Utilizing quantitative proteomic approaches to define HIV host-pathogen interactions in primary T lymphocytes and T cell lines

About This Grant

Abstract The human immunodeficiency virus (HIV) remains as an incurable pandemic pathogen that lacks an effective vaccine. While antiretroviral therapies (ART) can effectively suppress HIV replication, it does not eradicate the virus. As a result, people living with HIV (PLWH) remain vulnerable to AIDS in part due to viral immune evasion strategies. The HIV accessory protein Nef plays a crucial role in this process. Nef enhances viral replication and immune evasion by altering the trafficking and promoting the degradation of immune receptors. One of Nef’s most well characterized functions is the disruption of the major histocompatibility class I (MHC-I) trafficking to target it for lysosomal degradation. This Nef function prevents cytotoxic T lymphocyte (CTL) recognition and clearance of infected cells thereby enabling evasion of the immune response. While some pathways targeted by Nef are well studied, the full breadth of Nef’s host interactions— especially in the context of other HIV accessory proteins— remains poorly understood. One approach to address this gap in knowledge is unbiased proteomic analysis of Nef-host protein interactions. Three prior studies have utilized proteomic approaches to investigate Nef functions. However, their conclusions have been limited by using systems that express Nef in isolation, the use of potentially disruptive affinity tags, and failure to block degradative pathways, utilized by Nef. Our preliminary data as well as the work proposed here address these limitations. In our preliminary studies and proposed aims we will use both T cell lines and primary T cells expressing all HIV accessory proteins (Vif, Vpu, and Vpr, Rev and Tat) and a novel functionally validated tagged Nef protein. Using this system, we identified the Nef interactome in a T cell line in the presence and absence of lysosomal inhibition by TMT-based quantitative proteomics. Furthermore, with our approach we have identified novel Nef binding proteins associated with inflammatory, innate immune response and viral sensing pathways. Subsequent analyses have confirmed these novel interactions in both a T cell line and primary T cells. Based on our strong preliminary data, we will test the overarching hypothesis that novel Nef interacting proteins that have not previously been reported will reveal new pathways targeted by Nef that promote viral pathogenesis. We will evaluate our hypothesis with the following aims. In Aim 1, we will confirm our preliminary data and extend the characterization of the Nef interactome to primary T cells, and we will identify the Nef binding domains responsible for novel Nef-host protein interactions. Furthermore, in Aim 2, we will determine the functional significance of novel Nef interactions with inflammatory and antiviral pathways. The work proposed in this study will provide a complete, unbiased characterization of the Nef interactome as well as reveal pathways that are essential for HIV pathogenesis. Furthermore, these findings hold promise for identifying novel therapeutic targets for HIV infected patients.