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IFI16 phase separation via multi-phosphorylation drives innate immune signaling

ABSTRACT: Fractionaion of nuclear periphery and core, followed by DDA in primary HFFs at 1 hour post infection with ICP0-RF HSV-1; IP-PRM of IFI16 in primary HFFs at 1 hpi; in vitro kinase assay with IFI16 and select kinases and quantification on phosphorlyated peptides by PRM.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human) Escherichia Coli

TISSUE(S): Cell Culture, Fibroblast

SUBMITTER: Dawei Liu

LAB HEAD: Ileana Cristea

PROVIDER: PXD034348 | Pride | 2023-05-22

REPOSITORIES: Pride

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			Action	DRS
	20210124_DL_GFPIFI16_DDA_PTM_sensitive_DDA.msf	Msf
	20210124_DL_GFPIFI16_DDA_PTM_sensitive_DDA.mzML	Mzml
	20210124_DL_GFPIFI16_DDA_PTM_sensitive_DDA.mzid.gz	Mzid
	20210124_IFI16_GFP_IP.blib	Other
	20210125_DL_GFPIFI16_PTM_PRM_RF_1hpi_1.raw	Raw

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Project description:Rapid and dynamically shifting protein-protein interactions (PPIs) underlie the capacity of cells to recognize viral infections and ignite the downstream cascades that constitute the innate-immune response. Herpesviruses share an ancient history of coevolution with their hosts and have developed reciprocal methods to suppress or hijack immune response proteins — underscoring the biological complexity of host-pathogen interactions during the immune response. Accordingly, conventional approaches to studying PPIs downstream of innate immune sensing fail to capture both the global scope and the dynamic on-off behavior of protein complexes as they are altered throughout cellular space and time. To overcome this barrier, we have applied Thermal Proteome Profiling Mass Spectrometry (TPP-MS) to systemically characterize PPIs that coordinate the innate-immune response to Herpes Simplex Virus 1 (HSV-1) infection in primary human fibroblasts. Further, by advancing the power of thermal protein co ggregation analysis (TPCA) to infer associations de novo and to globally track these PPIs, we developed a time-resolved portrait of cellular and viral protein associations with IFI16, a nuclear DNA sensor that serves as a central platform for HSV-1 immune responses. Our TPCA analysis, along with high-resolution microscopy and molecular virological techniques, linked IFI16 sensing of viral DNA in the nuclear periphery to the master DNA damage response (DDR) regulatory kinase, DNA-PK — the activation of which we show to be necessary for the antiviral and inflammatory responses to infection. Finally, phospho-peptide enrichment and MS analysis of DNA-PK substrates revealed IFI16 to be targeted by DNA-PK after both DNA damage and viral infection. Functional analysis of this DDR-dependent IFI16 phosphorylation revealed the specific modified residue required for IFI16-driven cytokine responses. Altogether, our study represents the first systems-wide characterization of the global dynamics of PPIs during HSV-1 infection and uncovers a missing link in the immune signaling pathway that places IFI16 and DNA-PK at the center of herpesvirus innate immunity.

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IFI16 phase separation via multi-phosphorylation drives innate immune signaling

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