Abstract
Profiling integral plasma membrane proteins is of particular importance for the identification of new biomarkers for diagnosis and for drug development. We report in this study the identification of surface markers by performing comparative proteomics of established human immunodeficiency virus-1 (HIV-1) latent cell models and parental cell lines. To this end we isolated integral membrane proteins using a biotin-directed affinity purification method. Isolated proteins were separated by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) after in gel digestion. Seventeen different proteins were found to vary on the surface of T-cells due to HIV-1 infection. Of these proteins, 47% were integral membrane proteins, and 18% were membrane-associated. Through the use of complementary techniques such as Western blotting and fluorescent staining, we confirmed the differential expression of some of the proteins identified by MALDI-TOF including Bruton's tyrosine kinase and X-linked inhibitor of apoptosis. Finally, using phosphatidylinositol 3-kinase inhibitors and flavopiridol to inhibit Bruton's tyrosine kinase localization at the membrane and X-linked inhibitor of apoptosis protein expression, respectively, we showed that HIV-1 latently infected cells are more sensitive to these drugs than uninfected cells. This suggests that HIV-1 latently infected cells may be targeted with drugs that alter several pathways that are essential for the establishment and maintenance of latency.
Highlights
Ronment and play an important role in cellular homoeostasis and metabolic energy transduction
Surface Protein Biotinylation and Plasma Membrane Protein Isolation—We analyzed the differential expression of surface proteins in T-cells latently infected or uninfected with human immunodeficiency virus-1 (HIV-1) to identify surface markers for latency and targets for antibody or drug development
No proteins were isolated from cells that were not treated with biotin (Fig. 2A, lanes 2 and 4), demonstrating that the proteins isolated from cells treated with biotin (Fig. 2A, lanes 1 and 3) were pulled down by streptavidin beads
Summary
ACH2 (AIDS Research and Reference Reagents Program, National Institutes of Health) is an HIV-1-infected latent T-cell clone derived from the parental CEM (A3.01) cells that were initially infected with the LAV strain of HIV-1. ACH2, A3.01, and CEM cells were cultured in RPMI 1640 containing 10% fetal bovine serum, 1% penicillin/ streptomycin, and 1% L-glutamine (Quality Biological). HLM-1 cells (AIDS Research and Reference Reagents Program, NIH) are HeLa-T4ϩ cells containing one integrated copy of HIV-1 genome with a Tat-defective mutation at the first AUG of Tat gene. HLM-1 and HeLa-T4ϩ were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1% glutamine, and 1% penicillin/ streptomycin (Quality Biological) and G418 (200 g/ml) for selection. Isolated PBMC were counted and resuspended in Dulbecco’s modified Eagle’s medium with 20% fetal bovine serum and 10% Me2SO for storage in liquid nitrogen. The BTK-GFP plasmid was kindly provided by Dr Smith (Huddinge University Hospital, Sweden) and previously described [16]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
