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.

Graphical Abstract

KicGAS competes with the DNA-induced phase–separation of cGAS and inhibits its enzymatic activity. Oligomerization deficient mutant and the charge deficient mutant of KicGAS are unable to bind DNA and inhibit cGAS efficiently.

Figure 1.

KicGAS requires all the three domains for efficiently inhibiting cGAS. (A, B) Full length protein efficiently inhibits cGAS in a dose dependent manner. (A) cGAS activity assay was performed in absence and presence of different concentrations of KicGAS. (B) Fraction of cGAS enzymatic activity retained in presence of different concentrations of KicGAS. (C, D) Deletion of any domain results reduction in cGAS inhibition. (C) Diagram of the domain truncated constructs of KicGAS. (D) Plot of the fraction of inhibition of cGAS activity in presence different concentration of truncated constructs as inhibitor.

Figure 2.

Characterization of the DNA binding of KicGAS. (A, B) KicGAS binds efficiently to dsDNA. Electrophoretic mobility shift assay for the binding of (A) ISD25 and (B) ISD45 with KicGAS (concentration of KicGAS is shown in μM). The EMSA results in the top panel were fitted to the Hill equation to obtain dissociation constant (K_d) and the Hill coefficient (n). The fitted curves were shown in the bottom panel. (C–F) KicGAS shows specificity towards dsDNA and the binding is DNA length dependent but sequence independent. Binding curves of FL KicGAS with (C) different length of double stranded DNA, (D) different length of single stranded DNA, (E) RNA and DNA–RNA hybrid, (F) DNA containing different percentage of GC.

Figure 3.

Identification of the conserved residues in domain I, that are crucial for KicGAS oligomerization and cGAS inhibition. (A) Size exclusion chromatograph (Superose 6 increase) of the FL KicGAS and domain I mutants I21A, L24A, N28A and L31A (B) The domain III is disordered. Circular dichroic spectra of FL KicGAS, ΔD3 and D3. (C) Bar graph showing the fraction of cGAS activity retained in presence of domain I mutants (at cGAS:KicGAS/mutant = 1:6 molar ratio).

Figure 4.

The positively charged cluster in the disordered domain III is crucial for DNA binding and cGAS inhibition. (A) Binding curves for the interaction of Arginine mutants in domain III with ISD45. (B) Fraction of inhibition of cGAS activity in presence different concentration of these mutants. (C, D) The effect of the distance between the R-cluster (domain III) and the core domain on KicGAS DNA binding and cGAS inhibition. (C) DNA binding curves for distance mutants obtained from Fluorescence Polarization experiment. (D) Fraction of inhibition of cGAS activity in presence different concentrations of distance mutants of KicGAS.

Figure 5.

DNA binding promotes phase separation of KicGAS and DNA binding deficient mutants showed reduced ability of condensate formation. (A) DNA condensation assays of Cy3- ISD100 with KicGAS protein as monitored by spin down assay. (B) Western blot analysis for the supernatant and pellet obtained after spin-down assay. The number 1–3 corresponds to the respective samples in (A). T: Total protein before addition of DNA, S: Protein in the supernatant after spin down, P: Protein in the droplet after spin down, M: Protein size Markar (C) DNA condensation assay for all KicGAS domain I mutants with Cy3– ISD100. (D) The liquid-phase separation ability of KicGAS and its mutants as monitored by turbidity at different salt concentration.

Figure 6.

KicGAS underwent phase separation upon binding with DNA. (A) Representative fluorescent images of KicGAS–DNA phase separation. (B) Representative micrographs of FRAP experiments on partial bleaching of KicGAS–DNA condensates. (C) Quantification of FRAP of KicGAS–DNA liquid droplets in (B). Bleaching was performed at 30 min after KicGAS (30 μM) and DNA (30 μM) were mixed, and the recovery was allowed to occur at 25°C. Time 0 indicates the start of recovery after photobleaching. Shown are the means ± SD. n = 3 photobleaching bleaching areas in one liquid droplet.

Figure 7.

KicGAS competitively inhibited cGAS phase separation with DNA. (A) Representative fluorescent images of cGAS–DNA phase separation in the absence and presence of KicGAS at indicated concentrations. (B) Plot of Cy3 DNA, AF488 cGAS and AF647 KicGAS fluorescence intensities in the presence of increasing concentrations of KicGAS. Data were normalized by maximum value to 1. Values shown are means ± standard error. In both (A) and (B), cGAS: 10 μM, 3% Alexa Fluor 488-labeled; DNA: 10 μM, 2% Cy3-labeled. 200 nM Alexa Fluor 647 labeled KicGAS was supplemented for each titration.

Figure 8.

DNA binding deficient mutants of KicGAS have less inhibition on cGAS phase separation with DNA than WT KicGAS. (A) Representative fluorescent images of cGAS–DNA phase separation in presence of different mutants of KicGAS (10 μM). cGAS: 10 μM, 3% Alexa Fluor 488-labeled; 45-bp ISD: 10 μM, 2% Cy3-labeled. AF488, Alexa Fluor 488. (B) Quantification plot of AF488 cGAS and Cy3 DNA fluorescent intensities in (A). Data were normalized by maximum value to 1. Values shown are means ± standard error.