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. 2015 Sep 11:12:138.
doi: 10.1186/s12985-015-0365-6.

Proteomic analysis of HIV-1 Gag interacting partners using proximity-dependent biotinylation

Valerie Le Sage  1 , Alessandro Cinti  2   3 , Fernando Valiente-Echeverría  4   5   6 , Andrew J Mouland  7   8
Affiliations

Proteomic analysis of HIV-1 Gag interacting partners using proximity-dependent biotinylation

Valerie Le Sage et al. Virol J. .

Abstract

Background: The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein is necessary and sufficient to assemble non-infectious particles. Given that HIV-1 subverts many host proteins at all stages of its life cycle, it is essential to identify these interactions as potential targets for antiretroviral therapy.

Findings: This work demonstrates the use of proximity-dependent biotin identification (BioID) of host proteins and complexes that are proximal to the N-terminal domains of the HIV-1 Gag polyprotein. Two of the hits identified in the BioID screen were validated by immunoprecipation and confirmed the interaction of DDX17 and RPS6 with HIV-1 Gag.

Conclusions: Our results show that BioID is both a successful and complementary method to screen for nearby interacting proteins of HIV-1 Gag during the replicative cycle in different cell lines.

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Figures

Fig. 1
Fig. 1
Expression and localization of recombinant HIV-1 Gag. a Diagram of the expression cassette encoding MYC tagged BirA* alone and MYC-BirA* fused to the N-terminus of HIV-1. b Jurkat or HeLa cells were transfected with BirA* or BirA*-Gag for 24 h. The expression levels of BirA* and BirA*-Gag were evaluated by Western blot using anti-MYC (US Biological Life Sciences, M9601-30), anti-p24 (NIH, 183-H12-5C) and anti-actin (Abcam, ab8226) antibodies. c Immunofluorescence of Jurkat (left panels) or HeLa (right panels) cells transfected with BirA*, BirA*-Gag or the HIV-1-expressing plasmid (pNL4-3). Paraformaldehyde-fixed cells were incubated with primary anti-MYC and anti-p24 antibodies for 1 h at 37 °C and secondary antibodies Alexa Fluor 488 anti-mouse IgG and Alexa Fluor 594 anti-rabbit IgG (Molecular Probes). Coverslips were mounted in ProLong Gold antifade reagent (Molecular Probes) with DAPI to stain the nuclei. Images were acquired using a Wave FX Spinning disc confocal microscope with a 63x objective. Scale bar indicates 10 μm
Fig. 2
Fig. 2
Identification of potential Gag proximal and interacting partners. Jurkat cells were transfected with BirA* or BirA*-Gag in medium supplemented with 50 μM Biotin (Sigma). Cell lysates were harvested 24 h post-transfection. Dynabeads MyOne Streptavidin C1 (Life Technologies) were used to precipitate biotinylated proteins from 1 mg of lysate as described by the manufacturer. The eluted proteins were separated on 4-15 % SDS-PAGE (Bio-Rad), the bands excised and analyzed by LC-tandem MS at SAMS Centre for proteomics, University of Calgary. a Venn diagram depicting the number of overlapping hits between Gag-interacting proteins. b Genes families and subfamilies were grouped and classified, using PANTHER, according to their function in three different ways: biological process, molecular function and protein class. c GeneMANIA (used in automatically selected weighting method) was used to determine known interactions between input genes and how they interact. The relationships between genes in the network included physical and genetic interactions, pathways and protein domain similarity
Fig. 3
Fig. 3
Validation of BirA*-Gag interactions by immunoprecipitation and Western blot. The Gag coding sequence was cloned into the pCI-Neo-Flag (Flag) (Life Technologies) between XhoI and NotI to express Flag-Gag. HeLa cells were transfected with either GFP or Gag-GFP (left panels) or Flag or Flag-Gag (right panel) for 24 h. Cell lysates were washed with PBS and collected using NP40 lysis buffer (50 mM Tris pH 7.8; 150 mM NaCl; 0.5 mM EDTA; 0.5 % Nonidet P-40) supplemented with Complete protease inhibitor (Roche). Cell lysates were quantified by the Bradford assay (Bio-Rad) and 1 mg of protein was immunoprecipitated with anti-MYC tag mAb-Magnetic beads for 2 h as described by the manufacturer (MLB). The bound complexes were analyzed by SDS-PAGE using anti-p24 (NIH, 183-H12-5C), anti-DDX17 (Abcam, ab24601) and anti-S6 Ribosomal Protein (Cell Signalling 54D2) antibodies
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