Fig. 1: VUN100bv binds and inhibits US28 signaling. | Nature Communications

Fig. 1: VUN100bv binds and inhibits US28 signaling.

From: Targeting the latent human cytomegalovirus reservoir for T-cell-mediated killing with virus-specific nanobodies

Fig. 1

a ELISA binding of monovalent VUN100 and bivalent VUN100bv to membrane extracts of US28-expressing HEK293T cells. Representative figure of three independent experiments. b Displacement of 125I-CX3CL1 from US28-expressing membranes by unlabeled ligand or the nanobodies VUN100 and VUN100bv. Representative figure of three independent experiments. c Effect of nanobodies on US28-mediated NFAT (nuclear factor of activated T cells) activation. HEK293T cells expressing either NFAT-luciferase reporter only (Mock) or NFAT-luciferase reporter together with US28 wildtype receptor (WT), US28 Y16F mutant (Y16F), US28 ΔN (2–22) mutant (ΔN (2–22)) or US28 R129A mutant (R129A). Cells were untreated (untr) or treated with a non-targeting nanobody (NT Nb), VUN100, or VUN100bv for 24 h prior to luminescence measurement. Data were normalized to the untreated WT samples. Representative figure of three independent experiments. d Immunofluorescence microscopy of nanobody binding to US28-expressing THP-1 cells. US28 was detected using a polyclonal rabbit-anti-US28 antibody (US28 mAb). Cells were incubated without nanobody (No Nb), an NT Nb, VUN100, or VUN100bv. Bound nanobody was detected using the Myc-tag present on the nanobodies and an anti-Myc antibody (Nb). Representative figure of three independent experiments. e Western blot detection for total IFI16 levels of lysates of untreated THP-1 mock transduced cells (THP-1 Mock) or US28-expressing THP-1 cells (THP-1 US28 WT). THP-1 US28 WT cells were untreated (Untr) or treated with NT Nb, VUN100, or VUN100bv for 48 h. IFI16 protein levels were determined and normalized to actin protein levels. Relative IFI16 protein levels were normalized to untreated THP-1 mock cell lysates. n = 3 independent experiments from three independent biological replicates. All data are plotted as mean ± S.D. For all data, except for Fig. 1c, statistical analyses were performed using an unpaired two-tailed t test. For Fig. 1c, statistical significance was determined using the Holm–Sidak method (two-sided with alpha = 0.05). Source data are provided as a Source Data file.

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