Development of a novel HIV-1 nuclear localization assay

描述（由申请人提供）：一种新型HIV-1核定位试验的开发HIV核迁移和整合是在细胞条件不利时建立病毒感染或病毒潜伏期的决定性步骤。研究HIV核迁移对于理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有显著影响。然而，目前还没有方便的工具来测量HIV核迁移。目前，病毒的核定位可以通过一个替代制造者，2-LTR环来测量，它只聚集在细胞核中。病毒的核DNA也可以通过进行核分离来直接测量，以量化细胞核中的病毒DNA总量。这两种检测方法都有局限性。在早期时间点（12小时之前），2-LTR环的定量不敏感，特别是在静止的CD4 T细胞中，如果没有T细胞激活，2-LTR环很难检测到。通常，静止T细胞的早期核迁移只能通过核分离来测量，这需要多个分离控制，既耗时又容易产生实验误差。在本提案中，我们的目标是开发一种新的、方便的、更敏感的检测方法来直接测量HIV核迁移。在Specific Aim 1中，她将开发一种“基于bret的LMO2-Ldb1核迁移试验”（BLNMA），该试验基于LMO2和Ldb1两种核因子之间的核特异性和高亲和力相互作用。为了开发这种检测方法，HIV-1整合酶（IN）将融合到Nanoluc和LMO2中。同时，将融合蛋白Ldb1-EGFP引入靶细胞。in标记的HIV感染和核迁移会导致in - lmo2 - nanoluc和Lbd1-EGFP仅在细胞核内相互作用，产生BRET信号。这种新型检测方法有许多优点：(1)更快，在感染后数小时内测量信号；（ii）方便，直接测量信号，无需DNA提取和定量；（iii）更敏感，静止T细胞的核迁移可以在数小时内测量，并且不需要T细胞激活；（iv）直接，它是PIC核迁移的直接测量，而不是替代DNA圈的测量。此外，考虑到该试验是在活细胞中进行的，它将

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.