The Adeno-Associated Virus Type 2 Regulatory Proteins Rep78 and Rep68 Interact with the Transcriptional Coactivator PC4
The Adeno-Associated Virus Type 2 Regulatory Proteins Rep78 and Rep68 Interact with the Transcriptional Coactivator PC4
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DOI：
10.1128/jvi.73.1.260-269.1999
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发表时间：
1999-01
影响因子：
5.4
通讯作者：
S. Weger;Meike Wendland;J. Kleinschmidt;R. Heilbronn
中科院分区：
文献类型：
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作者：
S. Weger;Meike Wendland;J. Kleinschmidt;R. Heilbronn
腺相关病毒2型（AAV-2） Rep78/Rep68调节蛋白是病毒和细胞DNA复制、病毒和细胞癌基因的细胞转化以及同源和异源基因表达的多效效应器。为了寻找参与介导这些功能的细胞蛋白，我们在酵母双杂交系统中以Rep68为诱饵，并确定了转录共激活子PC4作为Rep的相互作用伙伴。PC4已被证明在体外介导多种序列特异性转录因子的转录激活。代表氨基酸172 ~ 530是足够的，氨基酸172 ~ 224是与PC4相互作用绝对必需的。相互作用所需的PC4结构域被映射到PC4的c端单链dna结合结构域。在谷胱甘肽s -转移酶（GST）下拉实验中，体外转录和翻译的Rep78或Rep68蛋白被GST- pc4融合蛋白特异性结合。同样，在大肠杆菌中表达的PC4被GST-Rep融合蛋白结合，证实了Rep与PC4在体外的直接相互作用。研究发现，Rep对非磷酸化、转录活性形式的PC4比磷酸化、转录无活性形式的PC4具有更高的亲和力。后者主要存在于HeLa或293细胞的核提取物中。在酵母系统中，而不是在体外，Rep-PC4的相互作用被Rep68假定的核苷酸结合位点的点突变破坏，这表明Rep和PC4在体内的稳定相互作用依赖于ATP。该突变也被证明损害Rep在AAV-2 DNA复制中的功能，并抑制基因表达和诱导DNA扩增。巨细胞病毒启动子驱动的PC4过表达导致非磷酸化PC4的短暂积累，同时在没有辅助病毒的情况下，所有三种AAV-2启动子的下调。在腺病毒存在的情况下，这种效果被缓解。这些结果表明，在没有辅助病毒的情况下，转录辅激活因子PC4参与了AAV-2基因表达的调控。
ABSTRACT The adeno-associated virus type 2 (AAV-2) Rep78/Rep68 regulatory proteins are pleiotropic effectors of viral and cellular DNA replication, of cellular transformation by viral and cellular oncogenes, and of homologous and heterologous gene expression. To search for cellular proteins involved in mediating these functions, we used Rep68 as bait in the yeast two-hybrid system and identified the transcriptional coactivator PC4 as a Rep interaction partner. PC4 has been shown to mediate transcriptional activation by a variety of sequence-specific transcription factors in vitro. Rep amino acids 172 to 530 were sufficient and amino acids 172 to 224 were absolutely necessary for the interaction with PC4. The PC4 domains required for interaction were mapped to the C-terminal single-stranded DNA-binding domain of PC4. In glutathione S-transferase (GST) pull-down assays, in vitro-transcribed and -translated Rep78 or Rep68 proteins were bound specifically by GST-PC4 fusion proteins. Similarly, PC4 expressed in Escherichia coli was bound by GST-Rep fusion proteins, confirming the direct interaction between Rep and PC4 in vitro. Rep was found to have a higher affinity for the nonphosphorylated, transcriptionally active form of PC4 than for the phosphorylated, transcriptionally inactive form. The latter is predominant in nuclear extracts of HeLa or 293 cells. In the yeast system, but not in vitro, Rep-PC4 interaction was disrupted by a point mutation in the putative nucleotide-binding site of Rep68, suggesting that a stable interaction between Rep and PC4 in vivo is ATP dependent. This mutation has also been shown to impair Rep function in AAV-2 DNA replication and in inhibition of gene expression and inducible DNA amplification. Cytomegalovirus promoter-driven overexpression of PC4 led to transient accumulation of nonphosphorylated PC4 with concomitant downregulation of all three AAV-2 promoters in the absence of helper virus. In the presence of adenovirus, this effect was relieved. These results imply an involvement of the transcriptional coactivator PC4 in the regulation of AAV-2 gene expression in the absence of helper virus.