Letter | Published:

Viral and metazoan poxins are cGAMP-specific nucleases that restrict cGAS–STING signalling

Abstract

Cytosolic DNA triggers innate immune responses through the activation of cyclic GMP–AMP synthase (cGAS) and production of the cyclic dinucleotide second messenger 2′,3′-cyclic GMP–AMP (cGAMP)1,2,3,4. 2′,3′-cGAMP is a potent inducer of immune signalling; however, no intracellular nucleases are known to cleave 2′,3′-cGAMP and prevent the activation of the receptor stimulator of interferon genes (STING)5,6,7. Here we develop a biochemical screen to analyse 24 mammalian viruses, and identify poxvirus immune nucleases (poxins) as a family of 2′,3′-cGAMP-degrading enzymes. Poxins cleave 2′,3′-cGAMP to restrict STING-dependent signalling and deletion of the poxin gene (B2R) attenuates vaccinia virus replication in vivo. Crystal structures of vaccinia virus poxin in pre- and post-reactive states define the mechanism of selective 2′,3′-cGAMP degradation through metal-independent cleavage of the 3′–5′ bond, converting 2′,3′-cGAMP into linear Gp[2′–5′]Ap[3′]. Poxins are conserved in mammalian poxviruses. In addition, we identify functional poxin homologues in the genomes of moths and butterflies and the baculoviruses that infect these insects. Baculovirus and insect host poxin homologues retain selective 2′,3′-cGAMP degradation activity, suggesting an ancient role for poxins in cGAS–STING regulation. Our results define poxins as a family of 2′,3′-cGAMP-specific nucleases and demonstrate a mechanism for how viruses evade innate immunity.

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Data Availability

Coordinates and structure factors of the VACV Poxin apo and Poxin–2′,3′-cGAMP complexes have been deposited in Protein Data Bank (PDB) under accession codes 6EA6, 6EA8 and 6EA9. All other data are available in the manuscript or the supplementary materials.

Change history

  • 13 May 2019

    In this Letter, Supplementary Fig. 1 was missing. This error has been corrected online.

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Acknowledgements

We thank A. Lee, K. Chat, R. Vance, S. Whelan, D. Knipe, N. Gray, V. Brusic, M. Bakkers, F. Ferguson and members of the Kranzusch laboratory for helpful comments and discussion, all collaborators for sharing virus-infected cells, W. Zhou, B. Lowey and K. McCarty for assistance with protein purification, R. Tomaino and the Harvard Medical School Taplin Mass Spectrometry Facility, and C. Miller for assistance with X-ray data collection. The work was funded by the Claudia Adams Barr Program for Innovative Cancer Research (P.J.K.), Richard and Susan Smith Family Foundation (P.J.K.), Charles H. Hood Foundation (P.J.K.), a Cancer Research Institute CLIP grant (P.J.K.), funding from the Parker Institute for Cancer Immunotherapy (P.J.K.), NIH grants R01 AI127654 and R01 AR062807 (T.S.K.) and support through an NIH T32 Training grant AI007245 (J.B.E.). X-ray data were collected at the Lawrence Berkeley National Laboratory Advanced Light Source beamline 8.2.1 and at the Northeastern Collaborative Access Team beamline 24-ID-C, which are funded by the NIGMS (P30 GM124165, P41 GM103403) and an NIH-ORIP HEI grant (S10 RR029205) and used resources of the DOE Argonne National Laboratory Advanced Photon Source (under contract number DE-AC02-06CH11357).

Reviewer information

Nature thanks A. Bowie, Z. J. Chen, M. A. Lee-Kirsch and O. Nureki for their contribution to the peer review of this work.

Author information

The project was conceived and experiments were designed by J.B.E. and P.J.K. All virology, biochemical and structural experiments were conducted by J.B.E. with assistance from P.J.K. Animal experiments were conducted by Y.P. and T.S.K. The manuscript was written by J.B.E. and P.J.K and all authors support the conclusions.

Correspondence to Philip J. Kranzusch.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 VACV-induced 2′,3′-cGAMP degradation is cell-line and tissue-type independent.

a, TLC analysis of the stability of 2′,3′-cGAMP (the 3′–5′ bond is radiolabelled) following incubation in human monocyte (THP-1) or kidney (HEK293T) cytosolic lysates. 2′,3′-cGAMP is highly stable with no degradation detected after >20 h of incubation. b, TLC analysis of VACV-induced 2′,3′-cGAMP degradation. Lysates were prepared from African green monkey (Chlorocebus aethiops (Vero)) and golden hamster (Mesocricetus auratus (BSR-T7)) cells. These lysates exhibit 2′,3′-cGAMP degradation activity after infection with VACV, but not after mock infection (M). c, Time-course analysis of 2′,3′-cGAMP-degradation activity following infection of BSC-40 cells (C. aethiops) with VACV. 2′,3′-cGAMP degradation activity is detectable early <1 h after infection and persists beyond 18 h post-infection. In all panels, the ‘−’ refers to a buffer-only control. All data are representative of three independent experiments.

