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Nuclear soluble cGAS senses DNA virus infection

Yakun Wu, Kun Song, Wenzhuo Hao, Lingyan Wang, Shitao Li

Preprint posted on August 28, 2021 https://www.biorxiv.org/content/10.1101/2021.08.27.457948v1

Viral sensing and its journey to the center of the cell

Selected by Roberto Amadio

 

Background. Mammalian cells detect viral infection by sensing incoming pathogen DNA thanks to germline-encoded receptors. According to the entry strategy, viral DNA can be spotted by either the endosomal toll-like receptor 9 (TLR9) or by different cytosolic sensors. Among cytosolic receptors, research into cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) has gained popularity in recent years owing to the broad expression and relevance of this sensor in multiple cell types and tissues.

After identifying cGAS as a cytosolic sensor, the scientific community has been puzzled by the fact that a considerable fraction of this protein resides within nuclei, meaning it could be exposed to self-DNA. Much effort has been made to understand the molecular mechanisms that keep cGAS silent against host genomic DNA. Intact nucleosomes sequester cGAS in an inactive state, and Barrier-to-Autointegration-Factor (BAF) competes with cGAS for naked genomic DNA binding (1, 2). When cells divide through mitosis and nuclear envelope disassembles, cGAS phosphorylation at multiple sites inhibits its DNA binding affinity and once again prevents activation against host DNA. (3, 4).

However, the presence and possible relevance of free (soluble) nuclear cGAS has not been considered until recently. To date, at least two cGAS Nuclear Localization Signals (NLS) have been mapped (5). A functional Nuclear Export Signal (NES) comprising the basic aminoacidic sequence 169LEKLKL174 and important to ensure cytosolic accumulation in response to DNA transfection, has also been described (6). This evidence points to a complex layer of cGAS nuclear-cytosolic shuttling regulation and suggests that the nucleus might be more than just a cGAS “sequester” to keep it silent against the self. Prompted by these notions, the authors of this preprint tested whether nuclear soluble cGAS exists and plays functional roles.

 

Key findings of this preprint

1) cGAS localization. The authors first tested cGAS localization in several cell types, confirming previous claims and detecting a nuclear fraction in every cell type tested. They nicely validated a novel cGAS antibody and found cGAS in multiple locations in H1299 human non-small cell lung carcinoma cell line (FIG. 1).

Figure 1 – Immunostaining of cGAS localization (green signal) in unstimulated H1299 cells. (i) nucleoli; (ii) micronucleus; (iii) chromosome; (iv) chromatin bridge; (v) perinuclear region. (From preprint fig. 1D)

2) Regulation of nuclear-cytosolic shuttling of cGAS. Deletion of the NES decreased but not completely impaired nuclear cGAS export, suggesting a complex layer of nuclear-cytosolic shuttling. Consistent with this idea, the disordered N-term domain of cGAS (AA 1-160) also plays a role in its localization. Overexpression of the N-term domain alone resulted in a near complete cytosolic localization, whereas deletion of the N-term yielded more chromatin tether, cytoskeletal and mitochondrial-associated cGAS signal (owing to the exposure of an otherwise cryptic mitochondrial targeting signal).

3) Some viral infections may induce nuclear soluble cGAS. Using biochemical fractionation the authors discriminated chromatin-bound (extracted in a high salt concentration) from nuclear soluble cGAS (extracted in a low salt concentration). Importantly, cell stimulation with a DNA virus that replicates inside nuclei (herpes simplex virus type 1, HSV-1), caused diffusion of nuclear cGAS to its soluble fraction. Strikingly, this did not happen upon infection with RNA viruses (i.e. influenza A virus and vesicular stomatitis virus), with a DNA virus that replicates into the cytosol (i.e. vaccinia virus), nor with DNA transfection. This suggests that the presence of viral DNA in the nucleus might be a signal for cGAS to overcome chromatin inhibition and block viral infection at an early stage.

4) Nuclear viral replication unleashes nuclear soluble cGAS. In search of the crucial step responsible for the release of cGAS from its chromatin tethering, the authors observed that inhibition of HSV-1 replication by Acyclovir blocked nuclear soluble cGAS accumulation, indicating that viral replication inside the nucleus initiates cGAS release.

5) Nuclear soluble cGAS is catalytically active. In an in vitro enzymatic assay performed after HSV-1 infection and subsequent subcellular fractionation, the authors detected higher cGAMP levels in nuclear extracts from HSV-1-infected cells compared to mock- treated counterparts. This result suggested the presence of an active nuclear soluble cGAS upon HSV-1 infection.

