Digard P, Williams K P, Hensley P, Brooks I S, Dahl C E, Coen D M
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1456-60. doi: 10.1073/pnas.92.5.1456.
The herpes simplex virus DNA polymerase consists of two subunits--a catalytic subunit and an accessory subunit, UL42, that increases processivity. Mutations affecting the extreme C terminus of the catalytic subunit specifically disrupt subunit interactions and ablate virus replication, suggesting that new antiviral drugs could be rationally designed to interfere with polymerase heterodimerization. To aid design, we performed circular dichroism (CD) spectroscopy and analytical ultracentrifugation studies, which revealed that a 36-residue peptide corresponding to the C terminus of the catalytic subunit folds into a monomeric structure with partial alpha-helical character. CD studies of shorter peptides were consistent with a model where two separate regions of alpha-helix interact to form a hairpin-like structure. The 36-residue peptide and a shorter peptide corresponding to the C-terminal 18 residues blocked UL42-dependent long-chain DNA synthesis at concentrations that had no effect on synthesis by the catalytic subunit alone or by calf thymus DNA polymerase delta and its processivity factor. These peptides, therefore, represent a class of specific inhibitors of herpes simplex virus DNA polymerase that act by blocking accessory-subunit-dependent synthesis. These peptides or their structures may form the basis for the synthesis of clinically effective drugs.
单纯疱疹病毒DNA聚合酶由两个亚基组成——一个催化亚基和一个辅助亚基UL42，后者可提高持续合成能力。影响催化亚基极端C末端的突变会特异性破坏亚基间的相互作用并消除病毒复制，这表明可以合理设计新的抗病毒药物来干扰聚合酶异源二聚化。为辅助设计，我们进行了圆二色性（CD）光谱分析和分析型超速离心研究，结果显示，一段与催化亚基C末端对应的36个残基的肽折叠成具有部分α-螺旋特征的单体结构。较短肽段的CD研究与一个模型相符，即α-螺旋的两个独立区域相互作用形成类似发夹的结构。该36个残基的肽段以及一个对应C末端18个残基的较短肽段，在一定浓度下可阻断UL42依赖的长链DNA合成，而该浓度对单独的催化亚基或小牛胸腺DNA聚合酶δ及其持续合成因子的合成没有影响。因此，这些肽段代表了一类单纯疱疹病毒DNA聚合酶的特异性抑制剂，其作用方式是阻断依赖辅助亚基的合成。这些肽段或其结构可能构成临床有效药物合成的基础。
