Bogner E, Radsak K, Stinski M F
Department of Microbiology, College of Medicine, University of Iowa, Iowa City 52242, USA.
J Virol. 1998 Mar;72(3):2259-64. doi: 10.1128/JVI.72.3.2259-2264.1998.
Using the cis-acting human cytomegalovirus (HCMV) packaging elements (pac 1 and pac 2) as DNA probes, specific DNA-protein complexes were detected by electrophoretic mobility shift assay (EMSA) in both HCMV-infected cell nuclear extracts and recombinant baculovirus-infected cell extracts containing the HCMV p130 (pUL56) protein. DNA-binding proteins, which were common in uninfected and infected cell extracts, were also detected. Mutational analysis showed that only the AT-rich core sequences in these cis-acting motifs, 5'-TAAAAA-3' (pac 1) and 5'-TTTTAT-3' (pac 2), were required for specific DNA-protein complex formation. The specificity of the DNA-protein complexes was confirmed by EMSA competition. Furthermore, a specific endonuclease activity was found to be associated with lysates of baculovirus-infected cells expressing recombinant p130 (rp130). This nuclease activity was time dependent, related to the amount of rp130 in the assay, and ATP independent. Nuclease activity remained associated with rp130 after partial purification by sucrose gradient centrifugation, suggesting that this activity is a property of HCMV p130. We propose a possible involvement of p130 in HCMV DNA packaging.
使用顺式作用的人巨细胞病毒（HCMV）包装元件（pac 1和pac 2）作为DNA探针，通过电泳迁移率变动分析（EMSA）在HCMV感染的细胞核提取物和含有HCMV p130（pUL56）蛋白的重组杆状病毒感染的细胞提取物中检测到特异性DNA-蛋白质复合物。还检测到未感染和感染细胞提取物中常见的DNA结合蛋白。突变分析表明，这些顺式作用基序中的富含AT的核心序列，5'-TAAAAA-3'（pac 1）和5'-TTTTAT-3'（pac 2），是特异性DNA-蛋白质复合物形成所必需的。通过EMSA竞争证实了DNA-蛋白质复合物的特异性。此外，发现一种特异性核酸内切酶活性与表达重组p130（rp130）的杆状病毒感染细胞的裂解物相关。这种核酸酶活性是时间依赖性的，与测定中rp130的量有关，并且不依赖于ATP。通过蔗糖梯度离心部分纯化后，核酸酶活性仍与rp130相关，表明这种活性是HCMV p130的特性。我们提出p130可能参与HCMV DNA包装。
