Identifizierung von Interaktionspartnern für HIV-1 Nef und ihre potentielle Relevanz bei der Entwicklung der HIV-assoziierten Demenz

Neuro-Aids as a consequence of infection with human immundeficiency virus (HIV), becomes increasingly important due to considerably more efficient anti-AIDS therapies that result in longer life expectancy. Besides HIV Tat the viral Nef protein is hypothesized to play a role in development of Neuro-AIDS. Till today the detailed function of HIV-1 Nef protein in the development of neurological manifestations of an HIV infection is not known. Possibly, Nef could contribute to open the blood-brain barrier by activating matrix metalloproteinase-9 allowing HIV to enter the central nervous system. Furthermore, Nef is known to induce chemokine CCL2/MCP1 expression, which causes infiltration of the brain by monocytes. It is also known that oligodendrocytes initiate vacuolar myelopathy. This is amongst others a disease pattern of HIV associated neuromanifestations. Thus, for elucidation of HIV associated neuromanifestations on the molecular level Nef is an interesting candidate. Aim of the present work was the identification of new cellular interaction partners for HIV-1 Nef protein with a potential role in development of HIV associated dementia (HAD). Knowledge of new brain specific interaction partners of Nef may lead to a better understanding of Nef cellular functions. Use of a modern yeast two-hybrid system yielded succeessful identification of proteins as so far unknown interaction partners of membrane associated Nef. A total of 21 proteins, including 17 membrane proteins, were verified as interaction partners of Nef in the yeast system. Some of them may be relevant for HIV/HAD. B-cell receptor-associated protein 31 (Bcap31) was the second most prevalent hit. The interaction of Nef with Bcap31 could be confirmed with coimmunprecipitation in yeast cells. Furthermore, colocalization in Neuro2A cells in structures at plasma membrane succeeded. The glycoprotein M6B (GPM6B) was the most dominant hit in the screen and its interaction with Nef was characterized in detail in the present work. The interaction of Nef with GPM6B could be verified by coimmunprecipitation from yeast cell extract and pulldown assays using rat brain. Furthermore, Nef and GPM6B colocalized in Neuro2A cells and the relevant binding region in GPM6B could be defined to a 16 amino acid residue region, which is very similar to the Nef-binding region of human CD4. Fluorescence measurements resulted in a submicromolar dissociation constant for this interaction. The results obtained in the present work deliver important contributions for a deeper understanding of Nef’s function in HAD

Direct binding to GABARAP family members is essential for HIV-1 Nef plasma membrane localization

HIV-1 Nef is an important pathogenic factor for HIV/AIDS pathogenesis. Studies have shown that the association of Nef with the inner leaflet of the plasma membrane and with endocytic and perinuclear vesicles is essential for most activities of Nef. Using purified recombinant proteins in pull-down assays and by co-immunoprecipitation assays we demonstrate that Nef binds directly and specifically to all GABARAP family members, but not to LC3 family members. Based on nuclear magnetic resonance (NMR) experiments we showed that Nef binds to GABARAP via two surface exposed hydrophobic pockets. S53 and F62 of GABARAP were identified as key residues for the interaction with Nef. During live-cell fluorescence microscopy an accumulation of Nef and all GABARAP family members in vesicular structures throughout the cytoplasm and at the plasma membrane was observed. This plasma membrane accumulation was significantly reduced after knocking down GABARAP, GABARAPL1 and GABARAPL2 with respective siRNAs. We identified GABARAPs as the first known direct interaction partners of Nef that are essential for its plasma membrane localization

Schematic of the Y2H screen and coimmunoprecipitation of selected preys show interaction with Nef.

<p>A. Schematic presentation of the split-ubiquitin Y2H screen with membrane-anchored Nef as bait. Wild type Nef myristoylation is replaced by the Ost4p transmembrane anchor and amino acids 39–76 of yeast ubiquitin (Cub) linked to the LexA-VP16 transcription factor carboxy-terminally to Nef. A human adult brain cDNA library (Dualsystems Biotech AG) cloned in pPR3-N expressing the cDNAs as fusions carboxy-terminally of amino acids 1–38 of yeast ubiquitin (Nub) and an HA-tag. Upon binding of the bait and prey both parts of the ubiquitin come together and are cleaved by the protease to activate reporter genes. If no interaction with the Nef bait protein is possible, the ubiquitin subunits stay apart and no reporter genes in the nucleus are turned on. B. Coimmunoprecipitation (CoIP) of Nef and preys. Yeast cell lysate proteins from different transfected cultures as well as a non-transfected control (NMY51) as indicated at the top were immunoprecipitated (IP) either with anti-HA or anti-LexA antibodies. For negative controls, cells were alternatively transfected with a bait expression vector coding for the SV40 large T antigen (largeT) instead of Nef. The resulting immunoprecipitates were electrophoretically separated, blotted (WB) on a PVDF-membrane as indicated at the right and detected with anti-Myc or anti-HA antibodies. The approximate molecular weights of the proteins are shown. Two additional experiments gave similar results.</p

Subcellular localization of the identified hits within a model cell.

