AbstractBackgroundEfficient HIV-1 replication depends on interaction of the viral capsid with the host protein cyclophilin A (CypA). CypA, a peptidylprolyl isomerase, binds to an exposed loop in the viral CA protein via the enzyme’s active site. Recent structural analysis of CypA in complex with CA tubes in conjunction with molecular dynamics simulations identified a secondary CA binding site on CypA that allows a bridging interaction with two hexameric subunits of the assembled CA lattice, leading to capsid stabilization (Liu et al. in Nat Commun 7:10714, 2016).ResultsWe performed mutational analysis of residues that have been proposed to mediate CA binding at the secondary binding site on CypA (A25, K27, P29 and K30) and tested the effects of the amino acid substitutions using interaction assays and HIV-1 infection assays in cells. The binding of recombinant CypA to self-assembled CA tubes or native HIV-1 capsids was measured in vitro using a quantitative fluorescence microscopy binding assay revealing that affinity and stoichiometry of CypA to the CA lattice was not affected by the substitutions. To test for functionality of the CypA secondary CA-binding site in HIV-1 infection, mutant CypA proteins were expressed in cells in which endogenous CypA was deleted, and the effects on HIV-1 infection were assayed. In normal HeLa-P4 cells, infection with HIV-1 bearing the A92E substitution in CA is inhibited by endogenous CypA and was inhibited to the same extent by expression of CypA mutants in CypA-null HeLa-P4 cells. Expression of the mutant CypA proteins in CypA-null Jurkat cells restored their permissiveness to infection by wild type HIV-1. ConclusionsThe amino acid changes at A25, K27, P29 and K30 did not affect the affinity of CypA for the CA lattice and did not impair CypA function in infection assays suggesting that these residues are not part of a secondary CA binding site on CypA.

中文翻译：

---

宿主蛋白亲环蛋白 A 中次级 HIV-1 衣壳结合位点的功能分析

摘要背景 HIV-1 的有效复制取决于病毒衣壳与宿主蛋白亲环蛋白 A (CypA) 的相互作用。CypA 是一种肽基脯氨酰异构酶，通过酶的活性位点与病毒 CA 蛋白中的暴露环结合。最近对 CypA 与 CA 管复合物的结构分析结合分子动力学模拟，确定了 CypA 上的次级 CA 结合位点，该位点允许与组装的 CA 晶格的两个六聚体亚基桥接相互作用，从而导致衣壳稳定（Liu 等人，在 Nat 中） Commun 7:10714, 2016）。结果我们对被提议介导 CypA 二级结合位点 CA 结合的残基（A25、K27、P29 和 K30）进行了突变分析，并使用相互作用测定测试了氨基酸取代的影响和细胞中的 HIV-1 感染测定。使用定量荧光显微镜结合测定在体外测量重组 CypA 与自组装 CA 管或天然 HIV-1 衣壳的结合，表明 CypA 对 CA 晶格的亲和力和化学计量不受取代的影响。为了测试 HIV-1 感染中 CypA 二级 CA 结合位点的功能，在删除了内源性 CypA 的细胞中表达突变型 CypA 蛋白，并分析了其对 HIV-1 感染的影响。在正常 HeLa-P4 细胞中，CA 中带有 A92E 取代的 HIV-1 的感染受到内源性 CypA 的抑制，并且在 CypA 无效的 HeLa-P4 细胞中受到 CypA 突变体表达的相同程度的抑制。CypA 缺失的 Jurkat 细胞中突变 CypA 蛋白的表达恢复了它们对野生型 HIV-1 感染的耐受性。结论 A25、K27、P29 和 K30 处的氨基酸变化不会影响 CypA 对 CA 晶格的亲和力，也不会损害感染测定中 CypA 的功能，表明这些残基不是 CypA 上第二 CA 结合位点的一部分。