Hwang Jae-Seon, Bogner Elke
Institut für Klinische und Molekulare Virologie, Schlossgarten 4, D-91054 Erlangen, Germany.
J Biol Chem. 2002 Mar 1;277(9):6943-8. doi: 10.1074/jbc.M108984200. Epub 2001 Dec 13.
Herpesviral DNA packaging is a complex process resulting in unit-length genomes packed into preformed procapsids. This process is believed to be mediated by two packaging proteins, the terminase subunits. In the case of double-stranded DNA bacteriophages, the translocation of DNA was shown to be an energy-dependent process associated with an ATPase activity of the large terminase subunit. In the case of human cytomegalovirus it was not known which protein has the ability to hydrolyze ATP. In this study we expressed human cytomegalovirus terminase subunits, pUL89 and the carboxyl-terminal half of pUL56, as GST fusion proteins and purified these by affinity chromatography. ATPase assays demonstrated that the enzymatic activity is exclusively associated with pUL56. The characterization of the ATP hydrolysis showed that the enzymatic reaction is a fast process, whereas the spontaneous ATP decay followed slow kinetics. Interestingly, although pUL89 did not show any ATPase activity, it was capable of enhancing the UL56-associated ATP hydrolysis. Furthermore, a specific association of in vitro translated pUL89 with the carboxyl-terminal half of GST-UL56C was detected. This interaction was confirmed by co-immunoprecipitations of infected cells. Our results clearly demonstrated that (i) both terminase subunits interact with each other and (ii) the subunit pUL56 has an ATPase activity.
疱疹病毒DNA包装是一个复杂的过程，会将单位长度的基因组包装到预先形成的原衣壳中。这个过程被认为是由两种包装蛋白，即末端酶亚基介导的。在双链DNA噬菌体的情况下，DNA的易位被证明是一个与大末端酶亚基的ATP酶活性相关的能量依赖过程。而对于人类巨细胞病毒，尚不清楚哪种蛋白具有水解ATP的能力。在本研究中，我们将人类巨细胞病毒末端酶亚基pUL89和pUL56的羧基末端一半作为GST融合蛋白进行表达，并通过亲和层析进行纯化。ATP酶分析表明，酶活性仅与pUL56相关。ATP水解的特性表明，酶促反应是一个快速过程，而自发的ATP衰变遵循缓慢的动力学。有趣的是，尽管pUL89没有显示出任何ATP酶活性，但它能够增强与UL56相关的ATP水解。此外，检测到体外翻译的pUL89与GST-UL56C的羧基末端一半有特异性结合。这种相互作用通过感染细胞的共免疫沉淀得到证实。我们的结果清楚地表明：（i）两个末端酶亚基相互作用；（ii）亚基pUL56具有ATP酶活性。
