cyclic-guanosine-monophosphate-adenosine-monophosphate and Vaccinia

Activation of the cyclic guanosine monophosphate (GMP)-AMP (cGAMP) sensor STING requires its translocation from the endoplasmic reticulum to the Golgi apparatus and subsequent polymerization. Using a genome-wide CRISPR-Cas9 screen to define factors critical for STING activation in cells, we identified proteins critical for biosynthesis of sulfated glycosaminoglycans (sGAGs) in the Golgi apparatus. Binding of sGAGs promoted STING polymerization through luminal, positively charged, polar residues. These residues are evolutionarily conserved, and selective mutation of specific residues inhibited STING activation. Purified or chemically synthesized sGAGs induced STING polymerization and activation of the kinase TBK1. The chain length and O-linked sulfation of sGAGs directly affected the level of STING polymerization and, therefore, its activation. Reducing the expression of Slc35b2 to inhibit GAG sulfation in mice impaired responses to vaccinia virus infection. Thus, sGAGs in the Golgi apparatus are necessary and sufficient to drive STING polymerization, providing a mechanistic understanding of the requirement for endoplasmic reticulum (ER)-to-Golgi apparatus translocation for STING activation.

Topics: Animals; Cell Line; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Cricetinae; Cytosol; Endoplasmic Reticulum; Glycosaminoglycans; Golgi Apparatus; HeLa Cells; Humans; Membrane Proteins; Mice; Nucleotides, Cyclic; Polymerization; Signal Transduction; Sulfate Transporters; Vaccinia; Vaccinia virus

Cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA during viral infection and catalyzes synthesis of the dinucleotide cGAMP, which activates the adaptor STING to initiate antiviral responses. Here we found that deficiency in the carboxypeptidase CCP5 or CCP6 led to susceptibility to DNA viruses. CCP5 and CCP6 were required for activation of the transcription factor IRF3 and interferons. Polyglutamylation of cGAS by the enzyme TTLL6 impeded its DNA-binding ability, whereas TTLL4-mediated monoglutamylation of cGAS blocked its synthase activity. Conversely, CCP6 removed the polyglutamylation of cGAS, whereas CCP5 hydrolyzed the monoglutamylation of cGAS, which together led to the activation of cGAS. Therefore, glutamylation and deglutamylation of cGAS tightly modulate immune responses to infection with DNA viruses.

Topics: Animals; Carboxypeptidases; Cytosol; DNA Virus Infections; DNA Viruses; DNA, Viral; Fluorescent Antibody Technique; Herpes Simplex; Immunoprecipitation; Interferon Regulatory Factor-3; Interferons; Mice; Mice, Knockout; Nucleotides, Cyclic; Nucleotidyltransferases; Peptide Synthases; Reverse Transcriptase Polymerase Chain Reaction; Simplexvirus; Vaccinia; Vaccinia virus

Infected cells detect viruses through a variety of receptors that initiate cell-intrinsic innate defense responses. Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) is a cytosolic sensor for many DNA viruses and HIV-1. In response to cytosolic viral DNA, cGAS synthesizes the second messenger 2'3'-cyclic GMP-AMP (cGAMP), which activates antiviral signaling pathways. We show that in cells producing virus, cGAS-synthesized cGAMP can be packaged in viral particles and extracellular vesicles. Viral particles efficiently delivered cGAMP to target cells. cGAMP transfer by viral particles to dendritic cells activated innate immunity and antiviral defenses. Finally, we show that cell-free murine cytomegalovirus and Modified Vaccinia Ankara virus contained cGAMP. Thus, transfer of cGAMP by viruses may represent a defense mechanism to propagate immune responses to uninfected target cells.

Topics: Animals; Chlorocebus aethiops; Cytosol; Dendritic Cells; Genetic Vectors; HeLa Cells; Herpesviridae Infections; HIV Infections; HIV-1; Humans; Immunity, Innate; Mice; Mice, Inbred C57BL; Muromegalovirus; Nucleotides, Cyclic; Second Messenger Systems; Vaccinia; Vaccinia virus; Vero Cells; Virion; Virus Assembly