Human immunodeficiency virus type 1 (HIV-1) is a lentivirus that causes acquired immunodeficiency syndrome (AIDS) in humans, a progressive failure of the immune system allowing life-threatening opportunistic infections and cancer development. The HIV Nef protein contributes essentially to the pathology of AIDS by a variety of protein-protein-interactions within the host cell. The versatile functionality of Nef is partially attributed to different conformational states and post-translational modifications, such as myristoylation. Up to now, many cellular interaction partners of Nef have been identified using classical yeast two-hybrid screens. Such screens rely on transcriptional activation of reporter genes in the nucleus to detect interactions. Thus, the identification of Nef interaction partners that are integral membrane proteins, membrane-associated proteins or other proteins that do not translocate into the nucleus is hindered.
We used a split-ubiquitin based yeast two-hybrid to identify novel membrane-localized interaction partners of Nef. More than 80% of the hereby identified interaction partners of Nef could be characterised as transmembrane proteins. The hits are involved in diverse cellular processes, like signaling, apoptosis, neurogenesis, cell adhesion and protein trafficking or quality control (Kammula et al., PLoSOne 2012;7(12):e51578).
Nef and Autophagy
Autophagy has been recognized to play an important role in cell-autonomous defence against microbes (Xenophagy) as well as in innate and adaptive immunity. Although autophagy is an important constituent of the host's antiviral defence, many viruses have developed counteractive strategies and even abuse parts of the autophagic machinery for their own benefit. This is also true for HIV-1, which seems to have distinct effects on autophagy in diverse cell types.
By applying a split-ubiquitin based yeast two-hybrid screen with a membrane attached HIV-1 Nef protein as bait, we also identified autophagy related proteins (Atgs) proteins as a putative Nef interacting protein when analyzing also further hits, which displayed a slight binding to the used control protein, in more detail. Interestingly, in a large scale siRNA screen for host proteins required by HIV-1, Atgs were among the ≈ 250 HIV dependency factors identified (Brass et al. Science 2008, 319(5865):921ff). Meanwhile we collected a huge amount of data that demonstrate that the interaction of Nef with certain, not yet recognized Atgs is specific and direct.
Our future studies will show, whether e.g. autophagosome formation and autophagic flux are influenced by a Nef-mediated alteration of certain Atgs function(s), whether the production of HIV-1 virions depends on the presence of specific Atgs, or whether Nef affects the activities of other membrane-trafficking systems, such as the endosomal and secretory pathways in an Atg mediated manner. The structural aspects of this project will be studied in tight colaboration with Melanie Schwarten.
Currently, we are performing more detailed experiments to explore the respective function and relevance of the observed Nef interactions.
Alexandra Boeske presented first results in a poster and an invited talk at the KEYSTONE CONFERENCE on Autophagy in Breckenridge, Colorado, USA June 19—24, 2015 (supported by a Barrie Hesp Scholarship). Title: "GABARAPs Mediate Anterograde Transport and Secretion of HIV-1 Nef via Mechanisms Related to Unconventional Protein Secretion".
This project reveives DFG-funding (SFB 1208).