Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
J Virol. 2012 Jun;86(12):6825-34. doi: 10.1128/JVI.00374-12. Epub 2012 Apr 4.
Herpes simplex virus 1 (HSV-1) ICP8 is a single-stranded DNA-binding protein that is necessary for viral DNA replication and exhibits recombinase activity in vitro. Alignment of the HSV-1 ICP8 amino acid sequence with ICP8 homologs from other herpesviruses revealed conserved aspartic acid (D) and glutamic acid (E) residues. Amino acid residue D1087 was conserved in every ICP8 homolog analyzed, indicating that it is likely critical for ICP8 function. We took a genetic approach to investigate the functions of the conserved ICP8 D and E residues in HSV-1 replication. The E1086A D1087A mutant form of ICP8 failed to support the replication of an ICP8 mutant virus in a complementation assay. E1086A D1087A mutant ICP8 bound DNA, albeit with reduced affinity, demonstrating that the protein is not globally misfolded. This mutant form of ICP8 was also recognized by a conformation-specific antibody, further indicating that its overall structure was intact. A recombinant virus expressing E1086A D1087A mutant ICP8 was defective in viral replication, viral DNA synthesis, and late gene expression in Vero cells. A class of enzymes called DDE recombinases utilize conserved D and E residues to coordinate divalent metal cations in their active sites. We investigated whether the conserved D and E residues in ICP8 were also required for binding metal cations and found that the E1086A D1087A mutant form of ICP8 exhibited altered divalent metal binding in an in vitro iron-induced cleavage assay. These results identify a novel divalent metal cation-binding site in ICP8 that is required for ICP8 functions during viral replication.
单纯疱疹病毒 1（HSV-1）ICP8 是一种单链 DNA 结合蛋白，它是病毒 DNA 复制所必需的，并在体外表现出重组酶活性。将 HSV-1 ICP8 的氨基酸序列与其他疱疹病毒的 ICP8 同源物进行比对，揭示了保守的天冬氨酸（D）和谷氨酸（E）残基。在分析的每个 ICP8 同源物中都保守了氨基酸残基 D1087，表明它可能对 ICP8 功能至关重要。我们采取遗传方法研究了 HSV-1 复制中保守的 ICP8 D 和 E 残基的功能。在互补测定中，ICP8 的 E1086A D1087A 突变形式未能支持 ICP8 突变病毒的复制。E1086A D1087A 突变 ICP8 结合 DNA，尽管亲和力降低，表明该蛋白没有全局错误折叠。这种突变形式的 ICP8 也被一种构象特异性抗体识别，进一步表明其整体结构完整。表达 E1086A D1087A 突变 ICP8 的重组病毒在 Vero 细胞中复制、病毒 DNA 合成和晚期基因表达均存在缺陷。一类称为 DDE 重组酶的酶利用保守的 D 和 E 残基在其活性位点中协调二价金属阳离子。我们研究了 ICP8 中的保守 D 和 E 残基是否也需要结合金属阳离子，并发现 ICP8 的 E1086A D1087A 突变形式在体外铁诱导切割测定中表现出改变的二价金属结合。这些结果确定了 ICP8 中的一个新的二价金属阳离子结合位点，该位点在病毒复制期间 ICP8 的功能中是必需的。
