Department of Biological Science, Florida State University, Tallahassee, Florida, USA.
J Virol. 2012 May;86(9):4811-22. doi: 10.1128/JVI.06641-11. Epub 2012 Feb 15.
Cyclophilin A (CyPA) and its peptidyl-prolyl isomerase (PPIase) activity play an essential role in hepatitis C virus (HCV) replication, and mounting evidence indicates that nonstructural protein 5A (NS5A) is the major target of CyPA. However, neither a consensus CyPA-binding motif nor specific proline substrates that regulate CyPA dependence and sensitivity to cyclophilin inhibitors (CPIs) have been defined to date. We systematically characterized all proline residues in NS5A domain II, low-complexity sequence II (LCS-II), and domain III with both biochemical binding and functional replication assays. A tandem cyclophilin-binding site spanning domain II and LCS-II was identified. The first site contains a consensus sequence motif of AØPXW (where Ø is a hydrophobic residue) that is highly conserved in the majority of the genotypes of HCV (six of seven; the remaining genotype has VØPXW). The second tandem site contains a similar motif, and the ØP sequence is again conserved in six of the seven genotypes. Consistent with the similarity of their sequences, peptides representing the two binding motifs competed for CyPA binding in a spot-binding assay and induced similar chemical shifts when bound to the active site of CyPA. The two prolines (P310 and P341 of Japanese fulminant hepatitis 1 [JFH-1]) contained in these motifs, as well as a conserved tryptophan in the spacer region, were required for CyPA binding, HCV replication, and CPI resistance. Together, these data provide a high-resolution mapping of proline residues important for CyPA binding and identify critical amino acids modulating HCV susceptibility to the clinical CPI Alisporivir.
亲环素 A（CyPA）及其肽基脯氨酰顺反异构酶（PPIase）活性在丙型肝炎病毒（HCV）复制中发挥着重要作用，越来越多的证据表明非结构蛋白 5A（NS5A）是 CyPA 的主要靶标。然而，迄今为止，既没有确定共识的 CyPA 结合基序，也没有确定调节 CyPA 依赖性和对亲环素抑制剂（CPIs）敏感性的特定脯氨酸底物。我们系统地研究了 NS5A 结构域 II、低复杂度序列 II（LCS-II）和结构域 III 中所有脯氨酸残基，通过生化结合和功能复制实验进行分析。鉴定出跨越结构域 II 和 LCS-II 的串联 CyPA 结合位点。第一个位点包含一个高度保守的共识序列基序 AØPXW（其中Ø是一个疏水性残基），该基序存在于 HCV 的大多数基因型中（七种中的六种；其余一种为 VØPXW）。第二个串联位点包含一个类似的基序，并且ØP 序列在七种基因型中的六种中也是保守的。与它们序列的相似性一致，代表两个结合基序的肽在斑点结合测定中竞争 CyPA 结合，并在与 CyPA 活性位点结合时诱导相似的化学位移。这两个基序中包含的两个脯氨酸（日本暴发性肝炎 1 型 [JFH-1] 的 P310 和 P341）以及间隔区中的保守色氨酸，对于 CyPA 结合、HCV 复制和 CPI 耐药性是必需的。这些数据共同提供了对亲环素结合重要的脯氨酸残基的高分辨率图谱，并确定了调节 HCV 对临床 CPI Alisporivir 敏感性的关键氨基酸。
