The Flexible Loop of the Human Cytomegalovirus DNA Polymerase Processivity Factor ppUL44 Is Required for Efficient DNA Binding and Replication in Cells
The Flexible Loop of the Human Cytomegalovirus DNA Polymerase Processivity Factor ppUL44 Is Required for Efficient DNA Binding and Replication in Cells
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DOI：
10.1128/jvi.00669-09
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发表时间：
2009-09-15
影响因子：
5.4
通讯作者：
Jans, David A.
中科院分区：
文献类型：
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作者：
Alvisi, Gualtiero;Roth, Daniela Martino;Jans, David A.
人巨细胞病毒(HCMV)DNA聚合酶的磷酸蛋白ppUL44在病毒复制中起重要作用，它通过将DNA聚合酶催化亚单位pUL54连接到DNA上而赋予其加工能力。在这里，我们首次在活细胞中研究了ppUL44(UL44-FL；第162至174位残基[PHTRVKRNVKKAP(174)])的高度灵活环的功能，该环被认为直接参与了ppUL44‘S与DNA的相互作用。特别是，我们在转基因细胞中使用了各种方法来详细表征ppUL44 Delta loop的行为，这是一种突变衍生物，其中UL44-FL中的五个碱性残基中的三个被非碱性氨基酸取代。我们的结果表明，与野生型ppUL44相比，ppUL44 Delta loop在二聚化和与pUL54结合方面具有功能，但在结合核内的核结构方面受到强烈损害，表现为不能形成核斑点，核积累减少，核内迁移率增加。此外，对洗涤剂和DNA酶处理后的细胞组分的分析表明，ppUL44 Delta loop与DNA结合能力显著降低，这与ppUL44-L86A/L87A类似，ppUL44-L86A/L87A是一种二聚化障碍的点突变衍生物。最后，ppUL44 Delta loop不能反式补充细胞中依赖于HCMV oriLyt的DNA复制，也能在野生型ppUL44存在的情况下抑制复制，可能是通过形成不能与双链DNA结合的异源二聚体。因此，UL44-FL首次成为细胞内HCMV复制的重要决定因素，对于通过靶向HCMV复制来开发新的抗病毒方法具有潜在的意义。
Phosphoprotein ppUL44 of the human cytomegalovirus (HCMV) DNA polymerase plays an essential role in viral replication, conferring processivity to the DNA polymerase catalytic subunit pUL54 by tethering it to the DNA. Here, for the first time, we examine in living cells the function of the highly flexible loop of ppUL44 (UL44-FL; residues 162 to 174 [PHTRVKRNVKKAP(174)]), which has been proposed to be directly involved in ppUL44's interaction with DNA. In particular, we use a variety of approaches in transfected cells to characterize in detail the behavior of ppUL44 Delta loop, a mutant derivative in which three of the five basic residues within UL44-FL are replaced by nonbasic amino acids. Our results indicate that ppUL44 Delta loop is functional in dimerization and binding to pUL54 but strongly impaired in binding nuclear structures within the nucleus, as shown by its inability to form nuclear speckles, reduced nuclear accumulation, and increased intranuclear mobility compared to wild-type ppUL44. Moreover, analysis of cellular fractions after detergent and DNase treatment indicates that ppUL44 Delta loop is strongly reduced in DNA-binding ability, in similar fashion to ppUL44-L86A/L87A, a point mutant derivative impaired in dimerization. Finally, ppUL44 Delta loop fails to transcomplement HCMV oriLyt-dependent DNA replication in cells and also inhibits replication in the presence of wild-type ppUL44, possibly via formation of heterodimers defective for double-stranded DNA binding. UL44-FL thus emerges for the first time as an important determinant for HCMV replication in cells, with potential implications for the development of novel antiviral approaches by targeting HCMV replication.