Identifying the Membrane Proteome of HIV-1 Latently Infected Cells

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.Biological membranes surround and compartmentalize cells. They provide a physical boundary between the cell and its environment and play an important role in cellular homoeostasis and metabolic energy transduction. According to the SingerNicolson "fluid-mosaic model" (1), plasma membranes are organized into lipid bilayers containing proteins that can diffuse rapidly throughout the two-dimensional surface of the membrane. Membrane proteins typically make up around a third of the proteome of a cell and associate with the membrane in different ways. Integral membrane proteins are contained within the bilayer through one or more transmembrane regions. They can also be associated with lipid anchors such as fatty acids and can cover large regions of a membrane with protein surfaces. Alternatively, proteins can be associated with the membrane through non-covalent interactions with integral membrane proteins or other membrane-associated proteins. The fluidity differences and the organization of protein and lipids within the plasma membrane depend upon the cholesterol content in the bilayer (2). The current evidence supports a novel concept of specific microdomains existing in the plasma membrane in vivo in regions rich in cholesterol (2, 3). These specific microdomains, termed "lipid rafts," are composed of tightly packed sphingolipids, gangliosides, and cholesterol (4). Lipid rafts are generally small, ranging from 25 to 700 nm depending on the activation state of the cell (5). In ad...

COMMD1/Murr1 Reinforces HIV-1 Latent Infection through IκB-α Stabilization

“…In fact, the levels of several ions in HIV/AIDS patients' peripheral blood are different from those of healthy subjects, as previously reported (58). XIAP is induced, which inhibits apoptosis, in latently HIV-1-infected cells (59). Further analysis of COMMD1 and proteins related to it is still required to show the importance of COMMD1 in HIV-1 latency.…”

Modulation of apoptosis and viral latency – an axis to be well understood for successful cure of human immunodeficiency virus

“…In addition, the anti-apoptotic XIAP (X-linked inhibitor of apoptosis protein) is upregulated in latently infected T-cell lines, macrophage/ monocytic cell lines and PBMCs. It was reported that chemical inhibition of XIAP sensitizes these cells to apoptosis (Berro et al, 2007). All these evidences suggest that apoptosis is inhibited during latency.…”

“…Several studies have reported that in latently infected cells, there is decreased HIV replication due to the increased expression of anti-apoptotic proteins (e.g. BCL-2, cFLIP, Mcl-1) (Aillet et al, 1998;Berro et al, 2007;Tan et al, 2013) or downregulation of pro-apoptotic proteins (e.g. BAX, FADD) Wang et al, 2011).…”

Viral proteomics: global evaluation of viruses and their interaction with the host

“…Interestingly, proteomics analysis indicated that HIV virions contain a high number of host cell proteins [35]. Multiple groups have reported different numbers of cellular proteins associating with HIV virions [5].…”