Development of a novel HIV-1 nuclear localization assay

描述（由申请人提供）： 一种新型HIV-1核定位检测方法的开发HIV核迁移和整合是建立病毒感染的决定性步骤，或当细胞条件不利时病毒潜伏。研究HIV核迁移对于了解病毒感染和潜伏期的调控至关重要，最近的研究表明趋化因子对病毒核迁移的显著影响。然而，目前还没有测量艾滋病毒核迁移的方便工具。目前，病毒核定位可以通过替代标记物2-LTR环来测量，2-LTR环仅在细胞核中积累。病毒核DNA也可以通过进行核分级分离以定量细胞核中的总病毒DNA来直接测量。这两种检测都有局限性。2-LTR环定量在早期时间点（12小时之前）不敏感，特别是在静息CD 4 T细胞中，其中在没有T细胞活化的情况下难以检测2-LTR环。通常，静息T细胞中的早期核迁移只能通过核分级分离来测量，这需要多次分级分离对照，并且既耗时又容易出现实验错误。在这项提案中，我们的目标是开发一种新的，方便的，更灵敏的检测方法来直接测量HIV核迁移。在特定目标1中，将开发基于LMO 2和Ldb 1两种核因子之间的核特异性和高亲和力相互作用的“基于BRET的LMO 2-Ldb 1核迁移测定”（BLNMA）。为了开发该检测试剂盒，将HIV-1整合酶（IN）与Nanoluc和LMO 2融合。同时，融合蛋白Ldb 1-EGFP将被引入靶细胞。IN标记的HIV感染和核迁移将导致IN-LMO 2-Nanoluc和Lbd 1-EGFP之间的相互作用，仅在细胞核中，这将产生BRET信号。这种新的测定法具有许多优点：（i）更快，在感染的数小时内测量信号;（ii）方便，直接测量信号而不需要DNA提取和定量;（iii）更灵敏，静息T细胞中的核迁移可以在数小时内测量，并且不需要T细胞活化;（iv）直接，它是PIC的核迁移的直接测量，而不是替代DNA环的测量。此外，鉴于该测定在活细胞中进行，其将 还允许在一段时间内监测核迁移。此外，该报告系统将适合于HIV-1核迁移抑制剂的高通量筛选.在特定目标2中，我们将进一步验证BLNMA方法在趋化因子处理或shRNA-Arp 3敲低细胞中定量HIV核迁移。这两种细胞调节直接影响HIV核迁移。这些验证对于改进该技术，使其适用于逆转录病毒学实验室的常规研究和药物筛选具有重要意义。

DESCRIPTION (provided by applicant): Development of a Novel HIV-1 Nuclear Localization Assay HIV nuclear migration and integration is a decisive step in the establishment of viral infection, or viral latency when cellular conditions are not favorable. Studying HIV nuclear migration is critically important for understanding regulations of viral infection and latency, as exemplified by recent studies showing dramatic effects on viral nuclear migration by chemokines. However, currently, no convenient tools for measuring HIV nuclear migration exist. At present, viral nuclear localization can be measured by a surrogate maker, the 2-LTR circle, which only accumulates in the nucleus. Viral nuclear DNA can also be directly measured by performing nuclear fractionation to quantify total viral DNA in the nucleus. Both of these assays have limitations. The 2-LTR circle quantification is insensitive at early time points (before 12 hours), and particularly in resting CD4 T cells, where 2-LTR circles are difficult to detect withou T cell activation. Frequently, early nuclear migration in resting T cells can only be measured by nuclear fractionation, which requires multiple fractionation controls, and is both time- consuming and prone to experimental errors. In this proposal, we aim to develop a novel, convenient, and more sensitive assay to directly measure HIV nuclear migration. In Specific Aim 1, will develop a "BRET-based LMO2-Ldb1 Nuclear Migration Assay" (BLNMA), which is based on the nucleus-specific and high-affinity interaction between two nuclear factors, LMO2 and Ldb1. To develop this assay, HIV-1 Integrase (IN) will be fused to Nanoluc and LMO2. Concomitantly, a fusion protein, Ldb1-EGFP, will be introduced into target cells. IN-tagged HIV infection and nuclear migration will lead to the interaction between IN-LMO2-Nanoluc and Lbd1-EGFP, exclusively in the nucleus, which will generate BRET signal. This novel assay has numerous advantages: (i) faster, signals are measured within hours of infection; (ii) convenient, signals are directly measured without the need for DNA extraction and quantification; (iii) more sensitive, nuclear migration in resting T cells can be measured within hours, and does not require T cell activation; (iv) direct, it is a direct measurement of the nuclear migration of PIC rather than a measurement of the surrogate DNA circles. In addition, given that the assay is performed in live cells, it will also allow monitoring nuclear migration over a time course. Furthermore, this reporter system would be amenable to high- throughput screening of HIV-1 nuclear migration inhibitors. In Specific Aim 2, we will further validate the BLNMA method for quantification of HIV nuclear migration in chemokine-treated or shRNA-Arp3 knockdown cells. Both cellular modulations directly affect HIV nuclear migration. These validations are important for improvements of the technology so that it is applicable to routine research and drug screening in retrovirology laboratories.