Research paper Intracellular detection of differential APOBEC3G, TRIM5alpha, and LEDGF/p75 protein expression in peripheral blood by flow cytometry Kim Mous a , Wim Jennes b , Ann De Roo c , Isabel Pintelon d , Luc Kestens b , Xaveer Van Ostade a, ⁎ a Laboratory for Proteinscience, Proteomics and Epigenetic Signaling (PPES), Department of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium b Laboratory of Immunology, Department of Microbiology, Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerp, Belgium c Department of Clinical Sciences, Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerp, Belgium d Laboratory for Cell Biology and Histology-Core Facility for Biomedical Microscopic Imaging, Department of Veterinary Sciences, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium article info abstract Article history: Received 24 March 2011 Received in revised form 24 June 2011 Accepted 24 June 2011 Available online 19 July 2011 Expression studies on specific host proteins predominantly use quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based assay to study intracellular expression levels of three important host proteins involved in HIV-1 replication: apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G), tripartite motif 5alpha (TRIM5α), and lens epithelium-derived growth factor (LEDGF/p75). An indirect intracellular staining (ICS) method was optimized using antibodies designed for other applications like enzyme-linked immunosorbent assay (ELISA), confocal imaging, and western blotting. The median fluores- cence intensity (MFI) value – a measure for the protein expression level – increased upon higher antibody concentration and longer incubation time, and was reduced following preincubation with recombinant proteins. Staining of stably transfected or knock-down cell lines supported the method's specificity. Moreover, confocal microscopy analysis of peripheral blood mononuclear cells (PBMC), when stained according to the ICS method, confirmed the localization of APOBEC3G and TRIM5α in the cytoplasm, and of LEDGF/p75 in the nucleus. Also, stimulation with mitogen, interferon-alpha, or interferon-beta resulted in detectable, albeit weak, increases in intracellular expression of APOBEC3G and TRIM5α. After optimization, the method was applied to healthy control and HIV-1 infected subjects. For all subjects studied, the memory subset of CD4+ T cells showed significantly higher expression levels of APOBEC3G, TRIM5α, and LEDGF/p75, while the CD16+ subset of monocytes was characterized by higher expression levels of LEDGF/p75. In addition, we observed that therapy-naïve HIV-1 patients tended to have lower expression levels of APOBEC3G and TRIM5α than HIV-1 negative controls. In summary, our data provide proof-of-principle for the detection of specific host factors at the level of a single cell, which may prove useful for our further understanding of their role in virus- host interactions. © 2011 Elsevier B.V. All rights reserved. Keywords: Protein expression Intracellular staining APOBEC3G TRIM5alpha LEDGF/p75 1. Introduction Human immunodeficiency virus type I (HIV-1) interacts with several cellular host proteins during its replication cycle. Some of these factors have antiviral activity whereas others are HIV-1 cofactors essential for HIV-1 replication. Recently, apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (Sheehy et al., 2002) (APOBEC3G) and tripartite motif 5alpha Journal of Immunological Methods 372 (2011) 52–64 ⁎ Corresponding author. Tel.: +32 3 265 2319; fax: +32 3 265 2339. E-mail addresses: kim.mous@ua.ac.be (K. Mous), wjennes@itg.be (W. Jennes), ann.deroo@so.antwerpen.be (A. De Roo), isabel.pintelon@ua.ac.be (I. Pintelon), lkestens@itg.be (L. Kestens), xaveer.vanostade@ua.ac.be (X. Van Ostade). 0022-1759/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2011.06.028 Contents lists available at ScienceDirect Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim