Interaction of rhadinoviral gH/gL complexes with Eph family receptors.

<p>(<b>A</b>) Binding of soluble gH-Fc/gL complexes to Eph family proteins. 293T cells were transfected with the indicated myc epitope-tagged Eph constructs. The cells were fixed and permeabilized, and both Eph expression and binding of soluble gH-Fc/gL complexes from RRV 26-95, RRV 17577 or KSHV at 10 nM was assayed by flow cytometry. Fc was used as a control. (<b>B</b>) As a semi-quantitative gauge of binding, the ratio of the geometric mean fluorescence intensities for Eph-expression and gH-Fc/gL binding was calculated. The whole area of the dot blot containing cells positive for Cy5 and/or Alexa488 except for the box gate containing negative cells in the lower left corner was analyzed, and the ratio of the geometric mean of the fluorescence intensity (MFI) for bound gH-Fc/gL (Alexa488) over the MFI for Eph expression (Cy5) was calculated. Values for the Fc control protein are shown to the left and represent the assay background.</p

Identification of cellular interaction partners of RRV gH/gL.

<p>(<b>A</b>) Purification of proteins interacting with the ectodomains of RRV 26-95, RRV 17577 and KSHV. Lysates of 293T cells were incubated with gH-Fc/gL (Strep epitope-tagged) complexes from RRV 26-95 or 17577 pre-coupled to Streptactin beads. After elution with desthiobiotin, the eluate was re-precipitated with Protein A sepharose, washed, and subjected to gel electrophoresis followed by colloidal coomassie staining. Arrows indicate Eph-containing bands. (<b>B</b>) Summary of proteins identified in the indicated band with the respective gH/gL complexes. The numbers of peptides identified for each protein in one sample are given in brackets. Where more than one number is given, several samples were analyzed and the first number is from the first sample, the second from the second sample. (<b>C</b>) Pairwise immunoprecipitation of individual Eph proteins with gH/gL of KSHV, RRV 26-95 and RRV 17577. Full length Eph proteins (myc epitope-tagged) were recombinantly expressed in 293T cells. The lysates were normalized for equal expression with the lysate of non-transfected 293T cells. The recombinant Eph proteins were immobilized with myc antibody on Protein G beads in a first round of immunoprecipitation. The immobilized proteins were then incubated with equal amounts of lysates from 293T cells transfected with expression constructs for the respective gH (V5 epitope-tagged)/gL complexes, followed by washing and Western Blot analysis.</p

Differential entry of KSHV and RRV into fibroblasts and endothelial cells.

<p>(<b>A</b>) RRV-GFP 26-95 was pre-incubated with the indicated Eph-Fc fusion proteins at 20 µg/ml or a mix of three at 6.66 µg/ml. The virus was then divided and inoculated onto primary rhesus fibroblasts or HUVEC cells. Entry was quantified by flow cytometry. (n = 3; error bars indicate sd) (<b>B</b>) KSHV was pre-incubated with the indicated Eph-Fc fusion proteins at 20 µg/ml prior to infection of rhesus fibroblasts or HUVEC and entry was quantified as above. (n = 3; error bars indicate sd) (<b>C</b>) HUVEC and rhesus fibroblasts were infected with RRV-GFP 26-95 in the presence of either EphB3-Fc or EGFR-Fc (control) at 10 µg/ml. Pictures were taken on day 1, 2 and 3 after infection (BF: brightfield, green: GFP).</p

Inhibition of KSHV cell-cell transmission into a B cell line by soluble Eph proteins and soluble Ephrins.

<p>BJAB B cells were co-cultured for 10 days (<b>A</b>) or 4 days (<b>B</b>) with lytically induced iSLK.219 cells in the presence of the indicated proteins at 5 µg/ml. The cells were then harvested and analyzed for expression of the GFP reporter gene by flow cytometry. BJAB cells were distinguished from iSLK.219 cells by a two-step gating strategy, first selecting by FSC/SSC for the B cell population and then by gating for CD20 expression. The percentage of green CD20-positive cells from co-culture with non-induced iSLK cells represents the assay background. PBS and EGFR-Fc serve as controls. (n = 3; error bars indicate sd).</p

Rhadinovirus entry is dependent on vesicle acidification.