6) Nuclear soluble cGAS activates an immune response and inhibits HSV-1 infection. Finally, the authors wanted to validate their hypothesis that the release of soluble nuclear cGAS is sufficient to inhibit HSV-1 infection. To do this, they generated a nuclear- restricted cGAS with inducible expression, to avoid potential cytosolic presence of newly synthetized cGAS molecules. NLS tagging alone was not sufficient, but adding a double mutation to transform the NES in a NLS (LL/RK) allowed nuclear-restricted cGAS expression in a time-controlled manner (FIG. 2).

Figure 2 – Schematic visualization of inducible vectors used. TRE: Tet Response Element; PGK: phosphoglycerate kinase promoter; Puro: puromycin; T2A: Thosea asigna virus 2A-like peptide; rTetR: reverse tet repressor. (From preprint fig. 3A)

After transfecting these constructs in cGAS KO RAW macrophages, the authors found that nuclear soluble cGAS can derive from chromatin-bound cGAS alone. Moreover, nuclear soluble cGAS synthetized cGAMP, which in turn activated downstream signaling and induced type I interferon production. The response generated by LL/RK-cGAS limited HSV-1 replication and infection, as visualized by decreased HSV-1-GFP positive cells in a reporter assay.

 

In summary, the authors provide evidence of a novel and previously unappreciated pool of nuclear soluble cGAS with a functional role against HSV-1. This finding moves forward our knowledge of how cells spot viral infections and brings cGAS-mediated viral sensing to “the center of the cell”.

 

Why I chose this preprint

Compartmentalization of DNA receptors is necessary to avoid reaction against the self. Regulation of cGAS beyond its “classical” cytosolic enzymatic activity is a fast moving and exciting field of research. In this preprint the authors cleverly merged experiments to corroborate previous findings and elucidated a new thought-provoking discovery in the viral-host interaction field. I liked the validation of a new antibody as part of the paper, the ability to put together so many new aspects of cGAS regulation, and the discussion the authors made about still-debated issues.

Several synthetic cGAS constructs and mutants are being established, the sharing of which will be important to improve reproducibility of experiments and to clarify contradictory or incomplete results that still exist in the field. In this direction, I think the inducible vectors and the tools for a detailed analysis of nuclear cGAS developed in this study are going to be helpful for future studies of this protein.

 

Questions to the authors

  • What other nuclear DNA sensors (i.e. IFI16) or factors may be involved in helping nuclear soluble cGAS activity or in limiting HSV-1 replication?
  • Would you advise enzymatic independent mechanisms for nuclear soluble cGAS in limiting HSV-1 infection and replication?
  • Did you encounter any difficulties in visualizing cGAS using a commercial kit for biochemical fractionation? Is post-lysis DNA binding of cGAS (7) a factor to consider while performing this kind of subcellular fractionation?
  • You clearly showed that nuclear soluble cGAS appears upon HSV-1 infection from a single pool of nuclear tethered cGAS. But what if a similar experiment would be done using a cytosolic-restricted cGAS construct? Would this be feasible or are there any limitations?
  • Do you think cytosolic-restricted cGAS could limit HSV-1 infection by some means?

 

The wider context of this study

Another paper in 2018 suggested a nuclear cGAS activation by a different angle (8). Lahaye et al. described NONO as a nuclear partner of cGAS in immune cells, important for cGAS nuclear accumulation and detection of HIV-2. This study further highlights an intriguing open question about whether other cellular factors might be also involved in nuclear HSV-1 sensing and which precise signal(s) triggered by HSV-1 replication allow a fraction of nuclear cGAS to become soluble and active in that setting.
I am aware of a report (still at its preprint stage) claiming that a fraction of STING, the downstream adaptor of cGAS, might localize not only to the endoplasmic reticulum but also to the inner and outer nuclear membrane in some cells (9). Giving the new evidence provided by Wu et al. that cGAMP can be synthetized from within nuclei, having STING near the nuclear envelope might be good to maximize detection of nuclear-derived cGAMP. Even though it is hasty to see the small fraction of “nuclear soluble” STING detected by Wu et al. as an indication that nuclear STING might exists, researchers will likely keep a closer eye on both cGAS and STING location in future studies.

 

