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

## TL;DR

Researchers developed a new treatment targeting Kaposi’s sarcoma-associated herpesvirus (KSHV) by using chemokine-based immunotoxins that selectively kill infected cells.

## Contribution

The study introduces fusion toxin proteins (FTPs) that exploit the KSHV-encoded ORF74 receptor for selective cell killing.

## Key findings

- FTPs based on inverse agonists like CXCL10 showed strong cell-killing activity and selectivity for ORF74 over endogenous receptors.
- Second-generation FTPs improved selectivity for ORF74 by 126-fold through a mutation in CXCL10-FTP.
- Inverse agonist-based FTPs effectively prevented KSHV reactivation.

## Abstract

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.

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.

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.

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.

•Kaposi’s sarcoma-associated herpesvirus (KSHV) is widespread and oncogenic.•Infections with KSHV and related diseases lack effective treatment.•KSHV encodes the constitutive active receptor ORF74, binding many CXC-chemokines.•Endogenous chemokines hijacking inspired the fusion toxin proteins (FTPs) design.•Our FTPs target ORF74, allowing selective killing of KSHV-infected cells.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is widespread and oncogenic.

Infections with KSHV and related diseases lack effective treatment.

KSHV encodes the constitutive active receptor ORF74, binding many CXC-chemokines.

Endogenous chemokines hijacking inspired the fusion toxin proteins (FTPs) design.

Our FTPs target ORF74, allowing selective killing of KSHV-infected cells.