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Investigation of cytoplasmic cGAS interactions reveals association with OASL during HSV-1 infection

ABSTRACT: Viral DNA sensing is an essential component of mammalian innate immune response. Upon binding viral DNA, the cyclic-GMP-AMP synthase (cGAS) catalyzes the production of cyclic dinucleotides to induce type I interferons. However, little is known about how cGAS is homeostatically maintained or regulated upon infection. Here, we define cytoplasmic cGAS interactions with cellular and viral proteins upon herpes simplex virus (HSV-1) infection in primary human fibroblasts. We compare several HSV-1 strains (wild-type, d109, d106) that induce cytokine responses and apoptosis, and place cGAS interactions in the context of temporal proteome alterations using isobaric-labeling mass spectrometry. Follow-up analyses establish a functional interaction between cGAS and 2’-5’-oligoadenylate synthase-like protein OASL. The OAS-like domain interacts with the cGAS Mab21 domain, while the OASL ubiquitin-like domain further inhibits cGAS-mediated interferon response. Our findings explain how cGAS may be inactively maintained in cellular homeostasis, with OASL functioning as a negative feedback loop for cytokine induction.

INSTRUMENT(S): LTQ Orbitrap, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Fibroblast

SUBMITTER: Joel Federspiel

LAB HEAD: Ileana M Cristea

PROVIDER: PXD010162 | Pride | 2018-11-21

REPOSITORIES: Pride

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			Action	DRS
	2016_10_13_mock_GFP_Cyto1_11.msf	Msf
	2016_10_13_mock_GFP_Cyto1_11.raw	Raw
	2016_10_13_mock_GFP_Cyto1_12.msf	Msf
	2016_10_13_mock_GFP_Cyto1_12.raw	Raw
	2016_10_13_mock_GFP_Cyto1_13.msf	Msf

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Investigation of cytoplasmic cGAS interactions reveals association with OASL during HSV-1 infection

Project description:Viral DNA sensing is an essential component of mammalian innate immune response. Upon binding viral DNA, the cyclic-GMP-AMP synthase (cGAS) catalyzes the production of cyclic dinucleotides to induce type I interferons. However, little is known about how cGAS is homeostatically maintained or regulated upon infection. Here, we define cytoplasmic cGAS interactions with cellular and viral proteins upon herpes simplex virus (HSV-1) infection in primary human fibroblasts. We compare several HSV-1 strains (wild-type, d109, d106) that induce cytokine responses and apoptosis, and place cGAS interactions in the context of temporal proteome alterations using isobaric-labeling mass spectrometry. Follow-up analyses establish a functional interaction between cGAS and 2â??-5â??-oligoadenylate synthase-like protein OASL. The OAS-like domain interacts with the cGAS Mab21 domain, while the OASL ubiquitin-like domain further inhibits cGAS-mediated interferon response. Our findings explain how cGAS may be inactively maintained in cellular homeostasis, with OASL functioning as a negative feedback loop for cytokine induction.

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Project description:DNA derived from the genetic material of pathogens or from cellular DNA damage provides a molecular pattern that can be sensed by pattern-recognition receptors of the mammalian innate immune system. In recent years, the cyclic GMP-AMP synthase (cGAS) protein has been characterized as a primary cytosolic DNA sensor during infection with bacteria, DNA viruses, or retroviruses. While the role of cGAS in downstream immune signaling through STING-TBK1-IRF3 proteins is well-defined, regulatory mechanisms of cGAS activity, such as through post-translational modifications (PTMs), are still an active area of research. Here, we report a comprehensive characterization of cGAS phosphorylations and acetylations in three different cell types. Data-dependent proteomic analyses of immunoaffinity purified cGAS was performed in HEK293T cells under control and DNA-challenged conditions (N = 3 each) and human fibroblasts (HFF) cells under control and HSV-1 infected conditions (N = 4 each) to generate candidate cGAS PTM sites. Using parallel reaction monitoring, a total of 11 PTMs (4 phosphorylations and 7 acetylations) were validated in HEK293T, HFF, and THP-1 cells. Of these, 3 phosphorylations and 5 acetylations have not been previously identified. The functions of these modifications were by generating a series of mutants and measuring cGAS-dependent apoptotic and immune signaling activities.

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