References

  1. B. Guey, M. Wischnewski, A. Decout, K. Makasheva, M. Kaynak, M. S. Sakar, A. Ablasser, BAF restricts cGAS on nuclear DNA to prevent innate immune activation. Science (80-. ). 369, 823–828 (2020).
  2. T. Kujirai, C. Zierhut, Y. Takizawa, R. Kim, L. Negishi, N. Uruma, S. Hirai, H. Funabiki, H. Kurumizaka, Structural basis for the inhibition of cGAS by nucleosomes. Science (80-. ). 0237, 1–9 (2020).
  3. L. Zhong, M.-M. Hu, L.-J. Bian, Y. Liu, Q. Chen, H.-B. Shu, Phosphorylation of cGAS by CDK1 impairs self-DNA sensing in mitosis. Cell Discov. 6 (2020), doi:10.1038/S41421-020-0162-2.
  4. T. Li, T. Huang, M. Du, X. Chen, F. Du, J. Ren, Z. J. Chen, Phosphorylation and Chromatin Tethering Prevent cGAS activation During Mitosis. Science. 371 (2021), doi:10.1126/SCIENCE.ABC5386.
  5. H. Liu, H. Zhang, X. Wu, D. Ma, J. Wu, L. Wang, Y. Jiang, Y. Fei, C. Zhu, R. Tan, P. Jungblut, G. Pei, A. Dorhoi, Q. Yan, F. Zhang, R. Zheng, S. Liu, H. Liang, Z. Liu, H. Yang, J. Chen, P. Wang, T. Tang, W. Peng, Z. Hu, Z. Xu, X. Huang, J. Wang, H. Li, Y. Zhou, F. Liu, D. Yan, S. H. E. Kaufmann, C. Chen, Z. Mao, B. Ge, Nuclear cGAS suppresses DNA repair and promotes tumorigenesis. Nature. 563, 131–136 (2018).
  6. H. Sun, Y. Huang, S. Mei, F. Xu, X. Liu, F. Zhao, L. Yin, D. Zhang, L. Wei, C. Wu, S. Ma, J. Wang,
    S. Cen, C. Liang, S. Hu, F. Guo, A Nuclear Export Signal Is Required for cGAS to Sense Cytosolic DNA. Cell Rep. 34, 108586 (2021).
  7. K. C. Barnett, J. M. Coronas-Serna, W. Zhou, M. J. Ernandes, A. Cao, P. J. Kranzusch, J. C. Kagan, Phosphoinositide Interactions Position cGAS at the Plasma Membrane to Ensure Efficient Distinction between Self- and Viral DNA. Cell. 176, 1432-1446.e11 (2019).
  8. X. Lahaye, M. Gentili, A. Silvin, C. Conrad, L. Picard, M. Jouve, E. Zueva, M. Maurin, F. Nadalin, G. J. Knott, B. Zhao, F. Du, M. Rio, J. Amiel, A. H. Fox, P. Li, L. Etienne, C. S. Bond, L. Colleaux, N. Manel, NONO Detects the Nuclear HIV Capsid to Promote cGAS-Mediated Innate Immune Activation. Cell. 175, 488-501.e22 (2018).
  9. L. Cheradame, I. C. Guerrera, J. Gaston, A. Schmitt, V. Jung, M. Pouillard, N. Radosevic- Robin, M. Modesti, J.-G. Judde, S. Cairo, V. Goffin, STING Promotes Breast Cancer Cell Survival by an Inflammatory-Independent Nuclear Pathway Enhancing the DNA Damage Response. bioRxiv, 1–44 (2020).

 

Tags: cgas, hsv-1, nucleus, viral sensing

Posted on: 16th September 2021

doi: https://doi.org/10.1242/prelights.30570

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Author's response

Shitao Li shared

 

Thank you for highlighting our work! Here are the responses to your questions:

  • What other nuclear DNA sensors (i.e. IFI16) or factors may be involved in helping nuclear soluble cGAS activity or in limiting HSV-1 replication?

IFI16 is known to interact with cGAS. Whether IFI16 or other factors regulate nuclear cGAS activity and the release of cGAS from chromatin tethering will be investigated in the future.

  • Would you advise enzymatic independent mechanisms for nuclear soluble cGAS in limiting HSV-1 infection and replication?

No, our data showed that the enzymatic activity was required for nuclear soluble cGAS-mediated antiviral activity.

  • Did you encounter any difficulties in visualizing cGAS using a commercial kit for biochemical fractionation? Is post-lysis DNA binding of cGAS (7) a factor to consider while performing this kind of subcellular fractionation?

We did not encounter any difficulties. Cytosolic and membrane fractions were isolated before intact nuclei were lysed.

  • You clearly showed that nuclear soluble cGAS appears upon HSV-1 infection from a single pool of nuclear tethered cGAS. But what if a similar experiment would be done using a cytosolic-restricted cGAS construct? Would this be feasible or are there any limitations?

The role of cytosolic cGAS in DNA sensing is well-established. A previous study showed the Y215E mutant of cGAS only localized in the cytoplasm (Sci Adv. 2020, doi:10.1126/ sciadv.abb8941).

  • Do you think cytosolic-restricted cGAS could limit HSV-1 infection by some means?

Our study does not challenge the current dogma that cGAS is a cytosolic sensor, which plays a major role in host defense to DNA viruses, including HSV-1. Instead, our findings further extend the role of cGAS to nuclear DNA sensing, probably during early HSV-1 infection. 

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