Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, 300 Chunchundong Jangangu, Suwon, Gyeonggido 440-746, Republic of Korea.
J Virol. 2010 Sep;84(17):8409-21. doi: 10.1128/JVI.00189-10. Epub 2010 Jun 10.
The human cytomegalovirus (HCMV) UL112-113 region encodes four phosphoproteins with common amino termini (p34, p43, p50, and p84) via alternative splicing and is thought to be required for efficient viral DNA replication. We have previously shown that interactions among the four UL112-113 proteins regulate their intranuclear targeting and enable the recruitment of the UL44 DNA polymerase processivity factor to viral prereplication foci. Here, we show that in virus-infected cells, the UL112-113 proteins form a complex with UL44 and other replication proteins, such as UL84 and IE2. In vitro assays showed that all four phosphoproteins interacted with UL44. Interestingly, p84 required both the shared amino-terminal region and the specific near-carboxy-terminal region for UL44 binding. UL44 required both the carboxy-terminal region and the central region, including the dimerization domain for p84 binding. The production of recombinant virus from mutant Towne bacterial artificial chromosome (BAC) DNA, which encodes intact p34, p43, and p50 and a carboxy-terminally truncated p84 defective in UL44 binding, was severely impaired compared to wild-type BAC DNA. A similar defect was observed when mutant BAC DNA encoded a carboxy-terminally truncated UL44 defective in p84 binding. In cotransfection replication assays using six replication core proteins, UL84, IE2, and UL112-113, the efficient replication of an HCMV oriLyt-containing plasmid required the regions of p84 and UL44 necessary for their interaction. Our data suggest that the UL112-113 proteins form a complex with other replication proteins such as UL44, UL84, and IE2 and that the specific interaction of UL112-113 p84 with UL44 is necessary for efficient viral DNA replication.
人巨细胞病毒 (HCMV) UL112-113 区域通过选择性剪接编码四个具有共同氨基末端的磷酸蛋白（p34、p43、p50 和 p84），被认为是病毒 DNA 复制所必需的。我们之前已经表明，四种 UL112-113 蛋白之间的相互作用调节它们的核内靶向，并使 UL44 DNA 聚合酶持续性因子募集到病毒复制前焦点。在这里，我们表明在受感染的细胞中，UL112-113 蛋白与 UL44 和其他复制蛋白（如 UL84 和 IE2）形成复合物。体外实验表明，所有四种磷酸蛋白都与 UL44 相互作用。有趣的是，p84 既需要共同的氨基末端区域，也需要特定位点的近羧基末端区域来结合 UL44。UL44 既需要羧基末端区域，也需要中央区域，包括二聚化结构域，用于与 p84 结合。与野生型 BAC DNA 相比，从突变型 Towne 细菌人工染色体 (BAC) DNA 产生的重组病毒的产量严重受损，突变型 BAC DNA 编码完整的 p34、p43 和 p50 以及羧基末端截断的 p84，该截断的 p84 不能结合 UL44。当突变型 BAC DNA 编码羧基末端截断的 UL44 时，该突变型不能结合 p84，也观察到类似的缺陷。在使用六个复制核心蛋白 UL84、IE2 和 UL112-113 的共转染复制实验中，包含 HCMV oriLyt 的质粒的有效复制需要 p84 和 UL44 的区域，这些区域对于它们的相互作用是必需的。我们的数据表明，UL112-113 蛋白与其他复制蛋白（如 UL44、UL84 和 IE2）形成复合物，UL112-113 p84 与 UL44 的特异性相互作用对于有效的病毒 DNA 复制是必需的。
