Activation of NF-kappaB by the latent vFLIP gene of Kaposi's sarcoma-associated herpesvirus is required for the spindle shape of virus-infected endothelial cells and contributes to their proinflammatory phenotype

Abstract

Kaposi's sarcoma (KS) is an inflammatory angioproliferative lesion induced by the infection of endothelial cells with the KS-associated herpesvirus (KSHV). Infected endothelial cells assume an elongated (spindle) shape that is one of the histologic signatures of KS. In vitro, latent viral infection of primary endothelial cells (but no other cell type) strikingly recapitulates these morphological findings. Here we report that the spindling phenotype involves major rearrangement of the actin cytoskeleton and can be attributed to the expression of a single viral protein, vFLIP, a known activator of NF-kappaB. Consistent with this, the inhibition of NF-kappaB activation blocks vFLIP-induced spindling in cultured endothelial cells. vFLIP expression in spindle cells also induces the production of a variety of proinflammatory cytokines and cell surface adhesion proteins that likely contribute to the inflammatory component of KS lesions.

FIG. 1.

Infection of endothelial cells with KSHV causes the formation of spindle cells. Primary HUVECs, LECs, and BECs were obtained as described in Materials and Methods. (a to f) HUVECs, LECs, and BECs were subjected to infection with KSHV at high MOIs for 2 h and rinsed and medium was replaced. (a to c) LANA, green; K8.1, red; DAPI (4′,6′-diamidino-2-phenylindole), blue. Images were taken at 3 days postinfection. (g to i) Mock- and KSHV-infected LECs and KSHV-infected BECs were stained with FITC-coupled phalloidin.

FIG. 2.

vFLIP expression alone causes spindle cell formation in HUVECs, LECs, and BECs. (a) HUVECs were transduced with retroviruses encoding the individual latency-associated genes and subjected to selection with 0.5 μg/ml puromycin. Images were taken at 3 days posttransduction. (b) LECs and BECs were transduced with retroviruses encoding vFLIP or empty vector. The cells were then selected with 0.25 μg/ml puromycin. Images were taken at 2 days posttransduction.

FIG. 3.

NF-κB activation correlates with spindle cell formation and upregulates inflammatory cytokines and markers of endothelial activation. (a) Bars 1 and 2, Spindled vFLIP-expressing HUVECs (1) and HUVECs expressing vector alone (2) were transfected with 0.5 μg NF-κB-luciferase reporter construct and 0.5 μg β-galactosidase-encoding plasmid to normalize for transfection efficiency. Forty-eight hours posttransfection, luciferase and β-galactosidase assays were performed; bars show normalized levels of luciferase activity. Bars 3, 4, and 5, 293 cells were transfected with equal amounts of vector (3), vFLIP (4), or E8 (5) plasmids along with an NF-κB-luciferase reporter and β-galactosidase construct. Error bars indicate standard deviations. Assays were performed at 48 h posttransfection. (b) HUVECs were transduced with retrovirus encoding either vFLIP, E8, or empty vector and selected with puromycin as described before. Images were taken 3 days posttransduction. (c) Supernatant conditioned for 24 h by fully spindled HUVECs expressing vFLIP was applied to the RayBio human cytokine antibody array V as per the manufacturer's indications. The cytokines indicated by arrows are those found to be increased over conditioned medium from vector-expressing HUVECs assayed in parallel. (d) Medium from vFLIP or empty vector-expressing HUVECs was assayed for content of IL-6 or granulocyte-macrophage colony-stimulating factor (GM-CSF) by ELISA. Error bars indicate standard deviations. (e) Spindle cells expressing vFLIP (thick line) or vector-transduced HUVECs (thin line) were stained for surface expression of VCAM-1 and examined by flow cytometry. Numbers next to histograms indicate geographic means of expression levels.

FIG. 4.

Inhibition of NF-κB prevents spindle cell formation. (a) HUVECs were pretreated with either 4 μM Bay 11-7082 (lower panels) or DMSO (top panels) for 2 h before being transduced at high MOIs with retroviruses encoding vFLIP. After transduction, medium containing either Bay 11-7082 or DMSO was added back. At 3 days posttransduction, the cells were photographed (right panels) or fixed and stained with a p65-specific antibody (left panels). (b) HUVECs were transduced at high MOIs with retrovirus coding for either the IκB superrepressor (lower panel) or empty vector (upper panel) and then selected in 0.5 μg/ml puromycin for several days. The cells were then superinfected at high MOIs with retrovirus encoding vFLIP, and their morphologies were assayed at 5 days posttransduction.