PhD thesis abstract by Dagmar Fæster Kildedal

Targeting ORF74 With Chemokine-based Immunotoxins

Kaposi’s sarcoma–associated herpesvirus (KSHV/HHV8) is an oncogenic herpesvirus that hijacks the host chemokine system to promote immune evasion, viral dissemination, and tumor development. A key mediator of this process is the virally encoded G protein–coupled receptor ORF74, which mimics host chemokine receptors, exhibits constitutive signaling and internalization, and binds a broad range of chemokines. Despite its central role in KSHV pathogenesis, ORF74 has remained largely unexplored as an antiviral target.

To address this gap, this thesis explores chemokine-guided delivery of cytotoxic payloads to ORF74-expressing cells as an antiviral strategy. The work is described in two manuscripts.

In Manuscript I, fusion toxin proteins (FTPs) were developed by coupling four human CXC chemokines, covering agonists, partial agonists, and inverse agonists, to the cytotoxic domain of Pseudomonas exotoxin A. All FTPs effectively eliminated ORF74-expressing cells with nanomolar to picomolar potency, independent of the chemokine pharmacology. The inverse agonist–based CXCL10-FTP showed the highest potency, mainly due to ORF74’s strong constitutive internalization, and was the only FTP to display selectivity for ORF74 over the endogenous receptor CXCR3. A single point mutation (R8D) further enhanced selectivity from 25-fold to 126-fold. In addition, inverse agonist–based FTPs suppressed viral reactivation.

In Manuscript II, the murine ORF74 homolog encoded by murine gammaherpesvirus 68 (MHV68) demonstrated cross-species compatibility, with high-affinity binding to both human and murine chemokines. Although MHV68-ORF74 lacked constitutive signaling, it exhibited robust constitutive internalization, and all FTPs retained high cytotoxic potency, validating MHV68 as a relevant in vivo model.

Together, these findings establish ORF74 as a viable antiviral target and position chemokine-based immunotoxins as a complementary strategy to traditional antivirals that inhibit viral enzymes by enabling the targeted elimination of infected cells.