Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Ave., SGMB 304, Boston, MA 02115, USA.
J Virol. 2010 Feb;84(4):1771-84. doi: 10.1128/JVI.01510-09. Epub 2009 Dec 9.
In the eukaryotic cell, DNA replication entails the interaction of multiple proteins with the DNA polymerase processivity factor PCNA. As the structure of the presumptive human cytomegalovirus (HCMV) DNA polymerase processivity factor UL44 is highly homologous to that of PCNA, we hypothesized that UL44 also interacts with numerous proteins. To investigate this possibility, recombinant HCMV expressing FLAG-tagged UL44 was generated and used to immunoprecipitate UL44 and associated proteins from infected cell lysates. Unexpectedly, nucleolin, a major protein component of the nucleolus, was identified among these proteins by mass spectrometry and Western blotting. The association of nucleolin and UL44 in infected cell lysate was confirmed by reciprocal coimmunoprecipitation in the presence and absence of nuclease. Western blotting and immunofluorescence assays demonstrated that the level of nucleolin increases during infection and that nucleolin becomes distributed throughout the nucleus. Furthermore, the colocalization of nucleolin and UL44 in infected cell nuclei was observed by immunofluorescence assays. Assays of HCMV-infected cells treated with small interfering RNA (siRNA) targeting nucleolin mRNA indicated that nucleolin was required for efficient virus production, viral DNA synthesis, and the expression of a late viral protein, with a correlation between the efficacy of knockdown and the effect on virus replication. In contrast, the level of neither global protein synthesis nor the replication of an unrelated virus (reovirus) was reduced in siRNA-treated cells. Taken together, our results indicate an association of nucleolin and UL44 in HCMV-infected cells and a role for nucleolin in viral DNA synthesis.
在真核细胞中，DNA 复制需要多种蛋白质与 DNA 聚合酶持续合成因子 PCNA 相互作用。由于假定的人巨细胞病毒 (HCMV) DNA 聚合酶持续合成因子 UL44 的结构与人 PCNA 高度同源，我们假设 UL44 也与许多蛋白质相互作用。为了研究这种可能性，我们构建了表达 FLAG 标签 UL44 的重组 HCMV，并用于从感染细胞裂解物中免疫沉淀 UL44 和相关蛋白。出乎意料的是，通过质谱和 Western blot 鉴定到核仁主要蛋白成分核仁磷蛋白是这些蛋白之一。在存在和不存在核酸酶的情况下，通过相互免疫沉淀进一步证实了核仁磷蛋白与 UL44 在感染细胞裂解物中的结合。Western blot 和免疫荧光检测表明，在感染过程中核仁磷蛋白的水平增加，并且核仁磷蛋白在整个细胞核中分布。此外，通过免疫荧光检测观察到核仁磷蛋白和 UL44 在感染细胞核中的共定位。用针对核仁磷蛋白 mRNA 的小干扰 RNA (siRNA) 处理 HCMV 感染的细胞的检测表明，核仁磷蛋白对于病毒的有效产生、病毒 DNA 合成和晚期病毒蛋白的表达是必需的，敲低的效果与病毒复制之间存在相关性。相比之下，siRNA 处理的细胞中，总的蛋白质合成水平或不相关病毒（呼肠孤病毒）的复制并没有降低。总之，我们的结果表明在 HCMV 感染的细胞中核仁磷蛋白和 UL44 之间存在关联，并且核仁磷蛋白在病毒 DNA 合成中起作用。
