Infectious Diseases Unit, Massachusetts General Hospital, 55 Fruit Street, Jackson 504, Boston, MA 02114, USA.
J Virol Methods. 2010 Oct;169(1):39-46. doi: 10.1016/j.jviromet.2010.06.012. Epub 2010 Jun 25.
A phenotypic assay to determine coreceptor usage of HIV-1 has been developed for rapid testing of clinical samples. The assay is based on the synthesis of viral stock from full-length env amplicons isolated from patient's plasma. Pseudoviral stock is generated rapidly by using an overlapping PCR method to assemble a CMV promoter to env, followed by co-transfection into producer cells with a HIV plasmid (pNL4-3.Luc.R(-)E(-)) containing a non-functional env. The coreceptor used by the viral quasispecies is tested by infection into U87.CD4.CCR5 and U87.CD4.CXCR4 cells. Viral entry is indicated by the expression of the luciferase gene in relative light units (RLU). The use of CXCR4 coreceptor by minor variants is confirmed with sufficient suppression of RLU by a CXCR4 inhibitor. Two statistical tests are employed to confirm viral entry. This assay accurately assigned coreceptor usage of isolates of various subtypes and in the majority of samples of various viral loads. The sensitivity to detect minor species of CXCR4-using env is 1% at higher viral loads and 5% at less than 1,000 copies/ml. This assay provides a sensitive, efficient and relatively low-cost approach suitable for use by research laboratories for assessing HIV-1 coreceptor usage of plasma samples.
已经开发出一种用于快速检测临床样本的 HIV-1 辅助受体使用表型测定法。该测定法基于从患者血浆中分离的全长 env 扩增子合成病毒 stock。通过使用重叠 PCR 方法将 CMV 启动子组装到 env 上来快速生成假病毒 stock，然后将其与含有非功能 env 的 HIV 质粒（pNL4-3.Luc.R(-)E(-)）共转染到产生细胞中。通过感染 U87.CD4.CCR5 和 U87.CD4.CXCR4 细胞来测试病毒准种使用的辅助受体。病毒进入由相对光单位（RLU）中 luciferase 基因的表达来指示。通过 CXCR4 抑制剂对 RLU 的充分抑制来确认次要变体对 CXCR4 辅助受体的使用。使用两种统计检验来确认病毒进入。该测定法准确地分配了各种亚型的分离物和各种病毒载量的大多数样本的辅助受体使用情况。在较高病毒载量下，检测 CXCR4 辅助受体使用的 env 次要物种的灵敏度为 1%，在低于 1,000 拷贝/ml 时为 5%。该测定法提供了一种灵敏、高效且相对低成本的方法，适合研究实验室用于评估血浆样本中的 HIV-1 辅助受体使用情况。
