Nef dimerization defect abrogates HIV viremia and associated immune dysregulation in the Bone Marrow-Liver-Thymus-Spleen (BLTS) humanized mouse model.

Loss of function mutations in the human immunodeficiency virus (HIV)-negative factor (Nef) gene are associated with reduced viremia, robust T cell immune response, and delayed acquired immunodeficiency syndrome (AIDS) progression in humans. Additionally, in vitro studies have shown that mutations in Nef's dimerization interface may play a significant role in modulating viral replication and impairing host defense. However, in vivo, mechanistic studies on the role of Nef dimerization in HIV infection are lacking. Humanized rodent models with human immune cells are robust platforms for investigating the interactions between HIV and the human immune system. We recently developed the bone marrow-liver-thymus-spleen (BLTS) humanized mouse model, which carries human immune cells, as well as primary and secondary lymphoid tissues that facilitate anti-viral immune responses. Here, we employed the BLTS-humanized mouse model to demonstrate that preventing Nef dimerization abrogates HIV viremia and the associated induction of immune dysregulation (immune activation and exhaustion). This suggests that Nef dimerization may be a therapeutic target for future HIV cure strategies, which can be explored in humanized mouse models. Human immune cells reconstituted BLTS humanized mice were infected with HIV-WT/ HIV dNef/ HIV-Y115D/ PBS. Three mice were assigned per group. HIV infected, and PBS-inoculated humanized mice were sacrificed at 5-7 weeks post HIV infection and total RNA was isolated from engrafted spleen tissue of humanized mice using Qiagen RNA isolation kit Cat# 74104. Nanostring profiling of host response was performed at the University of Pittsburgh Genomic Core facility using the nCounter Human Immunology v2 Panel, Cat #-XT-CSO-HIM2-12. Total RNA (50ng) was hybridized to reporter and capture probe sets at 65°C for 24 h. Hybridized samples were loaded on the nCounter cartridge and post-hybridization steps and scanning was performed on the nCounter Profiler. RCC files were analyzed using nSolver analysis software (Version 4.0) as per the manufacturer's protocols. Negative and positive controls included in probe sets were used for background thresholding, and normalizing samples for differences in hybridization or sample input respectively.