Targeting of Kaposi's sarcoma-associated herpesvirus by immunotoxins directed against the viral G protein-coupled receptor, ORF74.

BACKGROUND AND PURPOSE: Kaposi's sarcoma-associated herpesvirus (KSHV) is a common virus with severe outcome and no effective antiviral treatment. KSHV encodes the constitutive active chemokine receptor ORF74 with broad-spectrum CXC-chemokine binding. Here, we leverage ORF74's mimic of endogenous receptors to design chemokine-based immunotoxins for selective killing of KSHV-infected cells. EXPERIMENTAL APPROACH: Four CXC-chemokines with high affinity to ORF74 were fused to domain II, IB, and III of Pseudomonas exotoxin A to generate fusion toxin proteins (FTPs). FTP-induced cell killing was tested in cells expressing ORF74 or one of four chemokine receptors (CXCR1-4). Internalization of all receptors was probed using SNAP-tagged receptors. Second-generation FTPs were designed from receptor structures and molecular modelling to increase selectivity for ORF74 over CXCR1-4. Finally, antiviral activity of FTPs was tested using genetically engineered KSHV. KEY RESULTS: FTPs, based on the agonists (CXCL1, and -8) and inverse agonists (CXCL10 and -12) of ORF74 potently killed ORF74-expressing cells. The inverse agonist based FTPs leveraged constitutive internalization for efficient toxin delivery via ORF74, whereas agonists increased internalization further. CXCL10-FTP had the strongest cell-killing and, as the only FTP, selectivity for ORF74 over its endogenous receptor, CXCR3. Second-generation FTPs improved this selectivity from 25-fold to 126-fold by the mutation (R8D) in CXCL10-FTP, designed to lose ionic interaction within CXCR3's main binding pocket. Both inverse agonist-based FTPs effectively prevented KSHV-reactivation. CONCLUSION AND IMPLICATIONS: Our findings highlight the versatility of FTPs in precise delivery of toxin payloads and provide a foundation for potential applications in antiviral and anticancer therapies targeting KSHV-associated diseases.