Viewpoint Cite This: Biochemistry XXXX, XXX, XXX−XXX
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A Poxin on Both of Your Houses: Poxviruses Degrade the Immune Signal cGAMP Andrew B. Dippel†,‡ and Ming C. Hammond*,†,‡ †
Department of Chemistry, University of California, Berkeley, California 94720, United States Department of Chemistry and Henry Eyring Center for Cell and Genome Science, University of Utah, Salt Lake City, Utah 84112, United States
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t has long been known that cytosolic DNA serves as a potent immune stimulant; however, it was only recently discovered that this immune response occurs through the activation of cyclic GMP-AMP synthase (cGAS) and production of the second messenger 2′,3′-cyclic GMP-AMP (cGAMP)1 (Figure 1). Low nanomolar levels of cGAMP can bind to and activate the stimulator of interferon genes (STING) receptor, which initiates interferon (IFN) and NFκB immune responses. In this way, the presence of any aberrant cytosolic dsDNA, whether it is host-derived (from nuclear or mitochondrial leakage) or pathogen-derived (from DNA viruses, retroviruses, or bacteria) triggers the innate immune response. Given its central role in innate immune signaling, the cGAS-cGAMP-STING pathway has rapidly become an attractive target for the development of cancer immunotherapy and autoimmune disease treatments. Cytosolic cGAMP produced by cGAS can be spread to neighboring cells through cellular gap junctions or by being packaged into viral particles during egress, leading to immune activation in these cells. While the extracellular host enzyme ENPP1 has been shown to be capable of degrading cGAMP,2 surprisingly, no intracellular nucleases have been identified that are able to specifically target and degrade cGAMP to restrict downstream immune signaling. In a recent report in Nature,3 Eaglesham et al. hypothesized that viruses may encode the elusive cytosolic cGAMP nuclease as a strategy for the pathogen to silence the innate immune response. A biochemical screen was set up to test for virusinduced degradation of cGAMP by 24 different mammalian viruses representing 13 viral families. Lysates of infected cells were incubated with radiolabeled cGAMP and analyzed via PEI-cellulose TLC to look for degradation products. It was discovered that cGAMP was completely degraded by lysates of cells infected with the dsDNA vaccinia virus (VACV), a member of the Poxviridae family. Using activity-guided fractionation and mass spectrometry protein analysis, the VACV nuclease was found to be encoded by the previously uncharacterized B2R gene and was named VACV poxvirus immune nuclease, or VACV poxin. The degradation activity of the VACV poxin was specific for cGAMP, and no activity was observed against other cyclic dinucleotides, suggesting that the VACV poxin is indeed specialized for degradation of cGAMP. Although poxin deletion had no effect on VACV replication in interferon-deficient cells in culture, Δpoxin VACV replication was more than 40-fold attenuated compared to the wild type in mice, showcasing that poxin function was important for viral fitness in vivo. Interestingly, when tested in a cell culture model of replication where the multiplicity of © XXXX American Chemical Society
infection is high, no increase in IFN-β signaling was observed for VACV Δpoxin over the wild type. The authors suggest that the key function of poxin likely relates to preventing the spread of cGAMP to new cells, rather than the prevention of interferon signaling in the primary infected cell. To determine the mechanism of this enzyme, a series of Xray crystal structures of VACV poxin in the apo, prereactive (bound to nonhydrolyzable cGAMP), and postreactive (bound to reaction product) states were solved. These structures revealed that the enzyme makes extensive contacts with both the purine bases and noncanonical 2′−5′ linkage of cGAMP, explaining the selectivity of the enzyme for cGAMP over other cyclic dinucleotides. The active site contains no metal ions and appears to function via a catalytic triad of lysine, tyrosine, and histidine to cleave the 3′−5′ linkage of cGAMP and produce a 2′−3′ cyclic phosphate intermediate on adenosine, which is then resolved to the Gp[2′−5′]Ap[3′] product (Figure 2). A similar catalytic triad is used by tRNA-splicing endonucleases, further suggesting that poxin-catalyzed