Imaging of HIV-1 envelope-induced virological synapse and signaling on synthetic lipid bilayers—Prins 2012

Cell-to-cell transmission of HIV-1 is most effective(2,10,11). A virological synapse (VS), an F-actin-dependent cell-cell junction, is formed when HIV-1 envelope gp120 on the infected cell engages CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell (8). In addition to gp120 and its receptors, other membrane proteins, such as the adhesion molecule LFA-1 and its ligands, the ICAM family, are present on virus-infected donor cells, target cells, and HIV-1 virions (1,4,5,6,7,13). VS generation involves intracellular signals transduced by gp120-receptor interaction. We recently found that CD4(+) T cell contact with gp120 recruits and phosphorylates TCR signalosome signaling molecules Lck, CD3, ZAP70, LAT, SLP-76, Itk, and PLC (15). We describe a way to view supramolecular organization and membrane-proximal signaling during VS generation. We use the glass-supported planar bi-layer system to model HIV-infected cells with gp120 and ICAM-1 on their surfaces. The protocol involves bi-layer preparation and assembly in a flow cell, cell injection and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, image acquisition by TIRF microscopy, and data analysis. This method produces high-resolution pictures of the VS interface by detecting different clusters of VS molecular components and specific signaling molecules recruited to these sub-domains.

Prins, K. C., Vasiliver-Shamis, G., Cammer, M., Depoil, D., Dustin, M. L., & Hioe, C. E. (2012). Imaging of HIV-1 envelope-induced virological synapse and signaling on synthetic lipid bilayers. Journal of visualized experiments : JoVE, (61), 3757. https://doi.org/10.3791/3757