Promiscuous binding of CXC chemokines to ORF74 receptors enables immunotoxin approaches in Gammaherpesvirus models

Kaposi sarcoma-associated herpesvirus (KSHV) encodes the constitutively active chemokine receptor ORF74, a key factor in viral pathogenesis. Murine γ-herpesvirus 68 (MHV68) acts as an in vivo model for KSHV biology, but the ligand-binding and signaling functions of its ORF74 homolog are not fully understood. To address this knowledge gap, we studied interactions between MHV68-ORF74 and human and murine chemokines through binding assays and signaling tests. Additionally, we assessed the cytotoxicity of human chemokine-based fusion toxin proteins (FTPs) targeting ORF74. We found that MHV68-ORF74 bound multiple human and murine chemokines with similar affinities; CXCL10 showed the highest affinity in the low nanomolar range and displaced CXCL1. Although it lacked constitutive G_i/G_q signaling, MHV68-ORF74 responded to ELR chemokines as agonists and to CXCL10 as an antagonist, confirming conserved ligand-dependent signaling. The receptor exhibited strong constitutive internalization, enabling intracellular delivery of the FTPs. Among the tested FTPs, CXCL10-FTP was the most potent, specifically killing MHV68-ORF74-expressing cells with a logEC₅₀ of -10.00 ± 0.22, which was 51-fold more potent than in cells without the receptor. In conclusion, we confirmed that human chemokines can effectively target MHV68-ORF74, supporting the use of KSHV-ORF74-directed FTPs in mouse models. However, the similar sensitivity of MHV68-ORF74 and endogenous murine (m)CXCR3 underscores the need for mCXCR3-knockout systems for receptor-specific targeting in vivo.