Extended Data Fig. 2 Biochemical fractionation and mass spectrometry identification of VACV poxin.

a, Schematic of purification process developed to enrich VACV poxin from infected cell lysates. Lysates were fractionated using Q IEX and S200 size-exclusion chromatography (purification scheme 1, left) or ammonium sulfate precipitation followed by phenyl hydrophobic interaction and S75 size-exclusion chromatography (purification scheme 2, right). Fractions were tested for 2′,3′-cGAMP degradation activity at each stage of purification and active fractions were pooled for subsequent purification steps. Fractions with peak activity after size exclusion were analysed with mass spectrometry. Fold enrichment of proteins in the IEX active fraction compared to two inactive fractions was calculated using label-free mass spectrometry quantification. b, List of VACV proteins identified in each purification scheme. VACV poxin is encoded by the B2R gene (green).

Extended Data Fig. 3 Purification and biochemical characterization of VACV poxin.

a, Purification of recombinant VACV poxin from E. coli. VACV poxin was expressed as an N-terminal 6 × His-SUMO fusion, the tag was proteolytically removed and VACV poxin was isolated using S75 size-exclusion chromatography. VACV poxin migrates at around 50 kDa, consistent with a homodimeric complex. b, SDS–PAGE Coomassie stain analysis of purified recombinant VACV poxin. c, Reaction time-course analysis of recombinant VACV poxin 2′,3′-cGAMP-degradation activity. VACV poxin rapidly cleaves 2′,3′-cGAMP through a slower-migrating intermediate product, identical to the activity observed in VACV-infected cell lysates. The ‘−’ refers to a buffer-only control incubated for 120 min. Data are representative of three independent experiments. d, e, pH titration of the 2′,3′-cGAMP-degradation activity of recombinant VACV poxin and VACV-infected cell lysate. Recombinant VACV poxin and VACV-infected cell lysates share an alkaline pH optimum of 8.2–10.6. d, The ‘−’ refers to a buffer-only control at pH 7.5. Data are representative of three independent experiments.

Extended Data Fig. 4 Construction and validation of poxin-expressing cells and poxin knockout virus.

a, TLC analysis of lysates from HEK293T cells after transduction with poxin(WT) or the poxin(H17A) catalytically inactive construct, and selection with puromycin. HEK293T poxin(WT) cells but not control cells show degradation of 2′,3′-cGAMP after a 1-h reaction. Data are representative of two independent experiments. The ‘−’ refers to a buffer-only control. b, Western blot analysis of poxin-transduced cell lines demonstrating expression of both VACV poxin(WT) and poxin(H17A) proteins. Data are representative of two independent experiments. Gel source data are available in Supplementary Fig. 1. c, Schematic demonstrating strategy for poxin (B2R) knockout by homologous recombination and replacement with sfGFP. Coloured arrows depict primers used for PCR and sequencing validation of selected viral clones. d, PCR analysis of parental VACV and VACV ΔPoxin confirming removal of B2R and replacement with the sfGFP gene. Data are representative of two independent experiments. e, Sequencing trace confirming replacement of B2R with sfGFP in the genome of VACV ΔPoxin. f, Bright-field and fluorescence microscopy showing Vero cells infected with wild-type VACV poxin or VACV ΔPoxin after 20 h at MOI = 1. VACV ΔPoxin-infected cells express sfGFP. Data are representative of three independent experiments. g, TLC analysis of 2′,3′-cGAMP after incubation with lysates of cells infected with wild-type or ΔPoxin viruses. VACV ΔPoxin-infected cells lack detectable 2′,3′-cGAMP degradation activity. The ‘−’ refers to a buffer-only control; M refers to a mock-infection control. Data are representative of three independent experiments. h, Multiple cycle growth curve (MOI = 0.01) of wild-type and ΔPoxin VACV strains in Vero cells, demonstrating that poxin knockout has no effect on viral growth kinetics in interferon-deficient cells in cell culture (n = 2). Data are mean ± s.e.m. i, qRT–PCR analysis of the transcriptional induction of IFNβ and an interferon-stimulated gene (CXCL10) following infection of A549 cells with wild-type or ΔPoxin VACV after 5 h at MOI = 5 (n = 2). Poxin deletion does not increase IFNβ-dependent signalling in cell culture under these conditions. As a positive control, STING-dependent signalling in A549 cells was stimulated with 2′,3′-cGAMP and digitonin permeabilization. Source data

Extended Data Fig. 5 Structural analysis of VACV poxin.

