Pyrin oligomerization nucleates antiviral IFI16 sensing of herpesvirus DNA
The formation of multimerized protein assemblies has emerged as a core component of immune signal amplification, yet the biochemical basis of this phenomenon remains unclear for many mammalian proteins within host defense pathways. The interferon-inducible protein 16 (IFI16) is a viral DNA sensor that oligomerizes upon binding to nuclear viral DNA and induces downstream antiviral responses. Here, we first generated oligomerization-incompetent IFI16 mutants that exhibit severely reduced ability to induce antiviral cytokine expression, suppress herpes simplex virus 1 (HSV-1) protein levels, and restrict viral progeny production. Using immunoaffinity purification and targeted mass spectrometry, we establish that oligomerization promotes IFI16 interactions with several proteins involved in transcriptional regulation, including PAF1C, UBTF, and ND10
Sample Processing Protocol
Cells expressing IFI16-GFP (HEK293Ts for DDA, HFFs for PRM) were lysed by detergent lysis and IPs were performed using GFP-Trap_MA beads (Chromotek). For DDA, proteins were digested with trypsin via overnight filter aided sample preparation. Samples were then desalted with SDB-RPS membranes. For PRM, digestion was performed with trypsin and suspension trapping columns (S-Trap, Protifi) for 1 hour. Peptides were analyzed by nano-liquid chromatography coupled to tandem mass spectrometry with a Q Exactive HF Hybrid Quadrupole-Orbitrap intrument (Thermo Scientific) using data-dependent acquisition (DDA) or parallel-reaction monitoring (PRM) modes. Peptides (2 µL injections) were separated with a 3% solvent B to 30% solvent B gradient (solvent A: 0.1% FA, solvent B: 0.1% FA, 97% ACN) over 60 min at a flow rate of 250 nL/min on an EASYSpray C18 column (75 µm x 50 cm) heated to 50 °C. For DDA, the full scan range was set to 350-1800 m/z at 120,000 resolution and recorded in profile. The top 15 most intense precursors were subjected to HCD fragmentation – normalized collision energy (NCE) of 28 – for MS2 analysis at 30,000 resolution
Data Processing Protocol
Tandem MS spectra collected from DDA mode were analyzed by Proteome Discoverer v2.2. MS spectra were searched using the Sequest HT algorithm against a UniProt human database containing herpesvirus sequences and common contaminants (22,349 sequences, downloaded 2016-04). The Spectrum RC node was used to perform offline mass recalibration and the Minora Feature Detector node was used for label-free MS1 quantitation. Posttranslational modifications (PTMs) including static carbamidomethylation of cysteine, dynamic oxidation of methionine, dynamic deamidation of asparagine, dynamic loss of methionine plus acetylation of the protein N-terminus, and dynamic phosphorylation of serine, threonine, and tyrosine were all allowed. The Percolator node was then used to perform peptide spectrum match (PSM) validation and ptmRS node was used for assigning PTM sites.
Lum KK, Howard TR, Pan C, Cristea IM. Charge-Mediated Pyrin Oligomerization Nucleates Antiviral IFI16 Sensing of Herpesvirus DNA. MBio. 2019 10(4) PubMed: 31337724