in early gene induction (i.e., ptkCAT was induced), whereas the ICP4 derived from tsB32 was slightly inhibitory. Cotransfection of tsB32 ICP4 simultaneously with other immediate-early genes resulted in a marginal increase in ptkCAT induction. This induction was enhanced when the gene for ICP4 was inactivated by restriction enzyme cleavage, substantiating the inhibitory effect of the tsB32 form of ICP4. The two mutant ICP4 genes (tsB32 and tsL14) were unable to trans-activate either of the late CAT constructs (p5CAT and pL42CAT) tested. Cotransfecting tsL14 ICP4 with the other immediate-early genes resulted in activation of p5CAT but not pL42CAT. Taken together, these studies demonstrate that (i) low levels of wild-type ICP4 have stimulatory effect on immediate-early promoters and that higher concentrations of wild-type ICP4 have an inhibitory effect on these promoters, (ii) isolated mutant form of ICP4 exhibit activities that reflect the phenotypes of the mutants from which they were isolated, and (iii) immediate-early gene products other than ICP4 are involved in determining the distinct phenotypes of the two mutants at 39 degrees Celsius.
为了更明确温度敏感型和野生型转录调节蛋白ICP4对单纯疱疹病毒1型基因表达的作用，将单纯疱疹病毒即刻早期、早期和晚期基因的调控序列与氯霉素乙酰转移酶（CAT）基因融合。这些构建体用于与ICP4的野生型和温度敏感突变等位基因进行反式诱导及共转染实验。本研究中使用的ICP4基因是从KOS株（野生型）以及两个表型不同的温度敏感ICP4突变体tsB32和tsL14（DeLuca等人，《病毒学杂志》52:767 - 776，1984）中克隆得到的，这些基因单独使用以及与其他三个即刻早期基因一起使用。后一系列质粒用于评估在给定ICP4等位基因存在的情况下，其他即刻早期基因产物对基因表达的影响。本研究结果表明，在非允许温度下在细胞培养中观察到的这些ICP4突变体的表型部分由与突变ICP4多肽相关的活性决定，并且这些活性与野生型ICP4的活性不同。低水平的野生型ICP4对即刻早期CAT基因表达有轻微但可重复的刺激作用，尤其是对pIE4/5CAT嵌合体。随着ICP4量的增加，这种作用减弱，表明该蛋白的野生型形式具有抑制作用。ICP4突变体对即刻早期CAT表达有强烈的刺激作用，与其在39℃时的表型一致。ICP4多肽的突变形式在从早期嵌合基因诱导CAT活性的能力上有所不同。因此，ICP4的tsL14形式在早期基因诱导中有效（即ptkCAT被诱导），而源自tsB32的ICP4则有轻微抑制作用。将tsB32 ICP4与其他即刻早期基因同时共转染导致ptkCAT诱导略有增加。当通过限制性酶切使ICP4基因失活时，这种诱导增强，证实了tsB32形式的ICP4的抑制作用。两个突变ICP4基因（tsB32和tsL14）均无法反式激活所测试的任何晚期CAT构建体（p5CAT和pL42CAT）。将tsL14 ICP4与其他即刻早期基因共转染导致p5CAT激活，但pL42CAT未激活。综上所述，这些研究表明：（i）低水平的野生型ICP4对即刻早期启动子有刺激作用，而较高浓度的野生型ICP4对这些启动子有抑制作用；（ii）分离的ICP4突变形式表现出反映其来源突变体表型的活性；（iii）除ICP4外的即刻早期基因产物参与决定这两个突变体在39摄氏度时的不同表型。
