Kuhmann Shawn E, Pugach Pavel, Kunstman Kevin J, Taylor Joann, Stanfield Robyn L, Snyder Amy, Strizki Julie M, Riley Janice, Baroudy Bahige M, Wilson Ian A, Korber Bette T, Wolinsky Steven M, Moore John P
Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021, USA.
J Virol. 2004 Mar;78(6):2790-807. doi: 10.1128/jvi.78.6.2790-2807.2004.
We have described previously the generation of an escape variant of human immunodeficiency virus type 1 (HIV-1), under the selection pressure of AD101, a small molecule inhibitor that binds the CCR5 coreceptor (A. Trkola, S. E. Kuhmann, J. M. Strizki, E. Maxwell, T. Ketas, T. Morgan, P. Pugach, S. X. L. Wojcik, J. Tagat, A. Palani, S. Shapiro, J. W. Clader, S. McCombie, G. R. Reyes, B. M. Baroudy, and J. P. Moore, Proc. Natl. Acad. Sci. USA 99:395-400, 2002). The escape mutant, CC101.19, continued to use CCR5 for entry, but it was at least 20,000-fold more resistant to AD101 than the parental virus, CC1/85. We have now cloned the env genes from the the parental and escape mutant isolates and made chimeric infectious molecular clones that fully recapitulate the phenotypes of the corresponding isolates. Sequence analysis of the evolution of the escape mutants suggested that the most relevant changes were likely to be in the V3 loop of the gp120 glycoprotein. We therefore made a series of mutant viruses and found that full AD101 resistance was conferred by four amino acid changes in V3. Each change individually caused partial resistance when they were introduced into the V3 loop of a CC1/85 clone, but their impact was dependent on the gp120 context in which they were made. We assume that these amino acid changes alter how the HIV-1 Env complex interacts with CCR5. Perhaps unexpectedly, given the complete dependence of the escape mutant on CCR5 for entry, monomeric gp120 proteins expressed from clones of the fully resistant isolate failed to bind to CCR5 on the surface of L1.2-CCR5 cells under conditions where gp120 proteins from the parental virus and a partially AD101-resistant virus bound strongly. Hence, the full impact of the V3 substitutions may only be apparent at the level of the native Env complex.
我们之前描述过在小分子抑制剂AD101的选择压力下，人免疫缺陷病毒1型（HIV-1）逃逸变体的产生，AD101可结合CCR5共受体（A. 特科拉、S. E. 库曼、J. M. 斯特里茨基、E. 麦克斯韦、T. 凯塔斯、T. 摩根、P. 普加奇、S. X. L. 沃伊奇克、J. 塔加特、A. 帕拉尼、S. 夏皮罗、J. W. 克拉德、S. 麦康比、G. R. 雷耶斯、B. M. 巴鲁迪和J. P. 摩尔，《美国国家科学院院刊》99:395 - 400，2002年）。逃逸突变体CC101.19继续利用CCR5进入细胞，但它对AD101的抗性比亲代病毒CC1/85至少高20000倍。我们现在已从亲代和逃逸突变体分离株中克隆了env基因，并构建了嵌合感染性分子克隆，这些克隆完全重现了相应分离株的表型。对逃逸突变体进化的序列分析表明，最相关的变化可能发生在gp120糖蛋白的V3环中。因此，我们构建了一系列突变病毒，发现V3环中的四个氨基酸变化赋予了对AD101的完全抗性。当将每个变化单独引入CC1/85克隆的V3环时，它们各自都引起了部分抗性，但其影响取决于它们所在的gp120背景。我们推测这些氨基酸变化改变了HIV-1 Env复合物与CCR5相互作用的方式。也许出乎意料的是，鉴于逃逸突变体进入细胞完全依赖CCR5，在亲代病毒和部分抗AD101病毒的gp120蛋白能强烈结合的条件下，从完全抗性分离株的克隆中表达的单体gp120蛋白未能与L1.2 - CCR5细胞表面的CCR5结合。因此，V3替换的全部影响可能仅在天然Env复合物水平才明显。
