Three-Dimensional Imaging of CD63 Recruitment at the Virological Synapse: HIV-1

The infection of cells by human immunodeficiency virus-1 (HIV-1) occurs most commonly by direct virion transfer via cell–cell contact. ¹ The particles pass through cell contact-induced structures that in analogy to the immunological synapses are called virological synapses. ² Interaction between infected and uninfected cells is the main route of viral spread in vitro and therefore it is likely that it also plays an important role in vivo in infected humans. Contact with the target cells results in relocalization of HIV-1 to the site of interaction and release of the virus at this site. At the virological synapses the viral envelope (Env) protein, the cellular receptor CD4, and the coreceptors colocalize. This requires cytoskeletal rearrangements; in addition, lipid raft domains are enriched at the synapse and cholesterol is critical. Tetraspanin proteins such as CD81 and CD63 were characterized by four transmembrane domains, and they were also found to accumulate at the virological synapse. ³

Working in the field of cell–cell transfer of HIV-1 and protein–protein interactions at the virological synapse, we demonstrated by three-dimensional (3D) confocal imaging that the tetraspanin CD63 is predominantly present at the virological synapse (Fig. 1). Jurkat cells were nucleofected with the vector pHIV-1_JR-FL Gag-iGFP expressing the Gag-internal green fluorescent protein (iGFP) and uninfected Jurkat cells were added. Endogenous expressed CD63 was immunostained and localized by confocal laser microscopy in order to obtain a 3D image from cells in contact. To note, we recently demonstrated that CD63 interacts with the transmembrane envelope protein gp41 of HIV-1. ⁴ We conclude that HIV virions are routed through the virological synapse utilizing host proteins such as CD63.

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FIG. 1.

Three-dimensional (3D) imaging of CD63 and HIV-1 shows the formation of a virological synapse between cells. Jurkat cells were nucleofected with the molecular clone HIV-1_JR-FL Gag-iGFP and produce viral particles (green). The endogenous CD63 was stained with anti-CD63 antibodies (magenta; the brightness level represents signal intensity) and nuclei were stained with DAPI (blue). HIV-1 cell–cell transfer occurs at the virological synapse, which is formed between HIV-1 producer and acceptor cells where CD63 accumulates. The image was obtained with an inverted confocal laser microscope (Zeiss cLSM 780) by taking a stack of optical sections through the sample (z-stack) and 3D projection using Zeiss ZEN software. We used a Gaussian filter as the unsharp mask (Adobe Photoshop) to produce a sharper image.