<p>(<b>A</b>) R8 endothelial cells or rhesus fibroblasts were pre-incubated for 1 h with MBCD or bafilomycin A at the indicated concentrations. The cells were then inoculated with rKSHV.219 (black line, circles) or RRV-GFP 26-95 (black line, diamonds). SIVmac329deltanef-GFP pseudotyped with A-MLV (dashed line, open circles) or VSV-G (dashed line, open boxes) envelope proteins was used in parallel as controls. Entry was assayed by GFP expression after 48 h. (<b>B</b>) The same assay as in (A) was performed with BJAB B cells with RRV-GFP 26-95 (black diamonds). An exclusion dye based cell viability assay (dashed line, open boxes) was included to control for toxicity. (n = 3; error bars indicate sd).</p

Inhibition of KSHV entry by soluble Eph proteins and soluble Ephrins.

<p>(<b>A</b>) rKSHV.219 was pre-incubated with the indicated soluble Eph-Fc fusion proteins at 1 µg/ml for 45 min. Entry was quantified by flow cytometry. (n = 3; error bars indicate sd) (<b>B</b>) Inhibition of KSHV entry by soluble Ephrins. Target cells were pre-incubated with the indicated Ephrin-Fc fusion proteins at 5 µg/ml for 30 min followed by infection with rKSHV.219. Entry was quantified as in (A). PBS and EGFR-Fc serve as controls. (n = 4 for HUVEC and R8, else n = 3; error bars indicate sd).</p

Inhibition of RRV entry by soluble Eph proteins.

<p>(<b>A</b>) Inhibition of RRV entry into cells of different lineages through block with soluble Eph proteins. RRV-GFP 26-95 was pre-incubated with EphA1-Fc, mEphA2-Fc, EphB2-Fc, EphB3-Fc or EGFR-Fc as a control prior to infection. All proteins were used at 1 µg/ml. (n = 4 for rhMVEC, R8 and HUVEC, else n = 3; error bars indicate sd) (<b>B</b>) Dose dependent inhibition of RRV entry by soluble Eph proteins. BJAB, rhesus fibroblasts and 293T cells were infected with RRV-GFP 26-95 that was pre-incubated at the indicated concentrations prior to infection with either murine EphA2-Fc (black boxes), EphB3-Fc (black triangles) or EGFR-Fc (open circles) as control. Viral entry as indicated by GFP expression was quantified by flow cytometry. (n = 3 per data point for 293T and BJAB, n = 2 for rhesus fibroblasts; error bars indicate sd for n = 3 or range for n = 2).</p

Block of RRV entry by soluble Ephrins.

<p>Primary cells and cell lines were incubated with the soluble Ephrin-Fc fusion proteins at 5 µg/ml for 30 minutes prior to infection with GFP encoding RRV 26-95. Entry was quantified by flow cytometry. EGFR-Fc or mEphA2-Fc were used as controls in addition to PBS. (n = 4 for rhMVEC , LEC, and R8, else n = 3; error bars indicate sd).</p

Nuclear delivery of RRV into HUVEC is Eph-dependent.

<p>(<b>A</b>) Western Blot analysis of nuclear fractions (nuclei) and whole cellular lysates (WCL) prepared from HUVEC and rhesus fibroblasts. Whole cellular lysate and nucleic fraction were probed for lamin B (nuclear marker) and tubulin (cytoskeleton). (<b>B</b>) Quantification of RRV genomes in the nuclear fraction 4 h post infection. HUVEC or rhesus fibroblasts were infected with RRV for 4 h. Virus was pre-incubated with EphB3-Fc or EGFR-Fc (control) for 45 minutes. The cells were washed, harvested by trypsinization, and nuclei were prepared. Total DNA was extracted from the nuclei and copy number of the viral genome and the cellular GAPDH locus was quantified by realtime PCR. The ratio of RRV/GAPDH copy number for the control infection (EGFR-Fc) in each cell type was set to 100%. The average of three independent experiments is shown, error bars represent the standard deviation. The difference in HUVEC is significant (p = 0.00003), the difference in rhesus fibroblasts is not (p = 0.125) (n = 3, two sided student's t-test with unequal variance).</p

Comparison of Env239 and Env316 mutations.

<p>*Relative to homologous wild-type Env. N.d., not determined.</p><p>Comparison of Env239 and Env316 mutations.</p