Van Baelen Kurt, Vandenbroucke Ina, Rondelez Evelien, Van Eygen Veerle, Vermeiren Hans, Stuyver Lieven J
Virco BVBA, Generaal De Wittelaan L11 B4, 2800 Mechelen, Belgium.
J Virol Methods. 2007 Dec;146(1-2):61-73. doi: 10.1016/j.jviromet.2007.06.003. Epub 2007 Jul 19.
Orally bioavailable CXCR4 and CCR5 coreceptor antagonists are being developed for the treatment of HIV-1 infection. A new tropism-testing platform, which offers various options depending on the needs, was established. Each option has specific characteristics in terms of sensitivity, information, throughput and cost. The platform consists of four assays, all based on a one-step RT-PCR of the main part of the HIV envelope glycoprotein gp120 (called 'NH(2)-V4'). Population-based sequencing of gp120's V3 loop is generally cheap and easy to run, and was chosen as the first test in the platform's cascade. Given its drawbacks such as limited sensitivity, additional tests were developed. A sensitive assay using NH(2)-V4 gp120 clonal sequencing and tropism prediction enabled us to demonstrate the quasispecies diversity present in 13 patient samples. For phenotyping, an eGFP-containing HIV backbone deleted for NH(2)-V4 was constructed and used for clonal and population tropism determination. As expected, clonal NH(2)-V4 gp120 phenotyping demonstrated significant correlation between prediction algorithms and phenotype-based classification. The absence of the N-linked glycosylation motif in V3 was associated with CXCR4 usage. Finally, population NH(2)-V4 gp120 phenotypic tropism determination appeared to be a promising tool for the detection of minority species present in the amplified envelope fragments.
口服生物可利用的CXCR4和CCR5共受体拮抗剂正在被开发用于治疗HIV-1感染。建立了一个新的嗜性检测平台，该平台可根据需求提供多种选择。每种选择在灵敏度、信息量、通量和成本方面都有特定的特点。该平台由四种检测方法组成，所有方法均基于HIV包膜糖蛋白gp120主要部分（称为“NH(2)-V4”）的一步RT-PCR。基于群体的gp120 V3环测序通常成本低廉且易于操作，被选作该平台级联检测中的首个检测方法。鉴于其灵敏度有限等缺点，又开发了其他检测方法。一种使用NH(2)-V4 gp120克隆测序和嗜性预测的灵敏检测方法使我们能够证明13份患者样本中存在的准种多样性。为了进行表型分析，构建了一个缺失NH(2)-V4的含eGFP的HIV骨架，并用于克隆和群体嗜性测定。正如预期的那样，克隆的NH(2)-V4 gp120表型分析表明预测算法与基于表型的分类之间存在显著相关性。V3中N-连接糖基化基序的缺失与CXCR4的使用相关。最后，群体NH(2)-V4 gp120表型嗜性测定似乎是检测扩增包膜片段中存在的少数物种的一种有前景的工具。
