A major determinant of cyclophilin dependence and cyclosporine susceptibility of hepatitis C virus identified by a genetic approach.
A major determinant of cyclophilin dependence and cyclosporine susceptibility of hepatitis C virus identified by a genetic approach.
复制标题
DOI：
10.1371/journal.ppat.1001118
复制
发表时间：
2010-09-23
期刊：
影响因子：
6.7
通讯作者：
Tang H
中科院分区：
文献类型：
--
作者：
Yang F;Robotham JM;Grise H;Frausto S;Madan V;Zayas M;Bartenschlager R;Robinson M;Greenstein AE;Nag A;Logan TM;Bienkiewicz E;Tang H
自从全基因组小干扰RNA筛选技术出现以来，大量对病毒感染很重要的细胞辅因子被迅速发现，但许多这些辅因子的病毒靶点和作用机制仍不明确。一种这样的辅因子是亲环素A（CyPA），丙型肝炎病毒（HCV）复制严重依赖于该因子。在这里，我们报告了一个新的遗传选择方案，确定了HCV对CyPA的依赖性和对环孢素A的敏感性的主要病毒决定因素。我们选择的突变体病毒，能够感染CyPA敲低细胞，这是难感染的野生型HCV在细胞培养中产生的。五个独立的选择揭示了位于NS 5A结构域II的富含脯氨酸的区域中的单个二肽基序（D316和Y317）中的相关突变，该结构域II与CyPA结合有关。将突变工程化到野生型HCV中完全重现了CyPA非依赖性和CsA耐药表型，并且CyPA的四种推定脯氨酸底物被定位到DY基序附近。野生型和突变型NS 5A肽的圆二色性分析表明，D316 E/Y317 N突变（DEYN）诱导的构象变化在一个主要的CyPA结合位点。此外，核磁共振实验表明，NS 5A与DEYN突变采用了更广泛的，功能构象在推定的CyPA底物网站域II。最后，在GT 2a以外的其他基因型（GT）中证实了这一主要CsA敏感性决定因素的重要性。这项研究描述了一种新的遗传学方法，以确定细胞辅因子的病毒靶点，并确定了HCV对CsA及其衍生物的易感性的主要调节因子，目前正在临床试验中。细胞辅因子及其作用机制的鉴定是病毒-宿主相互作用研究的一个基本方面。筛选全基因组小干扰RNA文库已成为系统发现病毒生命周期各个方面所必需的细胞辅因子的有效途径。我们和其他人最近已经证明，亲环素A（CyPA）是丙型肝炎病毒（HCV）感染的一个重要的辅因子，并作为一类新的临床抗HCV化合物，环孢素A（CsA）及其衍生物，缺乏免疫抑制功能的直接目标。在这里，我们报告了一种新的遗传筛选方法，可以潜在地应用于额外的细胞辅因子和其他病毒的HCV的依赖CyPA和CsA的易感性的关键调节器的识别。这种方法的有效性，称为辅因子独立突变体（Cofactor-independent mutant，Cofactor-independent）筛选，进一步得到了使用其他HCV基因型的平行CsA选择结果的支持。本文报道了一种新技术，我们发现和表征HCV对CyPA抑制剂敏感性的主要决定因素，目前正在临床试验中进行测试。
Since the advent of genome-wide small interfering RNA screening, large numbers of cellular cofactors important for viral infection have been discovered at a rapid pace, but the viral targets and the mechanism of action for many of these cofactors remain undefined. One such cofactor is cyclophilin A (CyPA), upon which hepatitis C virus (HCV) replication critically depends. Here we report a new genetic selection scheme that identified a major viral determinant of HCV's dependence on CyPA and susceptibility to cyclosporine A. We selected mutant viruses that were able to infect CyPA-knockdown cells which were refractory to infection by wild-type HCV produced in cell culture. Five independent selections revealed related mutations in a single dipeptide motif (D316 and Y317) located in a proline-rich region of NS5A domain II, which has been implicated in CyPA binding. Engineering the mutations into wild-type HCV fully recapitulated the CyPA-independent and CsA-resistant phenotype and four putative proline substrates of CyPA were mapped to the vicinity of the DY motif. Circular dichroism analysis of wild-type and mutant NS5A peptides indicated that the D316E/Y317N mutations (DEYN) induced a conformational change at a major CyPA-binding site. Furthermore, nuclear magnetic resonance experiments suggested that NS5A with DEYN mutations adopts a more extended, functional conformation in the putative CyPA substrate site in domain II. Finally, the importance of this major CsA-sensitivity determinant was confirmed in additional genotypes (GT) other than GT 2a. This study describes a new genetic approach to identifying viral targets of cellular cofactors and identifies a major regulator of HCV's susceptibility to CsA and its derivatives that are currently in clinical trials. Identification of cellular cofactors and their mechanisms of action is a fundamental aspect of virus-host interaction research. Screening of genome-wide small interfering RNA libraries has become an efficient way of systematically discovering cellular cofactors essential for various aspects of viral life cycle. We and others have recently demonstrated that cyclophilin A (CyPA) is an essential cofactor for hepatitis C virus (HCV) infection and serves as the direct target of a new class of clinical anti-HCV compounds, cyclosporine A (CsA) and its derivatives, that are devoid of immunosuppressive function. Here we report the identification of a key regulator of HCV's dependence on CyPA and susceptibility to CsA using a novel genetic screening approach that can potentially be applied to additional cellular cofactors and other viruses. The effectiveness of this approach, termed cofactor-independent mutant (CoFIM) screening, was further supported by results obtained with a parallel CsA-based selection using additional genotypes of HCV. This paper reports a new technology with which we discover and characterize the major determinant of HCV's sensitivity to CyPA inhibitors, which are currently being tested in clinical trials.
登录
查看更多内容
影响因子：
6.7
作者：
Kaul A;Stauffer S;Berger C;Pertel T;Schmitt J;Kallis S;Zayas M;Lohmann V;Luban J;Bartenschlager R
通讯作者：
Bartenschlager R
影响因子：
2.9
作者：
Liang, Yu;Ye, Hong;Yoon, Ho Sup
通讯作者：
Yoon, Ho Sup
影响因子：
5.4
作者：
Benedicto, Ignacio;Molina-Jimenez, Francisca;Majano, Pedro L.
通讯作者：
Majano, Pedro L.
影响因子：
5.7
作者：
KE, HM;MAYROSE, D;WALSH, CT
通讯作者：
WALSH, CT
影响因子：
13.5
作者：
Ciesek, Sandra;Steinmann, Eike;Pietschmann, Thomas
通讯作者：
Pietschmann, Thomas