TRIM56-mediated monoubiquitination of cGAS for cytosolic DNA sensing

Intracellular nucleic acid sensors often undergo sophisticated modifications that are critical for the regulation of antimicrobial responses. Upon recognition of DNA, the cytosolic sensor cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the second messenger cGAMP, which subsequently initiates downstream signaling to induce interferon-αβ (IFNαβ) production. Here we report that TRIM56 E3 ligase-induced monoubiquitination of cGAS is important for cytosolic DNA sensing and IFNαβ production to induce anti-DNA viral immunity. TRIM56 induces the Lys335 monoubiquitination of cGAS, resulting in a marked increase of its dimerization, DNA-binding activity, and cGAMP production. Consequently, TRIM56-deficient cells are defective in cGAS-mediated IFNαβ production upon herpes simplex virus-1 (HSV-1) infection. Furthermore, TRIM56-deficient mice show impaired IFNαβ production and high susceptibility to lethal HSV-1 infection but not to influenza A virus infection. This adds TRIM56 as a crucial component of the cytosolic DNA sensing pathway that induces anti-DNA viral innate immunity.

TRIM proteins have a number of biological functions including immunity, cell proliferation and differentiation, signal transduction and autophagy. Recent evidence indicates several TRIM proteins serve as regulators in several signalling pathways in natural immunity. One of the TRIM proteins, TRIM56, has been reported to be a crucial regulator for viral infection and interferon signals. In this paper, the authors showed that TRIM56 regulates the interferon signalling pathway. The authors found that TRIM56 interacts with cGAS and mono-ubiquitinates cGAS at the Lys335 and that TRIM56 exhibits dimerization of cGAS and DNA-binding activity and cGAMP production. These findings suggest that TRIM52 is a critical molecule for the cytosolic DNA sensing. These insights are relevant in understanding the novel roles of TRIM52 in regulation of the innate immune system. This manuscript contains some important issues, such as the molecular insight of TRIM52 in the ubiquitination on cGAS. Although the experiments have been well performed, there may be some lacks of important controls and overstatements of results that preclude publication of the manuscript in the present form.
Specific points 1. Fig. 3a: The results is not clear. The authors should quantitate the monoubiquitinated forms and analyze statistically using three individual experiments. 2. Fig. 3b: To confirm that the band or smear is monoubiquitinated cGAS, the authors should show the molecular weight markers. 3. Fig. 3d: We can see the slight poly(or multi)-ubiquitinated cGAS at lane 4. Can the authors rule out that polyubiquitinated cGAS by TRIM56 is important in DNA sensing system? 4. In this study, the authors did not directly show that monoubiquitinated cGAS by TRIM56 enhances the dimerization and the binding activity to target DNA. It is very important to show the data substantiating this molecular insights.
Minor points: 1. The Method section (in vitro cGAS activity assay and others): carefully check again. The authors put "space" between numeric values and unit.

Reviewer #1 (Remarks to the Author):
The manuscript entitle "TRIM56-meadiated monoubiquitination of cGAS for cytosolic DNA sensing" by Seo and collaborators explore the involvement of the E3 ligase TRIM56 on cGAS dependent DNA detection and antiviral function. The authors perform a systematic analysis of TRIM56 and cGAS interaction, identifying the domains involved. Also, they characterize the type of ubiquitination involved in the named mechanism and a clear anti HSV function in vitro and in vivo using TRIM56 KO mice. The experiments presented by the authors are very clear and show persuasive evidences of the proposed mechanism.
It is important to confirm that the proposed TRIM56/cGAS interaction is direct. For this the authors need to perform IP with bacteria-produced purified proteins in order to demonstrate the proposed interaction  According to the reviewer's suggestion, we further examined whether TRIM56 directly interacted with cGAS by using bacterially purified cGAS and TRIM56 purified from 293T cells. As shown in the figure below, full-length (FL) cGAS fused with Maltose-Binding Protein (MBP) interacted with TRIM56. However, both MBP-cGAS C-terminal region (a.a.161-522) and MBP alone failed to interact with TRIM56. We have included this result to the revised manuscript. White arrows indicate MBP (left), MBP-cGAS C-terminal 161-522aa (middle) and MBP-cGAS full-length (right), respectively. Strikingly, a publication by the Akira lab (which the authors reference in the manuscript) showed also an antiviral effect of TRIM56 but through a different mechanism that involves interaction with the adaptor STING and its ubiquitination, which they show to be crucial for its interaction with TBK1 and the induction of type I IFN response. Even though, Seo et al. propose a TRIM56 effect on cGAS only. They only show one experiment using cGAMPs as STING stimulator, showing no effect. In these experiments (Fig 2) the authors do not see an effect on cGAMP detection by STING. In order to confirm this hypothesis, they need to explore with more detail the effect of TRIM56 on the DNA sensing versus RNA sensing pathways, since both STING and cGAS have shown antiviral properties for DNA and RNA viruses, in particular STING which can crosstalk to the RIG-I like and DNA sensing pathways. Visualization of endogenous TRIM56 in mock, DNA transfection, DNA virus infection and RNA virus infection conditions would help to clarify this. Also co-localization of cGAS and STING will give more relevance to the experiment and the paper.
 To comprehensively understand the role of TRIM56 on the cGAS-STING pathway upon DNA or RNA virus infection, we observed the co-localization of exogenously expressed cGAS or STING with endogenous TRIM56. Both endogenous TRIM56 and exogenous GFP-cGAS effectively co-localized at the indicated foci upon HT-DNA stimulation or Herpes Simplex Virus-1 (HSV-1) infection, but not upon Sendai virus infection (please see the figure with arrowheads below). However, STING did not co-localize with either cGAS or TRIM56 in any conditions. We have included this result in the revised manuscript.
Regarding the role of TRIM56/cGAS on virus detection, the authors only use SeV and Influenza (negative ssRNA viruses) as control. It has been shown that cGAS and STING play a role not only for DNA viruses but also for positive sense RNA viruses, but not for negative sense RNA viruses (Charles Rice lab, Schoggins et al Nature 2013). The authors should add a positive sense RNA virus and reevaluate the role of TRIM56 on infection and antiviral response  As the reviewer suggested, we examined whether TRIM56 is involved in the host's response to positive-sense RNA viruses. As shown in Figure  WT and TRIM56 -/-BMDMs were infected with Sindbis virus and viral titers were measured at different time points using a standard plaque formation assay on Vero cells. It showed similar viral titers of Sindbis virus in WT and TRIM56 -/-BMDMs (Figure f). Finally, WT and TRIM56 -/-BMDMs also showed similar viral RNA loads of ZIKV MR766 strain and ZIKV H/PF2013 strain during 48h infection periods (Figure g). These results indicate that TRIM56 is not required for the host response to control positive-sense RNA viruses. We have included these results to our revised manuscript.
I also suggest to repeat the TRIM56 and STING immunoprecipitation experiment shown by the Akira lab and add it as supplementary figure.  As the reviewer suggested, we conducted a co-immunoprecipitation experiment to determine the interaction between cGAS or STING with TRIM56. TRIM56 co-immunoprecipitated with cGAS, not STING, even in stringent binding and washing conditions (500mM NaCl). This shows that cGAS specifically binds to TRIM56. We have included these results to the revised manuscript.