Cell adhesion through αV-containing integrins is required for efficient HIV-1 infection in macrophages

Abstract

IntroductionIntegrins are a family of transmembrane cell adhesion receptors that recognize cell-surface and extracellular matrix ligands. 1 All integrins are heterodimers composed of noncovalently linked ␣ and ␤ subunits. In humans, at least 18 ␣ and 8 ␤ subunits have been identified, forming more than 20 heterodimers, each of them with different cell expression patterns and ligand specificities. 2 Integrinmediated interactions are vital to the maintenance of normal cell functioning, as they mediate the interaction between cells and with the extracellular matrix, having therefore important implications for cell adhesion, migration, and proliferation. Integrins function as mediators for cell-cell interaction and adhesion but may also elicit signal transduction events leading to cell activation and response to the extracellular stimulus. 1 Different enveloped and nonenveloped viruses use integrins to enter and replicate in host cells. 3 Integrins may act as viral receptors that mediate virus attachment to the cell, a process that may also promote signaling responses influencing viral replication at a later step. In the case of human cytomegalovirus, ␣V␤3 has been described as an entry coreceptor but also as a trigger-mediator of intracellular signaling pathways. 4 The ␣4␤7 integrin, preferentially expressed in activated T cells homing to the gut, may interact with the HIV-1 envelope glycoprotein gp120, leading to increased virus replication. 5 Human blood monocytes, originated from hematopoietic precursors in the bone marrow, give rise to mature macrophages, which are distributed ubiquitously in all tissues. 6,7 Adhesion molecules, particularly integrins, have been shown to up-regulate when monocytes differentiate into macrophages, [8][9][10] suggesting that integrins play an important role in macrophage adhesion, migration, and tissue infiltration. Blood monocytes and tissue resident macrophages are important in vivo cell targets of HIV-1 infection. [11][12][13] Macrophages may become chronically infected or serve as reservoirs of latent virus. Furthermore, HIV may sequester macrophage immunoregulatory function, that is, inducing secretion of proinflammatory cytokines that lead to recruitment and activation of new target cells for HIV, including CD4 ϩ T cells. 14,15 Unlike monocyte-derived macrophages (MDMs), undifferentiated monocytes are not readily infected by HIV-1 in cell culture. 16,17 A productive viral infection may only occur after monocytes are differentiated after serum or cytokine stimulation, 18 a process that leads to the up-regulation of different cell-surface markers and receptors, including ␣V integrins 19,20 and, more specifically, an increase in the vitronectin ␣V␤3 receptor during the first 3 to 6 hours after infection. 14 We and others have shown the antiviral effect of specific anti-␣V antibodies in MDMs. 19,20 Here, we describe the involvement of ␣V integrin-mediated adhesion in HIV-1 replication of MDMs and HeLa cells. Our data support the idea that blocking integrin ␣V interaction with it...

Characterization of the Influence of Mediator Complex in HIV-1 Transcription

“…Plasmids-Tat-expressing plasmid pcTat has been previously described (21,22). To generate the p3ϫFLAG-CMV-Tat, RNA from TZM-bl cells infected with an HIV-1 NL4-3 strain was extracted using the NucleoSpin RNA II kit (Macherey-Nagel) in accordance with the manufacturer's instructions.…”

ZNRD1 (Zinc Ribbon Domain–Containing 1) Is a Host Cellular Factor That Influences HIV‐1 Replication and Disease Progression

“…Cells were harvested, washed, and lysed in chilled hypotonic buffer (cell lysis buffer; Invitrogen). After running the samples, membranes were blocked, incubated with primary antibodies (ZNRD1, 1:1000, Abnova; Actin, 1:2000, Santa Cruz Biotechnology) overnight at 4ЊC, and then incubated with a horseradish peroxidase-conjugated antibody [20]. Membranes were revealed with SuperSignal West Pico chemiluminescent substrate (Pierce Biotechnology).…”

“…After 4 h, fresh DMEM was added [20,26]. Plasmid DNA was transfected in SupT1 cells by means of Amaxa Nucleofection technology (Lonza), in accordance with the manufacturer's recommendations [20,26]. Then, cells were recovered and seeded in prewarmed 24-well plates.…”

“…b-Galactosidase activity in 30 mL of cell extract was quantified by a colorimetric assay, as described elsewhere [20,26]. Absorbance was measured at 405-620 nm.…”

“…A day later, siRNA or plasmids were mixed with Lipofectamine 2000 reagent (Invitrogen) in serum-free medium (Invitrogen) and then added to previously washed cells. After 4 h, fresh DMEM was added [20,26]. Plasmid DNA was transfected in SupT1 cells by means of Amaxa Nucleofection technology (Lonza), in accordance with the manufacturer's recommendations [20,26].…”