<p>Overview of the subcellular localization of the interaction partners of Nef identified via the split-ubiquitin based Y2H system based on the data given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051578#pone.0051578.s001" target="_blank">Table S1</a>. Marked in magenta are the proteins studied in more detail.</p

Analysis of subcellular localization of BAP31, CD320/TCblR, CLDN10 and GPM6B with and without Nef.

<p>A. Confocal microscopy analysis of Cos-7 cells coexpressing GFP fusions of BAP31, CD320/TCblR, CLDN10 or GPM6B with Nef-DsRed. Cos-7 cells were transiently cotransfected with pNef-DsRed and pGFP-BAP31, pGFP-CD320/TCblR, pGFP-CLDN10 or pGFP-GPM6B and fixed 24 h posttransfection. Single images from the red (Nef-DsRed) and green (GFP-prey) channels were overlaid in the merged image. Yellow regions represent colocalization, details are given in the text. Arrows point out BAP31 accumulations and distinct areas of CLDN10 and Nef overlay. Scale bar: 10 µm. B. Negative controls for the colocalization images in Figure A. Confocal microscopy analysis of Cos-7 cells coexpressing GFP fusions of BAP31, CD320/TCblR, CLDN10 or GPM6B with DsRed. Cos-7 cells were transiently cotransfected with pDsRed and pGFP-BAP31, pGFP-CD320/TCblR, pGFP-CLDN10 or pGFP-GPM6B and fixed 24 h posttransfection. Single images from the red (DsRed) and green (GFP-prey) channels were overlaid in the merged image. The respective scatter grams, as well as the images and scatter grams from cotransfections with the pDsRed control vector are given.</p

Analysis of the bait dependency test.

<p>Bait dependency test for growth on minimal media (His^−/Ade^−/lacZ⁻) of the positive preys coexpressed with pDHB1-Nef or pDHB1-LargeT (negative control) and summarized CoIP results (last column). Only hits that passed the Nef dependency test are listed. The table is sorted by the number of hits, which is given beside the protein name. Putative Nef-interacting proteins prone to be false-positive interactors based on the components of the Y2H system itself (according to the Dualsystems support page) are written in italic.</p

Sequence analysis of Nef binding site and <i>in vitro</i> binding studies of GPM6B and Nef.

<p>A. Amino acid sequence of the Nef binding motif of CD4. B. Clustal W <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051578#pone.0051578-Larkin1" target="_blank">[70]</a> based alignment of the cytoplasmic loops of GPM6B, GPM6A and PLP1. Note, for GPM6B, GPM6A and PLP-DM20/PLP1 isoform 2, the sequences of the complete cytoplasmic loop regions are shown. Additionally, positions of the indicated proteolipid proteins with homologies to a sequence region in the cytoplasmic tail of T-cell surface glycoprotein CD4 (shown at the top) that includes the core Nef binding motif of CD4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051578#pone.0051578-Preusser1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051578#pone.0051578-Preusser2" target="_blank">[21]</a> are highlighted. Positions identical to the respective CD4 residue are shaded black, those with high similarity are shaded grey and those with lower similarity are boxed. The indicated residue numbers show the beginning and the end of the amino-terminally fluoresceinylated GPM6B and PLP1 peptides used for the Nef binding studies shown in D. Numbering of GPM6B is based on UniProtKB entry Q13491-1 throughout this figure. C. Sequence of the cytoplasmatic portion of CD320/TCblR, homologies to CD4 indicated as described above. D. Fluorescence titration of 0.5 µM of fluoresceinyl-labeled peptides, GPM6B_112–127, GPM6B_AA, or PLP_193–108 with recombinant HIV-1SF2 Nef_2–210 protein (prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051578#pone.0051578-Hoffmann1" target="_blank">[68]</a>). The fluorescence signals are shown as a function of the Nef_2–210 protein concentration. Values result from the fluorescence of the peptides in the presence of the indicated concentration of Nef_2–210 in comparison with a buffer control titration. Assuming a simple bimolar interaction between the peptide and Nef_2–210, the data were described by a model based solely on the law of mass action which accounts for ligand depletion <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051578#pone.0051578-Tran1" target="_blank">[71]</a>. Nonlinear curve fitting of the model to the fluorescence data (lines) yielded dissociation constants of 0.64±0.06 µM for GPM6B_112–127 and of 0.71±0.03 µM for PLP_193–108.</p