Analysis of in vitro activities of herpes simplex virus type 1 UL42 mutant proteins: correlation with in vivo function.
Analysis of in vitro activities of herpes simplex virus type 1 UL42 mutant proteins: correlation with in vivo function.
复制标题
1 型单纯疱疹病毒 UL42 突变蛋白的体外活性分析：与体内功能的相关性。
DOI：
10.1006/viro.2000.0506
复制
发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Parris,DS
中科院分区：
文献类型：
--
作者：
Thornton,KE;Chaudhuri,M;Monahan,SJ;Grinstead,LA;Parris,DS
单纯疱疹病毒1型的DNA聚合酶催化亚基UL30及其辅助因子UL42蛋白对病毒的复制都是必不可少的。由于UL42和Poll之间的稳定相互作用使Poll完全进行复制DNA合成，因此破坏这种相互作用是开发新型抗病毒化合物的一个潜在目标。为了更好地比较UL42蛋白突变对其已知功能的影响，将突变表达为谷胱甘肽-S-转移酶融合蛋白和用于亲和层析的融合蛋白。在这份报告中，我们展示了突变型UL42融合蛋白在体外结合Poll和刺激Pol1活性的能力与非融合突变型蛋白在病毒复制中的能力之间的关系。使用GST融合蛋白的POL刺激试验被发现比使用连接到固体基质的融合蛋白的POL结合试验更准确和敏感地衡量UL42蛋白在体外发挥作用的能力。我们还发现，纯化的GST融合蛋白在体外刺激Poll活性的能力与全长非融合UL42突变基因支持感染细胞DNA复制的能力之间存在很好的相关性。我们的结果表明，两个不连续的氨基酸延伸，从137到142和从274到282，是UL42在体内和体外发挥作用所必需的。虽然突变体d241-261在体外表现出接近野生型的刺激pol活性的能力，但它不能补充UL42缺失突变病毒的复制。UL42蛋白在241-261内或接近241-261的区域可以用来连接蛋白质的N-末端和C-末端部分中被认为是交错的必要区域。据推测，铰链区长度的缩短可能会改变UL42和/或其与Poll的复合体与感染细胞内DNA复制体中存在的一个或多个其他蛋白质发挥作用的能力。
The DNA polymerase (pol) catalytic subunit of herpes simplex virus type 1, encoded by UL30, and its accessory factor, UL42 protein, are both essential for the replication of the virus. Because the stable interaction between UL42 and pol renders the pol fully processive for replicative DNA synthesis, disruption of this interaction represents a potential goal in the development of novel antiviral compounds. To better compare the effects of mutations in UL42 protein on its known in vitro functions, mutations were expressed as glutathione-S-transferase (GST)-fusions and the fusion proteins used in affinity chromatography. In this report, we demonstrate the relationship between the abilities of mutant UL42 fusion proteins to bind pol and to stimulate pol activity in vitro, and the abilities of nonfusion mutant proteins to function in viral replication. The pol stimulation assay using GST fusion proteins was found to be a more accurate and sensitive measure of the ability of the UL42 protein to function in vitro than the pol binding assay using the fusion proteins linked to a solid matrix. We also found an excellent correlation between the ability of purified GST fusion proteins to stimulate pol activity in vitro and the ability of full-length nonfusion UL42 mutant genes to support DNA replication in infected cells. Our results demonstrate that two noncontiguous stretches of amino acids, from 137 to 142 and from 274 to 282, are essential for UL42 function in vivo and in vitro. Although mutant d241–261 exhibited close to wild-type abilities to stimulate pol activity in vitro, it was not capable of complementing the replication of a UL42 null mutant virus. The region of UL42 protein within or close to 241–261 may serve to hinge the essential regions within the N- and C-terminal portions of the protein which are thought to interdigitate. It is hypothesized that reduction in the length of the hinge region could alter the ability of UL42, and/or its complex with pol, to function with one or more of the other proteins present in the DNA replisome within infected cells.