Lohrengel Sabine, Hermann Felix, Hagmann Isabel, Oberwinkler Heike, Scrivano Laura, Hoffmann Caroline, von Laer Dorothee, Dittmar Matthias T
Abt. Virologie, Hygiene-Institut, Universität Heidelberg, D-69120 Heidelberg, Germany.
J Virol. 2005 Aug;79(16):10237-46. doi: 10.1128/JVI.79.16.10237-10246.2005.
The expression of a membrane-anchored gp41-derived peptide (M87) has been shown to confer protection from infection through human immunodeficiency virus type 1 (HIV-1) (Hildinger et al., J. Virol. 75:3038-3042, 2001). In an effort to characterize the mechanism of action of this membrane-anchored peptide in comparison to the soluble peptide T-20, we selected resistant variants of HIV-1(NL4-3) and HIV-1(BaL) by serial virus passage using PM1 cells stably expressing peptide M87. Sequence analysis of the resistant isolates showed different patterns of selected point mutations in heptad repeat regions 1 and 2 (HR1 and HR2, respectively) for the two viruses analyzed. For HIV-1(NL4-3) a single amino acid change at position 33 in HR1 (L33S) was selected, whereas for HIV-1(BaL) the majority of the sequences obtained showed two amino acid changes, one in HR1 and one in HR2 (I48V/N126K). In both selections the most important contiguous 3-amino-acid sequence, GIV, within HR1, associated with resistance to soluble T-20, was not changed. Site-directed mutagenesis studies confirmed the importance of the characterized point mutations to confer resistance to M87 as well as to soluble T-20 and T-649. Replication capacity and dual-color competition assays revealed that the double mutation I48V/N126K in HIV-1(BaL) results in a strong reduction of viral fitness, whereas the L33S mutation in HIV-1(NL4-3) did enhance viral fitness compared to the respective parental viruses. However, the selected point mutations did not confer resistance to the more recently described optimized membrane-anchored fusion inhibitor M87o (Egelhofer et al., J. Virol. 78:568-575, 2004), strengthening the importance of this novel antiviral concept for gene therapy approaches.
膜锚定的gp41衍生肽（M87）的表达已被证明可提供针对1型人类免疫缺陷病毒（HIV-1）感染的保护作用（Hildinger等人，《病毒学杂志》75:3038 - 3042，2001年）。为了与可溶性肽T - 20相比，表征这种膜锚定肽的作用机制，我们通过使用稳定表达肽M87的PM1细胞进行连续病毒传代，筛选出了HIV-1（NL4-3）和HIV-1（BaL）的抗性变体。对抗性分离株的序列分析显示，对于所分析的两种病毒，在七肽重复区域1和2（分别为HR1和HR2）中选择的点突变模式不同。对于HIV-1（NL4-3），在HR1的第33位选择了单个氨基酸变化（L33S），而对于HIV-1（BaL），获得的大多数序列显示有两个氨基酸变化，一个在HR1，一个在HR2（I48V/N126K）。在这两种筛选中，HR1内与可溶性T - 20抗性相关的最重要的连续3氨基酸序列GIV没有改变。定点诱变研究证实了所表征的点突变对于赋予对M87以及可溶性T - 20和T - 649抗性的重要性。复制能力和双色竞争试验表明，HIV-1（BaL）中的双突变I48V/N126K导致病毒适应性大幅降低，而HIV-1（NL4-3）中的L33S突变与各自的亲本病毒相比确实增强了病毒适应性。然而，所选的点突变并未赋予对最近描述的优化膜锚定融合抑制剂M87o（Egelhofer等人，《病毒学杂志》78:568 - 575，2004年）的抗性，这强化了这种新型抗病毒概念对基因治疗方法的重要性。
