Purification of Cyclic GMP-AMP from Viruses and Measurement of Its Activity in Cell Culture

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Abstract

Sensing of cytoplasmic DNA by cGAS is essential for the initiation of immune responses against several viruses. cGAS also plays important roles in some autoinflammatory and autoimmune diseases and may be involved in immune responses targeting cancer cells. Once activated, cGAS catalyzes the formation of the di-nucleotide 2′-3′-cyclic GMP-AMP (cGAMP), which propagates a signaling cascade leading to the production of type I interferons (IFNs). Interestingly, cGAMP is incorporated into enveloped viruses and is transferred to newly infected cells by virions. In this article, we describe a method to purify cGAMP from viral particles and a bioassay to measure its activity. This assay takes advantage of a reporter cell line that expresses the genes encoding green fluorescent protein (GFP) and firefly luciferase under the control of the IFNß promoter, allowing the testing of several samples in a single experiment taking not more than 3 days.

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Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING.

Xu Zhang, Heping Shi, Jiaxi Wu … (2013)

The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2'-OH of GMP and 5'-phosphate of AMP, and the other between 3'-OH of AMP and 5'-phosphate of GMP. This molecule, termed 2'3'-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2'3'-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation. Copyright © 2013 Elsevier Inc. All rights reserved.

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c-di-AMP secreted by intracellular Listeria monocytogenes activates a host type I interferon response.

Joshua Woodward, Anthony T. Iavarone, Daniel Portnoy (2010)

Intracellular bacterial pathogens, such as Listeria monocytogenes, are detected in the cytosol of host immune cells. Induction of this host response is often dependent on microbial secretion systems and, in L. monocytogenes, is dependent on multidrug efflux pumps (MDRs). Using L. monocytogenes mutants that overexpressed MDRs, we identified cyclic diadenosine monophosphate (c-di-AMP) as a secreted molecule able to trigger the cytosolic host response. Overexpression of the di-adenylate cyclase, dacA (lmo2120), resulted in elevated levels of the host response during infection. c-di-AMP thus represents a putative bacterial secondary signaling molecule that triggers a cytosolic pathway of innate immunity and is predicted to be present in a wide variety of bacteria and archea.

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Natural STING Agonist as an "Ideal" Adjuvant for Cutaneous Vaccination.

Ji. Wang, Peiyu Li, Mei Wu (2016)

A potent adjuvant that induces strong protective immunity without incurring any significant skin reactogenicity is urgently needed for cutaneous vaccination. Here, we report that a natural agonist of stimulator of interferon genes (STING), 2'3'- cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), robustly augmented and prolonged the cellular and humoral immune responses provoked by H5N1 and 2009 H1N1 pandemic influenza vaccines after a single dose of intradermal, but not intramuscular, immunization. The potency of cGAMP for cutaneous vaccination was ascribed to a large number of antigen-presenting cells resident in the skin and ready for immediate activation when cGAMP was injected. However, its potency was severely compromised in the muscle, because antigen-presenting cells could not be promptly recruited to the injection site before the injected cGAMP was diffused out. The superior adjuvant effect and safety of cGAMP were also confirmed in a more clinically relevant swine model of skin. The vigorous immune responses elicited by cGAMP with no overt skin irritation was attributable to its stay in the skin, which was brief but sufficient to activate dermal dendritic cells. This small and well-characterized self-molecule holds great promise as an ideal adjuvant for cutaneous vaccination.

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Author and article information

Contributors

Jan Rehwinkel: jan.rehwinkel@imm.ox.ac.uk

Journal

Journal ID (nlm-ta): Methods Mol Biol

Journal ID (iso-abbrev): Methods Mol. Biol

Title: Methods in Molecular Biology (Clifton, N.j.)

Publisher: Springer New York (New York, NY )

ISSN (Print): 1064-3745

ISSN (Electronic): 1940-6029

Publication date (Electronic): 15 August 2017

Publication date PMC-release: 15 August 2017

Publication date Collection: 2017

Volume: 1656

Pages: 143-152

Affiliations

[3 ]Medical Research Council Human Immunology Unit, Radcliffe Department of Medicine, Medical Research Council Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headley Way, Oxford, OX3 9DS UK

[4 ]ISNI 0000 0004 1936 8948, GRID grid.4991.5, Medical Research Council Human Immunology Unit, Radcliffe Department of Medicine, Medical Research Council Weatherall Institute of Molecular Medicine, , University of Oxford, ; Headley Way, Oxford, OX3 9DS UK

University of Hertfordshire, School of Life and Medical Sciences, Hatfield, Herts., United Kingdom

Article

Publisher ID: 8

DOI: 10.1007/978-1-4939-7237-1_8

PMC ID: 6126575

PubMed ID: 28808967

SO-VID: cea23410-fa74-460a-bc7f-7874056a7ccf

License:

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