Nano-Luciferase complementation assay of human herpesvirus 8 chemo/cytokine-receptor interactions

Some viruses encode proteins that mimic host chemokines and/or cytokines to modulate the host immune responses against infection. Human herpesvirus 8 (HHV-8), also termed Kaposi sarcoma-associated herpesvirus (KSHV), encodes three viral CC-type chemokines (vCCLs 1-3) and an interleukin-6 ortholog (vIL-6). Therapeutic targeting of the vCCLs and vIL-6 has yet to be established despite their importance in immune evasion and transformation by HHV-8. The present study aimed to design a robust and straightforward method to assay receptor binding and downstream signaling using a Nano-Luciferase Binary Technology reporter system (NanoBiT); with this approach, we verified the direct interactions of vCCLs 1-3 with their established receptors. Furthermore, screening with a comprehensive set of human chemokine receptors revealed additional vCCL-interacting receptors, including ACKR4. The vCCL-receptor interactions were validated using competition, antibody-mediated neutralization, or mutagenesis of vCCLs. We extended the utility of NanoBiT to assess the intracellular protein-protein interactions involved in vCCL-induced downstream signaling via b-arrestin. Likewise, we used NanoBiT to monitor vIL-6 binding to its receptor gp130 and downstream signaling via STAT3. In sum, we demonstrate the utility of NanoBiT for assessing viral chemo/cytokine-receptor interactions and signaling and its potential in screening for antibodies to treat HHV-8 infection and related diseases.