Characterization of Hepatitis C Virus Core Protein Multimerization and Membrane Envelopment: Revelation of a Cascade of Core-Membrane Interactions
Characterization of Hepatitis C Virus Core Protein Multimerization and Membrane Envelopment: Revelation of a Cascade of Core-Membrane Interactions
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DOI：
10.1128/jvi.00066-09
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发表时间：
2009-10-01
影响因子：
5.4
通讯作者：
Chen, Steve S. -L.
中科院分区：
文献类型：
--
作者：
Ai, Li-Shuang;Lee, Yu-Wen;Chen, Steve S. -L.
丙型肝炎病毒（HCV）核心蛋白成熟和形态发生的分子基础仍然难以捉摸。我们的特点与核心蛋白多聚化和膜相互作用的协同事件。从亚基因组系统表达的核心蛋白的分析表明，位于核心和包膜糖蛋白E1之间的信号序列是核心与内质网（ER）/晚期内体和核心的膜结合的关键，这是通过核心获得对蛋白酶K消化的抗性来判断的。尽管对核心与膜的结合施加抑制作用，但信号肽肽酶（SPP）的特异性抑制剂（Z-LL）（2）-酮不影响核心多聚体复合物的形成，表明寡聚核心复合物的形成在核心附着于膜之前或之后进行。在（Z-LL）（2）-酮存在下检测到含有先天蛋白和加工蛋白的蛋白酶抗性核心复合物，这意味着在膜内裂解后发生核心裂解。阻止信号肽切割或与SPP功能丧失D219 A突变体共表达的核心突变降低了核心的表达，表明SPP介导的切割是核心表达所需的。对结构域I缺失的核心突变体的分析表明，该结构域含有对核心突变至关重要的序列。Huh 7细胞中感染性JFH 1和Jc 1 RNA表达的核心蛋白也组装成多聚体复合物，与ER/晚期内体膜相关，并被膜包裹。用（Z-LL）（2）-酮处理或与D219 A突变SPP共表达干扰了核心表达和感染性HCV的产生，表明核心表达在HCV形态发生中起关键作用。这些结果提供了机制的见解，在关联的HCV核心蛋白与膜在病毒成熟和形态发生的早期阶段的顺序和协调的过程。
The molecular basis underlying hepatitis C virus (HCV) core protein maturation and morphogenesis remains elusive. We characterized the concerted events associated with core protein multimerization and interaction with membranes. Analyses of core proteins expressed from a subgenomic system showed that the signal sequence located between the core and envelope glycoprotein E1 is critical for core association with endoplasmic reticula (ER)/late endosomes and the core's envelopment by membranes, which was judged by the core's acquisition of resistance to proteinase K digestion. Despite exerting an inhibitory effect on the core's association with membranes, (Z-LL)(2)-ketone, a specific inhibitor of signal peptide peptidase (SPP), did not affect core multimeric complex formation, suggesting that oligomeric core complex formation proceeds prior to or upon core attachment to membranes. Protease-resistant core complexes that contained both innate and processed proteins were detected in the presence of (Z-LL)(2)-ketone, implying that core envelopment occurs after intramembrane cleavage. Mutations of the core that prevent signal peptide cleavage or coexpression with an SPP loss-of-function D219A mutant decreased the core's envelopment, demonstrating that SPP-mediated cleavage is required for core envelopment. Analyses of core mutants with a deletion in domain I revealed that this domain contains sequences crucial for core envelopment. The core proteins expressed by infectious JFH1 and Jc1 RNAs in Huh7 cells also assembled into a multimeric complex, associated with ER/late-endosomal membranes, and were enveloped by membranes. Treatment with (Z-LL)(2)-ketone or coexpression with D219A mutant SPP interfered with both core envelopment and infectious HCV production, indicating a critical role of core envelopment in HCV morphogenesis. The results provide mechanistic insights into the sequential and coordinated processes during the association of the HCV core protein with membranes in the early phase of virus maturation and morphogenesis.