Generation of infectious feline immunodeficiency virus (FIV) encoding FIV/human immunodeficiency virus chimeric protease.
Generation of infectious feline immunodeficiency virus (FIV) encoding FIV/human immunodeficiency virus chimeric protease.
复制标题
编码 FIV/人类免疫缺陷病毒嵌合蛋白酶的传染性猫免疫缺陷病毒 (FIV) 的产生。
DOI：
10.1128/jvi.00294-10
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发表时间：
2010
影响因子：
5.4
通讯作者：
Elder,JohnH
中科院分区：
文献类型：
--
作者：
Lin,Ying-Chuan;Torbett,BruceE;Elder,JohnH
猫免疫缺陷病毒（FIV）和人类免疫缺陷病毒1型（HIV-1）蛋白酶（PRs）只有23%的氨基酸相同，具有不同的特异性，但具有非常相似的三维结构。为了研究HIV特异性的分子基础，制备了在FIV PR中结构等效位置取代HIV残基的嵌合PR。先前的体外分析表明，这种替换极大地改变了突变pr的抑制剂特异性，但改变了Gag切割的速率和特异性，从而使嵌合fiv不具有传染性。将编码I37V、N55M、M56I、V59I、L97T、I98P、Q99V和P100N突变组合的嵌合PR克隆到FIV Gag-Pol中，选择最接近野生型FIV PR产生的时间切割模式，同时保持hiv样抑制剂特异性的构建体。两个突变M56I和L97T不耐受改变，导致NC-p2的低效率切割。然而，选择了6个替换的突变体PR (I37V、N55M、V59I、I98P、Q99V和P100N)，并将其置于全长FIV-34TF10的环境中。这种病毒被称为YCL6，在体内具有低水平的传染性，传代后，出现了具有较高生长速度的后代。六个突变之一I98P位置的残基在传递到P98H或P98S时进一步突变。这两种PR对HIV-1 PR抑制剂洛匹那韦（lopinavir， LPV）和达那韦（darunavir， DRV）以及基础广泛的抑制剂TL-3都敏感，50%的抑制浓度（IC50）为30至40 nM，与使用突变型fiv获得的体内结果一致。嵌合体提供了一种传染性系统，用于筛选具有广泛基础的PR抑制剂潜力的化合物，确定决定特异性的结构参数，并研究耐药发展途径。
Feline immunodeficiency virus (FIV) and human immunodeficiency virus type 1 (HIV-1) proteases (PRs) share only 23% amino acid identity and exhibit distinct specificities yet have very similar 3-dimensional structures. Chimeric PRs in which HIV residues were substituted in structurally equivalent positions in FIV PR were prepared in order to study the molecular basis of PR specificity. Previousin vitroanalyses showed that such substitutions dramatically altered the inhibitor specificity of mutant PRs but changed the rate and specificity of Gag cleavage so that chimeric FIVs were not infectious. Chimeric PRs encoding combinations of the I37V, N55M, M56I, V59I, L97T, I98P, Q99V, and P100N mutations were cloned into FIV Gag-Pol, and those constructs that best approximated the temporal cleavage pattern generated by wild-type FIV PR, while maintaining HIV-like inhibitor specificity, were selected. Two mutations, M56I and L97T, were intolerant to change and caused inefficient cleavage at NC-p2. However, a mutant PR with six substitutions (I37V, N55M, V59I, I98P, Q99V, and P100N) was selected and placed in the context of full-length FIV-34TF10. This virus, termed YCL6, had low-level infectivityex vivo, and after passage, progeny that exhibited a higher growth rate emerged. The residue at the position of one of the six mutations, I98P, further mutated on passage to either P98H or P98S. Both PRs were sensitive to the HIV-1 PR inhibitors lopinavir (LPV) and darunavir (DRV), as well as to the broad-based inhibitor TL-3, with 50% inhibitory concentrations (IC50) of 30 to 40 nM, consistent withex vivoresults obtained using mutant FIVs. The chimeras offer an infectivity system with which to screen compounds for potential as broad-based PR inhibitors, define structural parameters that dictate specificity, and investigate pathways for drug resistance development.