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.