Fig 2: EphA2 phosphorylation at Ser897 is critical for AR-mediated KSHV entry.(a) The Ser897Asn mutation abolishes the phosphorylation of EphA2 by the AR. Plasmids expressing WT EphA2 or its site-specific mutant (S897N) were transfected into 293T cells, with or without co-transfection with a plasmid expressing FLAG-tagged AR. (b, c) Increased nuclear accumulation of KSHV virions by ectopic WT EphA2 is abolished by the S897N mutant and is not rescued by co-transfection with a plasmid expressing FLAG-tagged AR. 293T cells in (a) were infected with KSHV infection (MOI = 1) for 20 min. Cells were processed for immunofluorescence using the indicated antibodies. Each reaction was repeated in, at least, triplicate. Scale bars represent 40 μm. Representative images are shown. (d) The internalized viral particles represented by RFP signal intensity in cells from (c) were analyzed and counted by using Image pro-primier software. (e, f) The EphA2 S897N mutant greatly impairs expression of KSHV viral genes and internalized virions compared with WT EphA2. Cellular RNA and genomic DNA in (b and c) were extracted, respectively, and were subjected to qRT-PCR detection of the expression of LANA, RTA and PAN, or were quantitated by amplification of the LANA gene by qRT-PCR. Data are shown as the mean±SEM; n = 3. One-way ANOVA analysis and unpaired Student’s t-tests were performed on (e) and (d, f) respectively. * p,0.05, ** p,0.01, n.s. p, no significance.

Fig 3: Knockdown of AR expression decreases the efficiency of KSHV infection in endothelial cells.(a) Endogenous AR expression in HUVECs and SLK cells is efficiently impaired by RNA interference of the AR. siRNAs were transfected into cells at final concentrations of 100 nM for HUVECs and 200 nM for SLK cells using Lipofectamine 2000. Cell lysates were subjected to immunoblotting using the indicated antibodies. (b, c). RNA transcription of the AR and its target genes is inhibited by AR siRNA treatment in both HUVECs (b) and SLK cells (c). Total RNA from the above samples was subjected to a qRT-PCR to determine the mRNA levels of the indicated genes. Values were normalized to the level of actin mRNA and calibrated by comparison to a control siRNA treatment. (d) AR inhibition leads to a significant reduction of the number of internalized KSHV DNA copies. The above cells were infected with KSHV for 2 h at 37°C, washed, and cultured for another 22 h, and the MOIs were 10 and 5 for HUVECs and SLK cells, respectively. Cellular genomic DNA was extracted, and internalized KSHV DNA was quantitated by amplification of the LANA gene by qRT-PCR. (e, f) The mRNA expression of KSHV genes is down-regulated by AR inhibition. Total RNA from the above HUVECs (e) and SLK cells (f) was subjected to qRT-PCR to determine the expression of LANA, RTA, and PAN. The data were normalized to the actin expression and then calibrated by the control siRNA treatment. (g, h) Knockdown of AR and EphA2 expression decreases nuclear LANA expression in SLK cells, as determined by an immunofluorescence assay and quantitative analysis by Image J software. The infection time was 48 h in this group. Scale bars represent 10 μm. The randomly selected fifty cells in each image were analyzed and the corrected total cell fluorescence (CTCF) of individual images was calculated as following: CTCF = Integrated Density–(Area of selected cells X Mean fluorescence of background readings). Representative images are shown. Data are shown as the mean±SEM; n = 3. Paired Student’s t-tests and one-way ANOVA analysis was respectively performed on (b, c, e and f) and (d, h). * p,0.05, ** p,0.01, n.s. p, no significance.

Fig 4: EPHA2 cataract mutations in the SAM domain.(A) Schematic diagram showing the domains of EPHA2 receptor and the locations of four SAM domain mutations found in human cataracts (c.2819C>T; c.2915_2916delTG; c.2826-9G>A; and c.2842G>T) in the EPHA2 gene. FN-III: fibronectin type-III domain; TM: transmembrane domain; Kinase: protein tyrosine kinase domain; SAM: sterile-α-motif domain; P: PDZ-binding motif. The SAM domain comprises 5 α-hecices (H1–5). (B) Reduction of mutant EPHA2 protein levels in transfected cells expressing EPHA2 mutants. Protein levels of EPHA2 mutants are decreased in both HEK293T and αTN4-1 cells. The blot was reprobed with anti-α-tubulin as a loading control. The graphs represent the quantification of relative band intensity of EphA2 as connected by the levels of α-tubulin from three independent experiments. Total EphA2 protein band intensity was determined using ImageJ software. Mean values are presented with S.D as indicated. Statistical differences between multiple groups were analyzed using one-way analysis of variance (ANOVA). ***, P<0.001; **, P<0.01; *, P<0.05; and ns, not significant. Values of P<0.05 were considered to be statistically significant. (C, D) No difference between wild-type and mutant EPHA2 genes in transcription levels. (C) Semi-quantitative RT-PCR and (D) Real-time PCR for wild-type and mutant EPHA2 genes were performed using total RNA, isolated from transfected HEK293T cells. GAPDH transcript levels are used as controls. The graphs represent the quantification of western blots from three independent experiments.

Fig 5: Subcellular localization of the wild-type and mutant EPHA2 receptors on transfected EphA2−/− MEF (E13.5) cells.EphA2 knockout MEF cells expressing wild-type or mutant EPHA2 were incubated with clustered ephrin-A5-Fc before fixation and treated with anti-Fc antibodies (red). After washing with PBS, cells were counterstained with anti-EphA2 antibody (green). Images were captured using a Nikon Eclipse C1 confocal microscope. Scale bar, 50 µm.