# Eph receptor tyrosine kinases are functional entry receptors for murine gammaherpesvirus 68

## TL;DR

This study shows that Eph receptors help murine gammaherpesvirus 68 (MHV68) enter cells, similar to how they help human viruses like KSHV and EBV, and suggests these receptors could be targets for vaccines.

## Contribution

The study identifies EphA4 and EphB3 as functional entry receptors for MHV68 and shows their role in viral infection and antibody neutralization.

## Key findings

- MHV68 gH/gL interacts with EphA4 and EphB3 in both human and mouse cells.
- Blocking Eph receptors with decoy proteins or antibodies reduces MHV68 infection.
- Antibodies targeting the gH N-terminal domain neutralize MHV68 in cell culture.

## Abstract

Interactions between viral glycoproteins and cellular receptors determine virus tropism and represent promising targets for vaccines. Eph receptor tyrosine kinases are conserved receptors for the human oncogenic gammaherpesviruses, Kaposi sarcoma herpesvirus (KSHV) and Epstein-Barr virus (EBV), and mediate entry into target cells by interaction with the viral gH/gL glycoprotein complex. To evaluate the use of murine gammaherpesvirus 68 (MHV68), a natural pathogen of rodents, as an in vivo model system for early events in gammaherpesvirus infection, we characterized the interaction of the MHV68 gH/gL complex with Eph receptors. We demonstrate a direct interaction of MHV68 gH/gL with EphA4 and EphB3, that is conserved between human and murine receptors. Pre-incubation of MHV68 inocula with soluble decoy receptors decreased infection of permissive fibroblasts. Ectopic expression of EphA4 and EphB3 enabled MHV68 to infect otherwise non-permissive human B cells, demonstrating EphA4 and EphB3 receptor function. Targeted mutations informed by protein structure predictions demonstrate that the MHV68 gH/gL-Eph interaction is determined by domain I (D-I) and follows structural motifs previously described in the KSHV gH/gL-EphA2 complex. The importance of gH D-I is further highlighted by the analysis of gH-targeting neutralizing antibodies. Antibody adsorption via the full gH ectodomain or gH D-I led to comparable reductions in neutralization capacity of serum from WT infected mice, indicating the Eph-binding domain is a major target for gH/gL-directed neutralizing antibodies. Our study characterizes Eph receptors as novel interaction partners and entry receptors for MHV68. Conservation of entry mechanisms provides the basis for future in vivo analyses of the contribution of Eph receptors to cell-type dependent MHV68 infection, as well as targeted strategies to prevent transmission and diseases associated with chronic infection.

Virus-specific intervention and prevention is needed to fight cancers and morbidity caused by the oncoviruses Kaposi sarcoma herpesvirus (KSHV) and Epstein-Barr Virus (EBV). The targeted design of such strategies requires a detailed knowledge of early events in the viral life cycle and the contribution of cellular receptors to infection and spread in vivo. Here, we characterize the role of Eph receptors in murine gammaherpesvirus 68 (MHV68) infection, a well-established mouse model for gammaherpesvirus (GHV) pathogenesis and preclinical therapeutic evaluations. As described for human GHVs, the interaction of Eph receptors with the gH/gL complex is mediated by a conserved motif comprised of the N-terminal domain of glycoprotein gH co-folded with gL and contributes to variable levels to infection of different target cells. We demonstrate that antibodies directed against the N-terminal domain of the gH/gL glycoprotein complex are generated upon MHV68 infection in mice and neutralize MHV68 infection in cell culture. Our data enables the analysis of the contribution of Eph receptor engagement to GHV infection in vivo and provides a promising target for subunit vaccination approaches.