Kerry J A, Priddy M A, Kohler C P, Staley T L, Weber D, Jones T R, Stenberg R M
Department of Microbiology and Immunology, Eastern Virginia Medical School, Norfolk 23501, USA.
J Virol. 1997 Feb;71(2):981-7. doi: 10.1128/JVI.71.2.981-987.1997.
The pp28 (UL99) gene of human cytomegalovirus is expressed as a true late gene, in that DNA synthesis is absolutely required for mRNA expression. Our previous studies demonstrated that pp28 promoter sequences from position -40 to +106 are sufficient for late gene expression in the context of the viral genome (C. P. Kohler, J. A. Kerry, M. Carter, V. P. Muzithras, T. R. Jones, and R. M. Stenberg, J. Virol. 68:6589-6597, 1994). To extend these studies, we have examined the sequences in the downstream leader region of the pp28 gene for their role in late gene expression. Deletion of sequences from position -6 to +46 (deltaSS) results in a threefold increase in gene expression in transient assays. In contrast, deletion of sequences from position +46 to +88 (deltaA) has little effect on gene expression. These results indicate that the sequences from position -6 to +46 may repress gene expression. To further analyze this region, site-directed mutagenesis was performed. Mutation of residues from either position +1 to +6 (SS1) or position +12 to +17 (SS2) duplicated the effect of the deltaSS deletion mutant, indicating that sequences from position +1 to +17 were important for the inhibitory effect. To assess the biological significance of these events, a recombinant virus construct containing the deltaSS mutant promoter regulating expression of the chloramphenicol acetyltransferase (CAT) reporter gene was generated. Analysis of this virus (RV delta SSCAT) revealed that deletion of sequences from position -6 to +46 does not alter the kinetic class of this promoter. However, the ratio of CAT protein to CAT mRNA levels in RV delta SSCAT-infected cells was 8- to 12-fold higher than that observed in the parental RV24/26CAT-infected cells. These results imply that the leader sequences within the pp28 gene can regulate the translation of this late gene.
人巨细胞病毒的pp28（UL99）基因作为一个真正的晚期基因表达，因为mRNA表达绝对需要DNA合成。我们之前的研究表明，从-40到+106位置的pp28启动子序列在病毒基因组背景下足以实现晚期基因表达（C.P.科勒、J.A.克里、M.卡特、V.P.穆齐瑟拉斯、T.R.琼斯和R.M.斯滕伯格，《病毒学杂志》68:6589 - 6597，1994年）。为了扩展这些研究，我们检查了pp28基因下游前导区的序列在晚期基因表达中的作用。从-6到+46位置的序列缺失（deltaSS）导致瞬时分析中基因表达增加了三倍。相比之下，从+46到+88位置的序列缺失（deltaA）对基因表达影响很小。这些结果表明，从-6到+46位置的序列可能抑制基因表达。为了进一步分析该区域，进行了定点诱变。从+1到+6位置（SS1）或+12到+17位置（SS2）的残基突变复制了deltaSS缺失突变体的效应，表明从+1到+17位置的序列对抑制作用很重要。为了评估这些事件的生物学意义，构建了一个重组病毒，其中包含调节氯霉素乙酰转移酶（CAT）报告基因表达的deltaSS突变启动子。对这种病毒（RV delta SSCAT）的分析表明，从-6到+46位置的序列缺失不会改变该启动子的动力学类别。然而，在感染RV delta SSCAT的细胞中，CAT蛋白与CAT mRNA水平的比率比在感染亲本RV24/26CAT的细胞中观察到的高8至12倍。这些结果表明，pp28基因内的前导序列可以调节这个晚期基因的翻译。
