Klinikum der Johann Wolfgang Goethe-Universität, Medizinische Klinik, Theodor-Stern-Kai, Frankfurt am Main, Germany.
J Viral Hepat. 2012 Nov;19(11):775-83. doi: 10.1111/j.1365-2893.2012.01605.x. Epub 2012 Apr 1.
The hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is assumed to function as a membrane anchor and protein hub for the viral replication complex. The aim of the current work was to modulate HCV replication efficacy in the subgenomic Con1 replicon by mutations of specific sites within the aminoterminal-located basic leucine zipper (bZIP), a candidate motif for protein-protein interactions involving NS4B. Mutational sites and amino acid substitutes were determined by in-silico sequence analyses of the NS4B-bZIP motif in 357 isolates of HCV genotype 1b from the euHCVdB and LosAlamos database and consecutive analysis of conserved physico-chemical properties at bZIP specific positions. Mutants with predicted minor, medium or major reduction of replication efficacy were tested in the pFKI389neo/NS3-3'/ET plasmid replicon model. Four sites (L25, T29, V39 and W43) of crucial importance for bZIP-mediated protein interaction with predicted apolarity of respective amino acid positions were selected for mutational studies. Substitutes with physico-chemical properties matching the predicted requirements either well (T29A), moderately (L25W, V39W), or insufficiently (T29E, W43E) were associated with slightly improved, moderate and marked decreased replication efficacy, respectively. Spontaneous (T29G) and adaptive (A28G, E40G) mutations occurred in the T29E mutation isolate only and were associated with marked reduction of replication efficacy. The bZIP motif region of NS4B is crucial for RNA replication in the subgenomic Con1 replicon system. RNA replication efficacy can be modulated by site-directed mutagenesis at specific bZIP functional sites. New adaptive amino acid mutations were identified within the HCV NS4B protein.
丙型肝炎病毒（HCV）非结构蛋白 4B（NS4B）被认为是病毒复制复合物的膜锚定蛋白和蛋白枢纽。本研究旨在通过突变位于氨基末端碱性亮氨酸拉链（bZIP）内的特定位点来调节亚基因组 Con1 复制子中的 HCV 复制效率，bZIP 是一种涉及 NS4B 的蛋白-蛋白相互作用的候选基序。通过对 euHCVdB 和 LosAlamos 数据库中 357 株 1b 型 HCV 的 NS4B-bZIP 基序进行计算机序列分析，并对 bZIP 特定位置的保守理化性质进行连续分析，确定了突变位点和氨基酸替代物。用预测复制效率有轻微、中度或显著降低的突变体在 pFKI389neo/NS3-3'/ET 质粒复制子模型中进行测试。选择四个对 bZIP 介导的蛋白相互作用至关重要的位点（L25、T29、V39 和 W43）进行突变研究，这些位点的氨基酸位置具有预测的非极性。具有与预测要求匹配的理化性质的替代物（T29A）、中等程度（L25W、V39W）或不足（T29E、W43E）分别与略微提高、中度和显著降低的复制效率相关。自发（T29G）和适应性（A28G、E40G）突变仅发生在 T29E 突变株中，与复制效率显著降低相关。NS4B 的 bZIP 基序区域对于亚基因组 Con1 复制子系统中的 RNA 复制至关重要。可以通过在特定 bZIP 功能位点进行定点诱变来调节 RNA 复制效率。在 HCV NS4B 蛋白中鉴定到新的适应性氨基酸突变。