a, VACV poxin consists of two domains and homodimerizes to form the active complex. The N-terminal domain (green) has structural homology to viral 3C-like proteases (r.m.s.d. of 2.9 Å). The norovirus NS6 protease and Bos taurus trypsin protease are coloured in green and presented in the same orientation for comparison49,50. Z-scores were obtained from the DALI server51. The C-terminal domain (cyan) of VACV poxin has no known structural homologues. b, Overlay of the apo (red) and 2′,3′-cGAMP bound pre-reactive (green/cyan/grey) poxin structures. 2′,3′-cGAMP binding induces a 4 Å movement of the clamp helix and repositions the active site for 3′–5′-bond hydrolysis. c, VACV poxin dimerization is mediated by antiparallel β-strand hydrogen bonding between monomers, as well as side-chain interactions within a hydrophobic core composed of M183, M185 and F187.

Extended Data Fig. 6 Structural analysis of poxin 2′,3′-cGAMP binding.

a, Overview of interactions in the VACV poxin–2′,3′-cGAMP complex that mediate substrate specificity. VACV poxin residues make three types of interactions with 2′,3′-cGAMP: sequence-specific contacts with the guanine base (left), hydrogen-bonding interactions with the 2′–5′ bond (middle) and sequence non-specific contacts with the adenine base (right). b, Simulated annealing omit maps showing electron density of 2′,3′-cGAMP before and after poxin cleavage. Base identities can clearly be assigned in both pre- and post-reactive structures. A clear gap exists in the post-reactive structure between the guanine 5′-OH and adenine 3′-phosphate confirming that the poxin product is Gp[2′–5′]Ap[3′]. c, tRNA splicing endoribonucleases are metal-independent enzymes that degrade ribonucleotide substrates through a 2′–3′-cyclic phosphate intermediate. These enzymes share the poxin catalytic triad composed of histidine, tyrosine and lysine, suggesting a related catalytic mechanism, despite the lack of sequence or structural homology.

Extended Data Fig. 7 VACV poxin degrades 2′,3′-cGAMP through hydrolysis of the 3′–5′ bond.

a, TLC analysis of poxin activity using 2′,3′-cGAMP radiolabelled at the 2′–5′-(α32P-A) or 3′–5′-(α32P-G) phosphodiester bonds. Radiolabelled 2′,3′-cGAMP was incubated with recombinant VACV poxin and then treated with phosphatase to remove exposed phosphates from the final product. Following hydrolysis, the guanosine phosphate is exposed for phosphatase removal, confirming the structural findings that VACV poxin specifically cleaves the 3′–5′ linkage of 2′,3′-cGAMP. The ‘−’ refers to a buffer-only control. b, Schematic of VACV poxin induced hydrolysis of 2′,3′-cGAMP. c, TLC analysis of VACV poxin 2′,3′-cGAMP-degradation activity in the presence of 5 mM EDTA metal chelation or divalent cation supplementation. Divalent cations were supplemented at the following concentrations: 5 mM Mg2+, 5 mM Ca2+, 1 mM Mn2+, 1 μM Co2+, 1 μM Ni2+, 1 μM Cu2+ or 1 μM Zn2+. Poxin activity is resistant to EDTA and divalent cations have no effect on the reaction, confirming the structural findings that VACV poxin activity is metal-independent. The ‘−’ refers to a buffer-only control; the ‘+’ refers to treatment with VACV poxin alone without metal addition. d, TLC analysis of mutants of the active site of VACV poxin that were incubated for 20 h with 2′,3′-cGAMP or 3′,3′-cGAMP demonstrates that all active-site mutants retain specificity for 2′,3′-cGAMP. The ‘−’ refers to a buffer-only control. All data are representative of three independent experiments.

Extended Data Fig. 8 Alignment of poxin proteins conserved in poxvirus representatives.

