Currently over 40 million individuals worldwide are infected with the human immunodeficiency virus (HIV) and will remain carriers and potential transmitters of HIV for the rest of their lives. The aim of our research is to identify the cellular locations of HIV persistence and to understand the mechanisms used by these cells to control HIV. A key aspect of our work is the identification of cellular mechanisms and factors that modulate HIV gene expression. We have generated multiple tools for quantitative, live-cell analysis of HIV gene expression and used these to evaluate the HIV-inhibitory potential of extracellular and intracellular molecules. Thus, we recently identified a novel cellular factor (Risp) that suppresses HIV gene expression by interfering with the activity of the HIV Rev regulatory factor. Important future aims include: (i) comprehensive identification of cellular HIV control factors; (ii) analysis of the influence of these factors on HIV replication in various HIV target cells; and (iii) evaluation of the potential of various cell types to form HIV reservoirs. The results of our work impact important medical aspects associated with HIV/AIDS, such as the monitoring of the burden of HIV-infected cells in patients and the expansion of current therapeutic options with novel antiviral strategies.
www.gsf.de/imv/rv_regul/index.html
The Technique
Extended bimolecular fluorescence complementation (exBiFC) is a powerful methodology for visualization of protein-protein interactions in living cells. Elucidation of interactions of cellular proteins with HIV proteins is essential to understanding the molecular mechanisms of HIV replication and has an enormous potential for the development of novel anti-HIV strategies. The HIV Rev and Tat proteins are currently unexploited targets for anti-retroviral therapy. These proteins are important key regulators of HIV replication and are essential for the activation of HIV-1 gene expression. One of our scientific aims is the identification of cellular proteins that are able to bind these HIV regulatory proteins. In the published study, we used exBiFC to confirm Rev-Rev homodimerization and interactions of Rev with known cellular partners (Risp and CRM1/Exportin). In our ongoing projects, exBiFC has proven to be an invaluable tool for the verification of interactors initially found with other assays, such as various two-hybrid assays, pulldowns, or functional screenings. In addition, we are adapting the exBiFC method for the primary identification of HIV protein interactors directly in HIV target cells.
Live-cell assay for simultaneous monitoring of expression and interaction of proteins, p. 688.
