Fig 2: NRP1 facilitates KSHV binding and internalization.(A) Binding and internalization assay of KSHV in PDLSC. PDLSC with EPHA2, NRP1, or TGFBR2 knockout were infected with KSHV at 4°C (for binding) for 3 hours or at 37°C (for internalization) for 4 hours followed by relative quantitative real-time PCR analysis for KSHV copy numbers. (B) Binding and internalization assay of KSHV in 293T cells. 293T cells transfected with expression plasmids for EPHA2, NRP1, or TGFBR1/2 were infected with KSHV at 4°C (for binding) for 3 hours or at 37°C (for internalization) for 4 hours followed by relative quantitative real-time PCR analysis for KSHV copy numbers. (C) Cell-based KSHV fusion assay. 293T cells transfected with expression plasmids for pT7-EMCLuc, pRL-SV40, and KSHV gB/gH/gL were cocultured with 293T cells cotransfected with expression plasmid for T7 polymerase and EPHA2, NRP1, or TGFBR1/2. The relative fusion activity was calculated as the ratio of firefly to Renilla luciferase activity 24 hours after coculture, with the empty vector (EV) controls set to 100%. (D) NRP1 colocalizes with KSHV. 293T cells transfected with PCR3.1-NRP1-myc plasmid and PDLSC were infected with biotin-labeled KSHV followed by immunofluorescence analysis. The NRP1 protein was stained using an anti–c-myc antibody (for 293T cells) or anti-NRP1 antibody (for PDLSC). KSHV were stained by streptavidin-Cy3. The colocalization profile on the right was analyzed by ImageJ software with the plot profile analysis function, which describe the intensity change of the fluorescence signal from left to right in the “Enlarge” image. Results are representative of two or three independent experiments. Data are means ± SEM. One-way ANOVA was carried out with Tukey’s correction for multiple comparisons. *P < 0.05; ***P < 0.001; ****P < 0.0001.

Fig 3: Loss-of-function and gain-of-function analysis show that EphA2 and NRP1 promote KSHV infection.(A to C) Knockout of NRP1, EPHA2, and TGFBR2 individually reduces KSHV infection. PDLSC, DPSC, and GMSC transduced with sgRNA targeting NRP1 (A), EPHA2 (B), or TGFBR2 (C) by lentivirus were analyzed for the protein expression by Western blotting (top) or for the KSHV infection efficiency by flow cytometry (bottom). (D) Effect of overexpression of NRP1, EPHA2, and TGFBR1/2 on KSHV infection. 293T cells transfected with expression plasmids for NRP1, EphA2, or TGFBR1 together with TGFBR2 (TGFBR1/2) were analyzed for the protein expression by Western blotting (top) or for the KSHV infection efficiency by flow cytometry (bottom). (E) Endogenous expression levels of NRP1, EPHA2, and TGFBR2 in PDLSC and human umbilical cord endothelial cells (HUVECs). Results are representative of two or three independent experiments. Data are means ± SEM. One-way analysis of variance (ANOVA) was carried out with Tukey’s correction for multiple comparisons (A to C). Comparison between two groups was performed by two-tailed unpaired Student’s t test (D). **P < 0.01; ****P < 0.0001; ns, no significance.