A conserved NS3 surface patch orchestrates NS2 protease stimulation, NS5A hyperphosphorylation and HCV genome replication.
A conserved NS3 surface patch orchestrates NS2 protease stimulation, NS5A hyperphosphorylation and HCV genome replication.
复制标题
DOI：
10.1371/journal.ppat.1004736
复制
发表时间：
2015-03
期刊：
影响因子：
6.7
通讯作者：
Tautz N
中科院分区：
文献类型：
--
作者：
Isken O;Langerwisch U;Jirasko V;Rehders D;Redecke L;Ramanathan H;Lindenbach BD;Bartenschlager R;Tautz N
丙型肝炎病毒（HCV）感染是世界范围内肝脏疾病的主要原因。HCV RNA基因组被翻译成单个多蛋白。非结构（NS）多蛋白区域中的大多数切割位点由NS 3/NS 4A丝氨酸蛋白酶加工。重要的NS 2-NS 3切割由NS 2自身蛋白酶催化。为了在NS 2/NS 3位点有效加工，NS 2半胱氨酸蛋白酶依赖于NS 3丝氨酸蛋白酶结构域。尽管它对病毒生命周期的重要性，但NS 3激活NS 2自身蛋白酶的分子细节知之甚少。在这里，我们报告了一个保守的疏水性NS 3表面补丁，是必不可少的NS 2蛋白酶激活的鉴定。该表面区域内的一个残基对于RNA复制和NS 5A过度磷酸化也是至关重要的，已知这两个过程依赖于功能性复制酶组装。NS 3表面补丁的这种双重功能促使我们重新研究NS 2-NS 3切割对NS 5A过度磷酸化的影响。有趣的是，NS 2-NS 3切割原来是NS 5A过度磷酸化的先决条件，表明这种切割必须在复制酶组装之前发生。基于我们的数据，我们提出了一个连续级联的分子事件：在未切割的NS 2-NS 3，疏水性NS 3表面补丁促进NS 2蛋白酶刺激，在NS 2-NS 3切割，这个表面区域成为可用于功能性复制酶组装。该模型解释了为什么有效的NS 2 -3切割对于HCV RNA复制至关重要。根据我们的模型，NS 3上的疏水性表面补丁代表了一个关键参与HCV复制酶组装时间协调的模块。丙型肝炎病毒（HCV）复制其基因组与细胞膜密切相关，细胞膜作为多亚基复制复合物的组装位点。复制复合物成熟的过程必须适当控制，以防止这些复合物的非功能性成熟/组装。在此过程中，病毒多蛋白加工的时间调节通常起关键作用，如HCV基因组复制对NS 2-NS 3切割的严格要求所例示。我们在这里证明，一个保守的疏水NS 3表面补丁激活NS 2蛋白酶，刺激NS 2-NS 3切割。通过剖析这些NS 3表面残基在病毒RNA复制中的作用，我们发现其中一个NS 3残基也是HCV基因组复制的关键决定因素，通过负调节NS 5A过度磷酸化。令人惊讶的是，进一步的实验揭示了NS 2-NS 3切割是NS 5A过度磷酸化的先决条件。为了满足逐渐组装成功能性复制复合物的要求，发生了有序的分子事件级联：在未切割的NS 2-NS 3中，疏水性NS 3表面补丁促进NS 2蛋白酶刺激;在NS 2-NS 3切割后，该表面区域可用于功能性复制酶组装。因此，游离NS 3上的疏水表面补丁可以促进NS 5A过度磷酸化，作为功能性复制酶组装的指示。
Hepatitis C virus (HCV) infection is a leading cause of liver disease worldwide. The HCV RNA genome is translated into a single polyprotein. Most of the cleavage sites in the non-structural (NS) polyprotein region are processed by the NS3/NS4A serine protease. The vital NS2-NS3 cleavage is catalyzed by the NS2 autoprotease. For efficient processing at the NS2/NS3 site, the NS2 cysteine protease depends on the NS3 serine protease domain. Despite its importance for the viral life cycle, the molecular details of the NS2 autoprotease activation by NS3 are poorly understood. Here, we report the identification of a conserved hydrophobic NS3 surface patch that is essential for NS2 protease activation. One residue within this surface region is also critical for RNA replication and NS5A hyperphosphorylation, two processes known to depend on functional replicase assembly. This dual function of the NS3 surface patch prompted us to reinvestigate the impact of the NS2-NS3 cleavage on NS5A hyperphosphorylation. Interestingly, NS2-NS3 cleavage turned out to be a prerequisite for NS5A hyperphosphorylation, indicating that this cleavage has to occur prior to replicase assembly. Based on our data, we propose a sequential cascade of molecular events: in uncleaved NS2-NS3, the hydrophobic NS3 surface patch promotes NS2 protease stimulation; upon NS2-NS3 cleavage, this surface region becomes available for functional replicase assembly. This model explains why efficient NS2-3 cleavage is pivotal for HCV RNA replication. According to our model, the hydrophobic surface patch on NS3 represents a module critically involved in the temporal coordination of HCV replicase assembly. Hepatitis C virus (HCV) replicates its genome in close association to cellular membranes which serve as assembly site of multi-subunit replication complexes. The process of replication complex maturation must be properly controlled to prevent the non-functional maturation/assembly of these complexes. In this process, the temporal regulation of viral polyprotein processing often plays a pivotal role as exemplified by the strict requirement for NS2-NS3 cleavage for HCV genome replication. We demonstrate here that a conserved hydrophobic NS3 surface patch activates the NS2 protease to stimulate NS2-NS3 cleavage. By dissecting the role of these NS3 surface residues in viral RNA replication, we show that one of these NS3 residues is also a critical determinant for HCV genome replication by negatively regulating NS5A hyperphosphorylation. Surprisingly, further experiments revealed that the NS2-NS3 cleavage is a prerequisite for NS5A hyperphosphorylation. To fulfill the requirements for gradual assembly into functional replication complexes, an ordered cascade of molecular events takes place: in uncleaved NS2-NS3, the hydrophobic NS3 surface patch promotes NS2 protease stimulation; upon NS2-NS3 cleavage, this surface region becomes available for functional replicase assembly. As a consequence, the hydrophobic surface patch on free NS3 can promote NS5A hyperphosphorylation as an indication of functional replicase assembly.
登录
查看更多内容
影响因子：
5.4
作者：
de la Fuente, Cynthia;Goodman, Zachary;Rice, Charles M.
通讯作者：
Rice, Charles M.
影响因子：
5.4
作者：
Appel, N;Pietschmann, T;Bartenschlager, R
通讯作者：
Bartenschlager, R
影响因子：
5.4
作者：
FAILLA, C;TOMEI, L;DEFRANCESCO, R
通讯作者：
DEFRANCESCO, R
影响因子：
5.4
作者：
Kolykhalov, AA;Mihalik, K;Rice, CM
通讯作者：
Rice, CM
影响因子：
5.4
作者：
Jones, Christopher T.;Murray, Catherine L.;Rice, Charles M.
通讯作者：
Rice, Charles M.