Background The continued persistence of HIV-1 as a public health concern due to the lack of a cure calls for the development of new tools for studying replication of the virus. Here, we used NanoLuc, a small and extremely bright luciferase protein, to develop an HIV-1 bioluminescent reporter virus that simplifies functional measurement of virus particle production. Results The reporter virus encodes a Gag protein containing NanoLuc inserted between the matrix (MA) and capsid (CA) domains of Gag, thereby generating virus particles that package high levels of the NanoLuc reporter. We observe that inserting the NanoLuc protein within HIV-1 Gag has minimal impact on Gag expression and virus particle release. We show that the reporter virus recapitulates inhibition of HIV-1 particle release by Gag mutations, the restriction factor tetherin, and the small-molecule inhibitor amphotericin-B methyl ester. Conclusion These results demonstrate that this vector will provide a simple and rapid tool for functional studies of virus particle assembly and release and high-throughput screening for cellular factors and small molecules that promote or inhibit HIV-1 particle production.

中文翻译：

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生成和验证高度敏感的生物发光 HIV-1 报告载体，可简化病毒释放的测量

背景 由于缺乏治愈方法，HIV-1 作为公共卫生问题的持续存在需要开发用于研究病毒复制的新工具。在这里，我们使用 NanoLuc（一种小而极亮的荧光素酶蛋白）开发了一种 HIV-1 生物发光报告病毒，可简化病毒颗粒生产的功能测量。结果报告病毒编码包含插入在 Gag 的基质 (MA) 和衣壳 (CA) 结构域之间的 NanoLuc 的 Gag 蛋白，从而产生包装高水平 NanoLuc 报告基因的病毒颗粒。我们观察到在 HIV-1 Gag 中插入 NanoLuc 蛋白对 Gag 表达和病毒颗粒释放的影响很小。我们表明，报告病毒概括了 Gag 突变、限制因子 tetherin 对 HIV-1 颗粒释放的抑制作用，和小分子抑制剂两性霉素-B甲酯。结论 这些结果表明，该载体将为病毒颗粒组装和释放的功能研究以及促进或抑制 HIV-1 颗粒产生的细胞因子和小分子的高通量筛选提供简单而快速的工具。