In studies of HIV-1, virus production is normally monitored by either a reverse transcriptase assay or a p24 antigen capture ELISA. However, these assays are costly and time-consuming for routine handling of a large number of HIV-1 samples. For example, sample dilution is always required in the ELISA procedure to determine p24 protein levels because of the very narrow range of detectable concentrations in this assay. Here, we establish a novel HIV-1 production assay system to solve the aforementioned problems by using a recently developed small peptide tag called HiBiT. This peptide is a fragment of NanoLuc luciferase and generates a strong luminescent signal when complemented with the remaining subunit. To employ this technology, we constructed a novel full-length proviral HIV-1 DNA clone and a lentiviral packaging vector in which the HiBiT tag was added to the C terminus of the integrase. Tagging the integrase with the HiBiT sequence did not impede the resultant virus production, infectivity, or susceptibility to an integrase inhibitor. EM revealed normal morphology of the virus particles. Most importantly, by comparing between ELISA and the HiBiT luciferase assay, we successfully obtained an excellent linear correlation between p24 concentrations and HiBiT-based luciferase activity. Overall, we conclude that HiBiT-tagged viruses can replace the parental HIV-1 and lentiviral vectors, which enables us to perform a super-rapid, inexpensive, convenient, simple, and highly accurate quantitative assay for HIV-1/lentivirus production. This system can be widely applied to a variety of virological studies, along with screening for candidates of future antiviral drugs.

中文翻译：

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对带有发光肽标签的 HIV-1 的产生进行超快速定量。

在 HIV-1 的研究中，通常通过逆转录酶测定或 p24 抗原捕获 ELISA 来监测病毒的产生。然而，这些检测对于大量 HIV-1 样本的常规处理来说既昂贵又耗时。例如，在 ELISA 过程中总是需要样品稀释来确定 p24 蛋白水平，因为该测定中可检测的浓度范围非常窄。在这里，我们建立了一个新的 HIV-1 生产分析系统，通过使用最近开发的称为 HiBiT 的小肽标签来解决上述问题。该肽是 NanoLuc 荧光素酶的片段，与剩余的亚基互补时会产生强烈的发光信号。为了运用这项技术，我们构建了一个新的全长前病毒 HIV-1 DNA 克隆和一个慢病毒包装载体，其中 HiBiT 标签被添加到整合酶的 C 末端。用 HiBiT 序列标记整合酶不会阻碍由此产生的病毒产生、感染性或对整合酶抑制剂的敏感性。EM显示病毒颗粒的正常形态。最重要的是，通过比较 ELISA 和 HiBiT 荧光素酶测定，我们成功地获得了 p24 浓度与基于 HiBiT 的荧光素酶活性之间极好的线性相关性。总的来说，我们得出的结论是，HiBiT 标记的病毒可以替代亲本 HIV-1 和慢病毒载体，这使我们能够对 HIV-1/慢病毒生产进行超快速、廉价、方便、简单和高度准确的定量分析。