Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics.
Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics.
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DOI：
10.1371/journal.ppat.1000546
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发表时间：
2009-08
期刊：
影响因子：
6.7
通讯作者：
Bartenschlager R
中科院分区：
文献类型：
--
作者：
Kaul A;Stauffer S;Berger C;Pertel T;Schmitt J;Kallis S;Zayas M;Lohmann V;Luban J;Bartenschlager R
病毒是专性的细胞内寄生虫，因此它们的复制完全取决于宿主细胞因素。对于丙型肝炎病毒(HCV)，一种在大多数感染中建立持久性的正链RNA病毒，亲环素被认为在RNA复制中发挥重要作用。在观察到已知的通过直接结合来隔离环孢素的环孢菌素能深刻地阻断培养的人肝癌细胞中的丙型肝炎病毒复制之后，关于病毒RNA复制所需的特定的亲环素(Cyp)和潜在的可能的作用模式，得到了相互矛盾的结果。通过使用一组稳定下调CypA或CypB的细胞系，我们在本工作中证明了CypA缺失可使不同基因类型的丙型肝炎病毒亚基因组复制子的复制减少1000倍，而CypB的下调则没有影响。野生型CypA的过度表达挽救了复制抑制，但缺乏异构酶活性的突变体不能拯救复制抑制。与亚基因组复制子相比，JFH1来源的全长基因组的复制对CypA耗尽甚至更敏感，病毒的产生完全被阻止。这些结果表明，CypA可能以最小复制酶外的另一个病毒因子为靶点，对RNA扩增和组装起作用，推测是非结构蛋白2。通过以剂量依赖的方式选择针对CypA的环孢素类似物Debio-025的抗性，我们发现了两个突变(V2440A和V2440L)，它们靠近非结构蛋白5A和非结构蛋白5B中依赖RNA的RNA聚合酶之间的切割位点，减缓了该位点的切割动力学，减少了对CypA的病毒复制的依赖。在同一切割位点的进一步氨基酸替换加速了加工过程，增加了对CypA的依赖。因此，我们的结果确定了丙型肝炎病毒多蛋白处理和CypA依赖的丙型肝炎病毒复制之间的意外关联。由于遗传信息有限，病毒必须利用宿主细胞来实现高效的病毒后代生产。因此，宿主细胞因子和途径对病毒复制起着重要作用，因此是抗病毒治疗的可能靶点。对于丙型肝炎病毒(HCV)，一种感染肝细胞并导致慢性肝病的RNA病毒，宿主细胞亲环素被证明在复制中发挥重要作用。亲环素是蛋白质折叠的催化剂，对其进行药理抑制可显著抑制丙型肝炎病毒的复制，但亲环素促进病毒复制的潜在机制和亲环素的确切性质尚不清楚。在这项研究中，我们证明了丙型肝炎病毒复制和病毒颗粒组装需要亲环素A(CypA)，环孢素A可以被环孢素类似物Debio-025阻断。我们确定了影响病毒多蛋白蛋白水解性切割的突变，这些突变使丙型肝炎病毒复制不那么依赖CypA，从而导致Debio-025耐药性。对更多突变体的研究揭示了多蛋白裂解动力学与CypA依赖之间的相关性。我们的结果支持一个模型，通过该模型，CypA激活病毒复制酶的方式取决于病毒多蛋白被切割的动力学。
Viruses are obligate intracellular parasites and therefore their replication completely depends on host cell factors. In case of the hepatitis C virus (HCV), a positive-strand RNA virus that in the majority of infections establishes persistence, cyclophilins are considered to play an important role in RNA replication. Subsequent to the observation that cyclosporines, known to sequester cyclophilins by direct binding, profoundly block HCV replication in cultured human hepatoma cells, conflicting results were obtained as to the particular cyclophilin (Cyp) required for viral RNA replication and the underlying possible mode of action. By using a set of cell lines with stable knock-down of CypA or CypB, we demonstrate in the present work that replication of subgenomic HCV replicons of different genotypes is reduced by CypA depletion up to 1,000-fold whereas knock-down of CypB had no effect. Inhibition of replication was rescued by over-expression of wild type CypA, but not by a mutant lacking isomerase activity. Replication of JFH1-derived full length genomes was even more sensitive to CypA depletion as compared to subgenomic replicons and virus production was completely blocked. These results argue that CypA may target an additional viral factor outside of the minimal replicase contributing to RNA amplification and assembly, presumably nonstructural protein 2. By selecting for resistance against the cyclosporine analogue DEBIO-025 that targets CypA in a dose-dependent manner, we identified two mutations (V2440A and V2440L) close to the cleavage site between nonstructural protein 5A and the RNA-dependent RNA polymerase in nonstructural protein 5B that slow down cleavage kinetics at this site and reduce CypA dependence of viral replication. Further amino acid substitutions at the same cleavage site accelerating processing increase CypA dependence. Our results thus identify an unexpected correlation between HCV polyprotein processing and CypA dependence of HCV replication. Owing to limited genetic information, viruses have to exploit host cells to achieve efficient production of virus progeny. Host cell factors and pathways therefore play an important role for virus replication and thus represent a possible target for antiviral therapy. In case of the hepatitis C virus (HCV), an RNA virus infecting liver cells and causing chronic liver disease, host cell cyclophilins were shown to play an important role in replication. Pharmacological inhibition of cyclophilins, which are catalysts of protein folding, causes profound inhibition of HCV replication, but neither the underlying mechanism by which cyclophilins contribute to viral replication, nor the exact nature of the cyclophilin are known. In this study we demonstrate that HCV replication and presumably also virus particle assembly requires cyclophilin A (CypA), which can be blocked by the cyclosporine analogue DEBIO-025. We identify mutations affecting proteolytic cleavage of the viral polyprotein that render HCV replication less dependent on CypA and thus cause DEBIO-025 resistance. Studies with additional mutants reveal a correlation between polyprotein cleavage kinetics and CypA dependence. Our results support a model by which CypA activates the viral replicase in a manner that depends on the kinetics with which the viral polyprotein is cleaved.
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影响因子：
5.8
作者：
Heck, Julie A.;Meng, Xiao;Frick, David N.
通讯作者：
Frick, David N.
EUHCVDB：欧洲丙型肝炎病毒数据库。
影响因子：
14.9
作者：
Combet, Christophe;Garnier, Nicolas;Charavay, Celine;Grando, Delphine;Crisan, Daniel;Lopez, Julien;Dehne-Garcia, Alexandre;Geourjon, Christophe;Bettler, Emmanuel;Hulo, Chantal;Le Mercier, Philippe;Bartenschlager, Ralf;Diepolder, Helmut;Moradpour, Darius;Pawlotsky, Jean-Michel;Rice, Charles M;Trepo, Christian;Penin, Francois;Deleage, Gilbert
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Deleage, Gilbert
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：
Koutsoudakis, George;Kaul, Artur;Bartenschlager, Ralf
通讯作者：
Bartenschlager, Ralf
影响因子：
13.5
作者：
Flisiak, Robert;Horban, Andrzej;Scalfaro, Pietro
通讯作者：
Scalfaro, Pietro