Winkler M, aus Dem Siepen T, Stamminger T
Institut für Klinische und Molekulare Virologie der Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.
J Virol. 2000 Sep;74(17):8053-64. doi: 10.1128/jvi.74.17.8053-8064.2000.
The phosphoprotein pUL69 of human cytomegalovirus (HCMV), which is a herpesvirus of considerable medical importance in immunosuppressed patients and newborns, has previously been identified as an early-late viral protein that can stimulate several viral and cellular promoters and thus exerts a rather broad activation pattern. To gain insight into the mechanism of this transactivation process, we looked for cellular factors interacting with pUL69 in a yeast two-hybrid screen. Using a B-lymphocyte cDNA library fused to the GAL4 activation domain, we identified 34 clones, 11 of which comprised one distinct gene. Interaction with this gene turned out to be very strong, producing beta-galactosidase levels 100-fold greater than the background as measured in an ONPG (o-nitrophenyl-beta-D-galactopyranoside) assay. Sequencing identified this gene as the human homolog of the yeast factor SPT6, which is thought to be involved in the regulation of chromatin structure. A direct interaction of pUL69 and the carboxy terminus of hSPT6 could be demonstrated using in vitro pull-down experiments. After having generated a specific antiserum that is able to detect the endogenous hSPT6 protein, we were able to observe an in vivo interaction of both proteins by coimmunoprecipitation analysis. The interaction domain within pUL69 was mapped to a central domain of this viral protein that is conserved within the homologous proteins of other herpesviruses such as the ICP27 protein of herpes simplex virus. Internal deletions within this central domain, as well as a single amino acid exchange at position C495, resulted in a loss of interaction. This correlated with a loss of the transactivation potential of the respective mutants, suggesting that the hSPT6 interaction of pUL69 is essential for stimulating gene expression. Furthermore, we demonstrate that the carboxy terminus of hSPT6 also binds to histon H3 and that this interaction can be antagonized by pUL69. This allows the deduction of a model by which pUL69 acts as an antirepressor by competing for binding of histones to hSPT6, thereby antagonizing the chromatin remodeling function of this cellular protein.
人巨细胞病毒（HCMV）的磷蛋白pUL69是一种在免疫抑制患者和新生儿中具有重要医学意义的疱疹病毒，此前已被鉴定为一种早期-晚期病毒蛋白，它可以刺激多种病毒和细胞启动子，因此具有相当广泛的激活模式。为了深入了解这种反式激活过程的机制，我们在酵母双杂交筛选中寻找与pUL69相互作用的细胞因子。使用与GAL4激活结构域融合的B淋巴细胞cDNA文库，我们鉴定出34个克隆，其中11个包含一个不同的基因。与该基因的相互作用非常强烈，在ONPG（邻硝基苯-β-D-吡喃半乳糖苷）测定中产生的β-半乳糖苷酶水平比背景高100倍。测序确定该基因为酵母因子SPT6的人类同源物，据认为它参与染色质结构的调节。使用体外下拉实验可以证明pUL69与hSPT6的羧基末端直接相互作用。在产生能够检测内源性hSPT6蛋白的特异性抗血清后，我们能够通过共免疫沉淀分析观察到两种蛋白在体内的相互作用。pUL69内的相互作用结构域被定位到该病毒蛋白的中央结构域，该结构域在其他疱疹病毒的同源蛋白如单纯疱疹病毒的ICP27蛋白中是保守的。该中央结构域内的内部缺失以及C495位置的单个氨基酸交换导致相互作用丧失。这与各个突变体反式激活潜力的丧失相关，表明pUL69与hSPT6的相互作用对于刺激基因表达至关重要。此外，我们证明hSPT6的羧基末端也与组蛋白H3结合，并且这种相互作用可以被pUL69拮抗。这允许推断出一个模型，通过该模型pUL69通过竞争组蛋白与hSPT6的结合而作为抗阻遏物起作用，从而拮抗这种细胞蛋白的染色质重塑功能。
