Eph-binding-negative RRV and KSHV mutants exhibit normal attachment and reduced specific infectivity.

A Attachment of KSHV on LECs is not affected by mutational ablation of the Eph interaction. Cells were incubated with cold virus at the indicated concentrations at 4°C for 30min followed by genomic DNA isolation. The ratio of viral to cellular DNA as a measurement for attached virus was calculated based on ΔCt values of a genomic (CCR5) and a viral locus (ORF59, KSHV or ORF73, RRV) as determined by qPCR and plotted against input viral genome number. B Attachment of RRV on LECs is not affected by mutational ablation of the Eph interaction. Attachment was determined as in A. C-D Eph-binding-negative RRV and KSHV mutants exhibit a reduced specific infection. Target cells were infected with KSHV wt and gH-ELAAN (C) or RRV wt, gH-AELAAN and ΔgL (D) at the indicated virus concentrations. GFP (KSHV) or YFP (RRV) expression as indicator of infection was measured by flow cytometry. Solid lines represent one representive experiment (triplicates, error bars indicate sd). Dotted lines represent non-linear fitting of combined representative experiments of three independent pairs (KSHV) or two independent triplets (RRV) of virus stocks. The ratio K_wt/K_mutant of the rate constant K of fitted curves for wt (K_wt) and mutant viruses (K_ELAAN, K_AELAAN, K_ΔgL) represents the effect of introduced mutations on specific infectivity. Bar graphs represent infections achieved by two specific input virus concentrations normalized to genome copies for one representative experiment per cell type. *: p-value < 0.05, **: p-value < 0.01, ***: p-value < 0.001.