Identifying the membrane proteome of HIV-1 latently infected cells.
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.
Berro
Reem
R
Genetics Program, Department of Biochemistry and Molecular Biology, The George Washington University, School of Medicine, Washington, DC 20037, USA.
de la Fuente
Cynthia
C
Klase
Zachary
Z
Kehn
Kylene
K
Parvin
Lida
L
Pumfery
Anne
A
Agbottah
Emmanuel
E
Vertes
Akos
A
Nekhai
Sergei
S
Kashanchi
Fatah
F
D000077329:Agammaglobulinaemia Tyrosine Kinase; D017209:Apoptosis; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000821:virology*; D002470:Cell Survival; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism / Q000821:virology; D008954:Models, Biological; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D014764:Viral Proteins / Q000737:chemistry*; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000378:metabolism
D014764:Viral Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase