Scheffczik Hanno, Savva Christos G W, Holzenburg Andreas, Kolesnikova Larissa, Bogner Elke
Institut für Klinische und Molekulare Virologie, Schlossgarten 4, D-91054 Erlangen, Germany.
Nucleic Acids Res. 2002 Apr 1;30(7):1695-703. doi: 10.1093/nar/30.7.1695.
Herpesvirus DNA packaging involves binding and cleavage of DNA containing the specific DNA-packaging motifs. Here we report a first characterization of the terminase subunits pUL56 and pUL89 of human cytomegalovirus (HCMV). Both gene products were shown to have comparable nuclease activities in vitro. Under limiting protein concentrations the nuclease activity is enhanced by interaction of pUL56 and pUL89. High amounts of 2-bromo-5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole partially inhibited the pUL89-associated nuclease activity. It was demonstrated that pUL56 is able to bind to nucleocapsids in vivo. Electron microscopy (EM) and image analysis of purified pUL56 revealed that the molecules occurred as a distinct ring-shaped structure with a pronounced cleft. EM analysis of purified pUL89 demonstrated that this protein is also a toroidal DNA-metabolizing protein. Upon interaction of pUL56 with linearized DNA, the DNA remains uncut while the cutting event itself is mediated by pUL89. Using biochemical assays in conjunction with EM pUL56 was shown to (i) bind to DNA and (ii) associate with the capsid. In contrast to this, EM analysis implied that pUL89 is required to effect DNA cleavage. The data provide the first insights into the terminase-dependent viral DNA-packaging mechanism of HCMV.
疱疹病毒的DNA包装涉及对含有特定DNA包装基序的DNA进行结合和切割。在此，我们首次对人巨细胞病毒（HCMV）的末端酶亚基pUL56和pUL89进行了表征。两种基因产物在体外均显示出相当的核酸酶活性。在蛋白质浓度有限的情况下，pUL56和pUL89的相互作用会增强核酸酶活性。高剂量的2-溴-5,6-二氯-1-β-D-呋喃核糖基苯并咪唑部分抑制了与pUL89相关的核酸酶活性。已证明pUL56在体内能够与核衣壳结合。对纯化的pUL56进行电子显微镜（EM）和图像分析显示，这些分子呈现为具有明显裂隙的独特环形结构。对纯化的pUL89进行EM分析表明，该蛋白也是一种环形DNA代谢蛋白。当pUL56与线性化DNA相互作用时，DNA保持未切割状态，而切割事件本身由pUL89介导。结合EM使用生化分析表明，pUL56能够（i）与DNA结合，（ii）与衣壳结合。与此相反，EM分析表明pUL89是实现DNA切割所必需的。这些数据首次揭示了HCMV依赖末端酶的病毒DNA包装机制。
