Kerry J A, Priddy M A, Staley T L, Jones T R, Stenberg R M
Department of Microbiology and Immunology, Eastern Virginia Medical School, Norfolk 23501, USA.
J Virol. 1997 Mar;71(3):2120-6. doi: 10.1128/JVI.71.3.2120-2126.1997.
Previous analysis of the human cytomegalovirus (HCMV) DNA polymerase (UL54) early gene promoter demonstrated that transcriptional activation of this gene is dependent upon the interaction of cellular transcription factors with viral transactivators (J. A. Kerry, M. A. Priddy, T. Y. Jervey, C. P. Kohler, T. L. Staley, C. D. Vanson, T. R. Jones, A. C. Iskenderian, D. G. Anders, and R. M. Stenberg, J. Virol. 70:373-382, 1996). A sequence element, IR1, was shown to be the primary regulatory element of this promoter in transient assays. However, assessment of this element in the context of the viral genome revealed IR1-independent activation at late times after infection. To extend these studies, we aim to identify additional sequence elements involved in the activation of the UL54 promoter. Our present studies demonstrate that the level of binding of proteins to the ATF site in the UL54 promoter is enhanced by viral infection. Furthermore this increase is sensitive to treatment with phosphonoacetic acid (PAA), a DNA synthesis inhibitor. These data suggest that the increase in the level of ATF binding activity is regulated, either directly or indirectly, by HCMV late gene expression. By using specific antibodies, we determined that ATF-1 was a major component of the proteins binding to the UL54 ATF site at late times. In addition, we have demonstrated direct binding of recombinant ATF-1 to the UL54 ATF site. To assess the biological significance of these events, a recombinant virus construct was generated that contained the UL54 promoter with a mutation in the ATF site regulating expression of the chloramphenicol acetyltransferase (CAT) reporter gene inserted between open reading frames US9 and US10. Analysis of this virus (RVATFmCAT) revealed that mutation of the ATF site does not alter the kinetics of UL54 promoter activation. However, levels of CAT mRNA and activity were reduced by 5- to 10-fold compared to those of the wild-type promoter at all stages of infection. These findings indicate that ATF-1 can regulate the levels of UL54 promoter activity at both early and late times. Furthermore, these results imply that HCMV can regulate the activity of cellular factors involved in early gene regulation.
先前对人巨细胞病毒（HCMV）DNA聚合酶（UL54）早期基因启动子的分析表明，该基因的转录激活依赖于细胞转录因子与病毒反式激活因子的相互作用（J. A. 克里、M. A. 普里迪、T. Y. 杰维、C. P. 科勒、T. L. 斯泰利、C. D. 万森、T. R. 琼斯、A. C. 伊斯肯德里安、D. G. 安德斯和R. M. 斯滕伯格，《病毒学杂志》70:373 - 382，1996年）。在瞬时分析中，一个序列元件IR1被证明是该启动子的主要调控元件。然而，在病毒基因组背景下对该元件的评估显示，在感染后期存在不依赖IR1的激活。为了扩展这些研究，我们旨在鉴定参与UL54启动子激活的其他序列元件。我们目前的研究表明，病毒感染可增强蛋白质与UL54启动子中ATF位点的结合水平。此外，这种增加对DNA合成抑制剂膦甲酸（PAA）的处理敏感。这些数据表明，ATF结合活性水平的增加是由HCMV晚期基因表达直接或间接调控的。通过使用特异性抗体，我们确定ATF - 1是感染后期与UL54 ATF位点结合的蛋白质的主要成分。此外，我们已经证明重组ATF - 1与UL54 ATF位点直接结合。为了评估这些事件的生物学意义，构建了一种重组病毒，其包含UL54启动子，该启动子在ATF位点发生突变，调控插入开放阅读框US9和US10之间的氯霉素乙酰转移酶（CAT）报告基因的表达。对这种病毒（RVATFmCAT）的分析表明，ATF位点的突变不会改变UL54启动子激活的动力学。然而，在感染的所有阶段，CAT mRNA水平和活性与野生型启动子相比降低了5至10倍。这些发现表明，ATF - 1在早期和晚期均可调控UL54启动子活性水平。此外，这些结果意味着HCMV可以调控参与早期基因调控的细胞因子的活性。
