Schwartz R, Helmich B, Spector D H
Department of Biology, University of California, San Diego, La Jolla, California 92093-0357, USA.
J Virol. 1996 Oct;70(10):6955-66. doi: 10.1128/JVI.70.10.6955-6966.1996.
The human cytomegalovirus (HCMV) immediate-early region 2 86-kDa protein (IE2 86) is the major transactivator of the promoter for the 2.2-kb class of early RNAs (open reading frame UL 112-113). Previously, we reported that a DNA segment on this promoter between nucleotides (nt) -113 and -59 was critical for activation by IE2 86 in vivo and could be bound by IE2 86 in vitro (R. Schwartz, M. H. Sommer, A. Scully, and D. H. Spector, J. Virol. 68:5613-5622, 1994). With a set of site-specific mutations within nt -84 to -61, we have localized the essential cis-acting sequences to nt -72 to -61, which contain an ATF/CREB-binding site. The IE2 86-binding site between nt -113 and -85 is not essential for activation of the promoter by IE2 86 in transient-expression assays, but its presence can enhance the level of activation mediated through the sequences located between nt -84 and -59. Electrophoretic mobility shift assays with a segment containing nt -84 to -59 and nuclear extracts from human cells permissive for the HCMV infection revealed a complex band pattern. However, by supershift analysis with specific antibodies, we were able to identify CREB as the major ATF/CREB family member in the protein-DNA complexes. Further evidence that CREB is a target for IE2 86-mediated induction, is provided by the finding that IE2 86 activates the somatostatin promoter to high levels. Although the binding of IE2 86 to nonphosphorylated full-length CREB or deltaCREB is minimal, IE2 86 does form complexes with p300 and the CREB-binding protein (CBP), which in turn bind to CREB and can serve as adaptor proteins for CREB function. In addition, the in vivo functional relevance of the interaction between IE2 86 and CBP is indicated by the ability of IE2 86 to enhance transcriptional activation mediated by a GAL4-CBP fusion protein brought to a promoter by GAL4-binding sites.
人巨细胞病毒（HCMV）即刻早期区域2的86 kDa蛋白（IE2 86）是2.2 kb类早期RNA（开放阅读框UL 112 - 113）启动子的主要反式激活因子。先前我们报道，该启动子上核苷酸（nt）-113至-59之间的一个DNA片段对于IE2 86在体内的激活至关重要，并且在体外可被IE2 86结合（R. Schwartz、M. H. Sommer、A. Scully和D. H. Spector，《病毒学杂志》68:5613 - 5622，1994年）。利用nt -84至-61内的一组位点特异性突变，我们已将必需的顺式作用序列定位到nt -72至-61，其包含一个ATF/CREB结合位点。在瞬时表达试验中，nt -113至-85之间的IE2 86结合位点对于IE2 86激活启动子并非必需，但它的存在可增强通过nt -84至-59之间序列介导的激活水平。用包含nt -84至-59的片段以及来自允许HCMV感染的人细胞的核提取物进行的电泳迁移率变动分析揭示了一个复杂的条带模式。然而，通过用特异性抗体进行超迁移分析，我们能够鉴定出CREB是蛋白质 - DNA复合物中主要的ATF/CREB家族成员。IE2 86激活生长抑素启动子至高水平这一发现，进一步证明了CREB是IE2 86介导诱导的靶点。尽管IE2 86与非磷酸化的全长CREB或δCREB的结合很微弱，但IE2 86确实与p300和CREB结合蛋白（CBP）形成复合物，而它们又与CREB结合并可作为CREB功能的衔接蛋白。此外，IE2 86增强由GAL4结合位点带到启动子的GAL4 - CBP融合蛋白介导的转录激活的能力，表明了IE2 86与CBP之间相互作用在体内的功能相关性。