a, The poxin protein is highly conserved in mammalian poxviruses. The alignment is shaded according to conservation of physiochemical amino acid property, and numbered above according the VACV poxin amino acid sequence. The determined VACV poxin secondary structure is depicted below, active-site residues are indicated with a red dot and boxed in red, and residues that contact 2′,3′-cGAMP are boxed in orange. VACV poxin residues 195–219 are not observed in the crystal structure. Sequences depicted in alignment are as follows: VACV WR (vaccinia virus strain Western Reserve, accession YP_233066.1), VACV Cop (vaccinia virus strain Copenhagen, accession P20999.1), VACV Dryvax (vaccinia virus strain Dryvax, accession AEY73716.1), VACV ACAM2000 (vaccinia virus strain ACAM2000, accession AAQ93281.1), VACV NYVAC (vaccinia virus strain NYVAC), RPXV Utr (rabbit poxvirus strain Utrecht, accession AY484669.1), HSPV MNR76 (horsepox virus strain MNR76, accession ABH08291.1), CPXV AUS1999 (cowpox virus strain AUS1999, accession ADZ24189.1), MPXV ZAR (monkeypox virus strain Zaire-96-I-16, accession NP_536592.1), CMLV CMS (camelpox virus strain CMS, accession AAG37679.1), TATV DAH68 (taterapox virus strain Dahomey 1968, accession YP_717493.1), CPXV GER2002 (cowpox virus strain GER2002, accession ADZ30373.1), CPXV BR (cowpox virus strain Brighton Red, accession NP_619978.1), ECTV MOS (ectromelia virus strain Moscow, accession NP_671672.1), VPXV (volepox virus, accession YP_009281928.1), SKPV (skunkpox virus, accession YP_009282874.1), RCNV (raccoonpox virus, accession YP_009143488.1), YKV (yokapox virus, accession YP_004821513.1), NY_014 (NY_014 virus, accession YP_009408559.1), Murmansk (Murmansk poxvirus, accession YP_009408359.1), EPTV (Eptesipoxvirus (Eptesicus fuscus), accession YP_009408111.1), PTPV (Pteropus scapulatus, accession YP_009268718.1), Melanoplus sanguinipes (M. sanguinipes entomopox virus, accession NP_048308.1). b, Schematized alignment of poxvirus genomic DNA showing the poxin B2RB3R locus. White boxes indicate predicted open reading frames beginning with the annotated start codons and ending with the first stop codon; deletion and nonsense mutations are shown as orange and red bars. CPXV encodes an intact poxin–schlafen fusion protein, whereas the VACV genome contains a stop codon immediately following the poxin coding region and a frameshift mutation in the schlafen (B3R) gene. Poxin is inactivated in MVA and VARV by serial mutation.

Extended Data Fig. 9 Conservation of poxin family members and 2′,3′-cGAMP-specific nuclease activity in Poxviridae, Baculoviridae and host Lepidoptera.

a, Phylogenetic conservation of poxin family members in Poxviridae, Baculoviridae and host Lepidoptera genomes. Poxin catalytic residues (red) and 2′,3′-cGAMP-interacting residues (black) are indicated on the right, shaded in blue according to conservation, and listed according to VACV poxin amino acid number (Poxviridae, top) or AcNPV poxin amino acid number (Baculoviridae, middle). The metazoan poxin sequences from moth and butterfly genomes (Lepidoptera, bottom) share homology throughout the entire poxin protein and exhibit identical 2′,3′-cGAMP degradation activity, but the alignment with viral poxins does not allow definitive assignment of the catalytic residues. Phylogram schematics are based on previous analyses52,53,54,55. b, Coomassie-stained SDS–PAGE analysis of recombinant SUMO2-tagged poxin homologue proteins. c, TLC analysis of recombinant viral and host cellular poxin activity after 20 h incubation with substrates. All viral and metazoan poxin family members are specific 2′,3′-cGAMP nucleases. No activity is detected using the chemically related cyclic dinucleotide 3′,3′-cGAMP. The ‘−’ refers to a buffer-only control. Data are representative of three independent experiments.

Extended Data Table 1 Summary of data collection, phasing and refinement statistics

Supplementary information

Supplementary Table 1

This table details the sources of all virus samples used for the biochemical screen in Figure 1b, acknowledges the individuals who provided the samples, and provides conditions under which these samples were produced.

Reporting Summary

Supplementary Figure 1

This file contains the original gel source data.

Source data

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Further reading

Fig. 1: A poxvirus nuclease disrupts 2′,3′-cGAMP immune signalling.
Fig. 2: VACV poxin is critical for evasion of cGAS-STING immunity.
Fig. 3: Structural basis of poxin activity and mechanism of 2′,3′-cGAMP degradation.
Fig. 4: Discovery of viral and host cellular poxin homologues.
Extended Data Fig. 1: VACV-induced 2′,3′-cGAMP degradation is cell-line and tissue-type independent.
Extended Data Fig. 2: Biochemical fractionation and mass spectrometry identification of VACV poxin.
Extended Data Fig. 3: Purification and biochemical characterization of VACV poxin.
Extended Data Fig. 4: Construction and validation of poxin-expressing cells and poxin knockout virus.
Extended Data Fig. 5: Structural analysis of VACV poxin.
Extended Data Fig. 6: Structural analysis of poxin 2′,3′-cGAMP binding.
Extended Data Fig. 7: VACV poxin degrades 2′,3′-cGAMP through hydrolysis of the 3′–5′ bond.
Extended Data Fig. 8: Alignment of poxin proteins conserved in poxvirus representatives.
Extended Data Fig. 9: Conservation of poxin family members and 2′,3′-cGAMP-specific nuclease activity in Poxviridae, Baculoviridae and host Lepidoptera.

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