Division of Infectious Diseases, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
J Int AIDS Soc. 2013 Sep 18;16(1):18723. doi: 10.7448/IAS.16.1.18723.
A dual split reporter protein system (DSP), recombining Renilla luciferase (RL) and green fluorescent protein (GFP) split into two different constructs (DSP1-7 and DSP8-11), was adapted to create a novel rapid phenotypic tropism assay (PTA) for HIV-1 infection (DSP-Pheno).
DSP1-7 was stably expressed in the glioma-derived NP-2 cell lines, which expressed CD4/CXCR4 (N4X4) or CD4/CCR5 (N4R5), respectively. An expression vector with DSP8-11 (pRE11) was constructed. The HIV-1 envelope genes were subcloned in pRE11 (pRE11-env) and transfected into 293FT cells. Transfected 293FT cells were incubated with the indicator cell lines independently. In developing the assay, we selected the DSP1-7-positive clones that showed the highest GFP activity after complementation with DSP8-11. These cell lines, designated N4R5-DSP1-7, N4X4-DSP1-7 were used for subsequent assays.
The env gene from the reference strains (BaL for R5 virus, NL4-3 for X4 virus, SF2 for dual tropic virus) subcloned in pRE11 and tested, was concordant with the expected co-receptor usage. Assay results were available in two ways (RL or GFP). The assay sensitivity by RL activity was comparable with those of the published phenotypic assays using pseudovirus. The shortest turnaround time was 5 days after obtaining the patient's plasma. All clinical samples gave positive RL signals on R5 indicator cells in the fusion assay. Median RLU value of the low CD4 group was significantly higher on X4 indicator cells and suggested the presence of more dual or X4 tropic viruses in this group of patients. Comparison of representative samples with Geno2Pheno [co-receptor] assay was concordant.
A new cell-fusion-based, high-throughput PTA for HIV-1, which would be suitable for in-house studies, was developed. Equipped with two-way reporter system, RL and GFP, DSP-Pheno is a sensitive test with short turnaround time. Although maintenance of cell lines and laboratory equipment is necessary, it provides a safe assay system without infectious viruses. With further validation against other conventional analyses, DSP-Pheno may prove to be a useful laboratory tool. The assay may be useful especially for the research on non-B subtype HIV-1 whose co-receptor usage has not been studied much.
我们对双分裂报告蛋白系统（DSP）进行了改造，将海肾荧光素酶（RL）和绿色荧光蛋白（GFP）重组为两个不同的构建体（DSP1-7 和 DSP8-11），从而创建了一种用于 HIV-1 感染的新型快速表型嗜性测定法（PTA）（DSP-Pheno）。
DSP1-7 在表达 CD4/CXCR4（N4X4）或 CD4/CCR5（N4R5）的神经胶质瘤衍生 NP-2 细胞系中稳定表达。构建了带有 DSP8-11 的表达载体（pRE11）。将 HIV-1 包膜基因亚克隆到 pRE11 中（pRE11-env），并转染 293FT 细胞。将转染的 293FT 细胞分别与指示细胞系孵育。在开发测定法时，我们选择了在与 DSP8-11 互补后显示出最高 GFP 活性的 DSP1-7 阳性克隆。将这些细胞系命名为 N4R5-DSP1-7 和 N4X4-DSP1-7，用于后续的测定。
用 pRE11 测试的参考株（R5 病毒的 BaL、X4 病毒的 NL4-3、双嗜性病毒的 SF2）的 env 基因与预期的共受体使用情况一致。测定结果有两种方式（RL 或 GFP）。通过 RL 活性获得的测定灵敏度与使用假病毒的已发表表型测定法相当。获得患者血浆后的最短周转时间为 5 天。融合测定中，所有临床样本在 R5 指示细胞上均产生阳性 RL 信号。低 CD4 组的中位 RLU 值在 X4 指示细胞上明显更高，表明该组患者中存在更多的双嗜性或 X4 嗜性病毒。与 Geno2Pheno [共受体] 测定的代表性样本比较结果一致。
我们开发了一种新的基于细胞融合的 HIV-1 高通量 PTA，适用于内部研究。该测定法配备了 RL 和 GFP 两种报告系统，是一种具有高灵敏度和短周转时间的检测方法。尽管需要维持细胞系和实验室设备，但它提供了一个安全的无传染性病毒的检测系统。与其他常规分析方法进一步验证后，DSP-Pheno 可能成为一种有用的实验室工具。该测定方法可能特别适用于对尚未研究过共受体使用情况的非 B 亚型 HIV-1 的研究。
