Cooperative DNA binding mediated by KicGAS/ORF52 oligomerization allows inhibition of DNA-induced phase separation and activation of cGAS

Abstract

Cyclic GMP-AMP synthase (cGAS) is a key DNA sensor that detects aberrant cytosolic DNA arising from pathogen invasions or genotoxic stresses. Upon binding to DNA, cGAS is activated and catalyzes the synthesis of cyclic GMP-AMP (cGAMP), which induces potent antimicrobial and antitumor responses. Kaposi sarcoma-associated herpesvirus (KSHV) is a human DNA tumor virus that causes Kaposi sarcoma and several other malignancies. We previously reported that KSHV inhibitor of cGAS (KicGAS) encoded by ORF52, inhibits cGAS enzymatic activity, but the underlying mechanisms remained unclear. To define the inhibitory mechanisms, here we performed in-depth biochemical and functional characterizations of KicGAS, and mapped its functional domains. We found KicGAS self-oligomerizes and binds to double stranded DNA cooperatively. This self-oligomerization is essential for its DNA binding and cGAS inhibition. Interestingly, KicGAS forms liquid droplets upon binding to DNA, which requires collective multivalent interactions with DNA mediated by both structured and disordered domains coordinated through the self-oligomerization of KicGAS. We also observed that KicGAS inhibits the DNA-induced phase separation and activation of cGAS. Our findings reveal a novel mechanism by which DNA viruses target the host protein phase separation for suppression of the host sensing of viral nucleic acids.

Structural basis of higher order oligomerization of KSHV inhibitor of cGAS

“…3 B ). Previously, we have shown that single mutation of I21, L24, N28, or L31 to alanine shifted KicGAS elution positions from oligomers to dimers ( 15 ). Here we found that change of L17 to the bulky arginine residue also shifted the peak elution position from oligomers to dimers, affirming the critical roles of these residues in forming α1-mediated dimer interface ( SI Appendix , Fig.…”

“…Although no homolog has been identified in betaherpesviruses, ORF52 is found to be structurally related to VP22 in alphaherpesviruses such as herpes simplex virus 1 (HSV-1) ( 14 ). We have reported that KicGAS and its homologs from other gammaherpesviruses inhibit cGAS activity in vitro ( 15 ). Recently, we and others found that KicGAS undergoes liquid–liquid phase separation upon binding with DNA ( 15 , 16 ).…”

“…We have reported that KicGAS and its homologs from other gammaherpesviruses inhibit cGAS activity in vitro ( 15 ). Recently, we and others found that KicGAS undergoes liquid–liquid phase separation upon binding with DNA ( 15 , 16 ). The DNA-induced phase separation of KicGAS correlates well with its ability to inhibit cGAS.…”

“…Any mutations that abrogate DNA-induced KicGAS phase separation also abolish the inhibition of cGAS phase separation and activity. Purified KicGAS protein exists as an oligomer and binds to DNA cooperatively ( 15 ). Mutations that disrupt KicGAS oligomerization also abolish DNA binding and cGAS inhibition, suggesting KicGAS oligomerization is critical for its functions ( 10 , 15 ).…”

“…Purified KicGAS protein exists as an oligomer and binds to DNA cooperatively ( 15 ). Mutations that disrupt KicGAS oligomerization also abolish DNA binding and cGAS inhibition, suggesting KicGAS oligomerization is critical for its functions ( 10 , 15 ). Collectively, oligomerization of KicGAS underlies cooperative and multivalent interaction with DNA that is essential for phase separation of KicGAS itself and the inhibition of cGAS phase separation and activity ( 15 , 16 ).…”