EBV gH/gL and KSHV gH/gL bind to different sites on EphA2 to trigger fusion.

Both Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are human γ-herpesviruses and are important in a variety of malignancies. Eph family receptor tyrosine kinase A2 (EphA2) is a cellular receptor for KSHV and EBV. Previous studies identified 5 conserved residues (ELEFN50-54) in the N-terminal domain of KSHV gH that are critical for Eph-binding and KSHV infection. However, the specific domains of EBV gH/gL important for EphA2 binding are not well described. We found that the KSHV gH (ELEFN50-54) motif is important for higher KSHV fusion and that EBV gH/gL does not utilize a similar motif for fusion activity. We previously identified that an EBV gL N-glycosylation mutant (gL-N69L/S71V) was hyperfusogenic in epithelial cells but not in B cells. To determine if this glycosylation site may be the binding region for EphA2, we compared the EphA2 binding activity of EBV gH/gL and the EBV gH/gL-N69L/S71V mutant. We found that EBV gH/gL-N69L/S71V had higher binding affinity for EphA2 indicating that the EBV gL N-glycosylation site might be responsible for inhibiting the binding of gH/gL to EphA2. Loss of N-glycosylation at this site may remove steric hindrance that reduces EBV gH/gL binding to EphA2. Additionally, the mutations located in the large groove of EBV gH/gL (R152A and G49C) also have decreased binding with EphA2. Taken together, our data indicates that the binding site of EphA2 on EBV gH/gL is at least in part proximal to the EBV gL glycosylation site, which in part accounts for differences in EphA2 binding affinity by KSHV.Importance Virus entry into target cells is the first step for virus infection. Understanding the overall entry mechanism including the binding mechanism of specific virus glycoproteins with cellular receptors can be useful for the design of small molecular inhibitors and vaccine development. Recently, EphA2 was identified as an important entry receptor for both KSHV and EBV. In our current study, we investigated the required binding sites within EphA2 and EBV gH/gL that mediate the interaction of these two proteins allowing entry into epithelial cells and found it differed in when compared to the interaction of KSHV gH/gL with EphA2. Our discoveries may uncover new potential interventional strategies that block EBV and KSHV infection of target epithelial cells.