degradation of cGAMP proceeds through the conventional mechanism of metalindependent ribonucleases. Poxins are conserved in most viruses in the genus Orthopoxvirus, and four divergent poxins were tested and found to retain cGAMP-specific hydrolysis activity. The structures of the VACV poxin and its conserved active site residues were used to guide a bioinformatic search for poxin family members outside of the Poxiviridae, and active poxin homologues were found both in the Alphabaculovirus genus of insect DNA viruses and in insects of the order Lepidoptera, which serve as exclusive hosts of α-baculoviruses. Given that the STING signaling pathway is functional in insects and that the lepidopteran poxin gene is induced upon pathogen infection, the authors suggest that poxins may have originally evolved in insects to regulate the immune response and then spread to insect viruses and mammalian pathogens as a mechanism to escape immune responses (Figure 2). The discovery of poxins reveals a novel viral strategy for restricting cGAS-STING signaling by directly targeting and preventing the spread of cGAMP and helps to explain earlier findings that virulent poxviruses are able to inhibit cGASSTING signaling.4 Interestingly, inactivation of the poxin gene in some members of Poxviridae (including Variola major virus, the causative agent of smallpox) suggests that these viruses may have adopted other, as yet unknown, strategies to evade or even exploit cGAS-STING immunity. Conservation of poxins Received: April 11, 2019
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DOI: 10.1021/acs.biochem.9b00325 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry
Figure 1. cGAS-cGAMP-STING immune signaling pathway in animals.
Figure 2. Mechanism of poxin-mediated cGAMP degradation. Poxins cleave the 3′−5′ linkage of cGAMP via a metal-independent mechanism to produce the linear product Gp[2′−5′]Ap[3′] and restrict downstream STING signaling. The structure of the VACV poxin in the prereactive state is shown (PDB 6EA8).
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between insects, insect viruses, and mammalian viruses provides further evidence for the ancient origins of cGAMP signaling in animals5 and highlights the broad range of host− virus conflicts that drive innate immune surveillance and evasion. Looking forward, it will be interesting to see if other classes of cGAMP-specific nucleases have evolved in other viruses and/or bacterial pathogens in order to evade the cGAS-STING immune signaling pathway. Further research into the spatiotemporal dynamics of poxin expression and activity may provide useful information for the development of poxvirus-based vaccines and therapeutics. This featured study and other recent papers have raised many interesting questions about the dynamics and localization of cGAS-STING signaling in response to different viruses and other pathophysiological conditions. We expect that the development of tools for directly imaging cGAMP in live cells will lead to further progress in understanding this important immune signaling pathway.
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REFERENCES
(1) Chen, Q., Sun, L., and Chen, Z. J. (2016) Regulation and function of the cGAS-STING pathway of cytosolic DNA sensing. Nat. Immunol. 17, 1142−1149. (2) Li, L., Yin, Q., Kuss, P., Maliga, Z., Millán, J. L., Wu, H., and Mitchison, T. J. (2014) Hydrolysis of 2’3′-cGAMP by ENPP1 and design of nonhydrolyzable analogs. Nat. Chem. Biol. 10, 1043−1048. (3) Eaglesham, J. B., Pan, Y., Kupper, T. S., and Kranzusch, P. J. (2019) Viral and metazoan poxins are cGAMP-specific nucleases that restrict cGAS−STING signalling. Nature 566, 259−263. (4) Georgana, I., Sumner, R. P., Towers, G. J., and Maluquer de Motes, C. (2018) Virulent Poxviruses Inhibit DNA Sensing by Preventing STING Activation. J. Virol. 92, 1−14. (5) Kranzusch, P. J., Wilson, S. C., Lee, A. S. Y., Berger, J. M., Doudna, J. A., and Vance, R. E. (2015) Ancient Origin of cGASSTING Reveals Mechanism of Universal 2’,3′ cGAMP Signaling. Mol. Cell 59, 891−903.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel: (801) 213-0892. ORCID
Ming C. Hammond: 0000-0003-2666-4764 Funding
This work was supported by the following grant: NIH R01 GM124589. Notes
The authors declare no competing financial interest. B
DOI: 10.1021/acs.biochem.9b00325 Biochemistry XXXX, XXX, XXX−XXX
