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 identify the pyrin domain (PYD) residues that mediate IFI16 oligomerization in a charge-dependent manner. Based on structure modeling, these residues are predicted to be surface-exposed within distinct α-helices. By generating oligomerization-deficient mutants, we demonstrate that IFI16 homotypic clustering is necessary for its assembly onto parental viral genomes at the nuclear periphery upon herpes simplex virus 1 (HSV-1) infection. Preventing oligomerization severely hampered the capacity of IFI16 to induce antiviral cytokine expression, suppress viral protein levels, and restrict viral progeny production. Restoring oligomerization via residue-specific charge mimics partially rescued IFI16 antiviral roles. We show that pyrin domains from PYHIN proteins are functionally interchangeable, facilitating cooperative assembly with the IFI16 HINs, highlighting an inherent role for pyrin domains in antiviral response. Using immunoaffinity purification and targeted mass spectrometry, we establish that oligomerization promotes IFI16 interactions with proteins involved in transcriptional regulation, including PAF1C, UBTF, and ND10 bodies. We further discover PAF1C as an HSV-1 restriction factor. Altogether, our study uncovers intrinsic properties that govern IFI16 oligomerization, which serves as a signal amplification platform to activate innate immune responses and to recruit transcriptional regulatory proteins that suppress HSV-1 replication.

Fibroblast cells expressing IFI16-GFP were lysed using detergent lysis. Immunoaffinity purifications were performed targeting GFP with GFP-Trap_MA beads (Chromotek). IP eluates were reduced and alkylated then subjected to tryptic digestion with suspension trapping columns (S-Trap, Protifi) for 1 hour. Peptide eluates were resuspended in 1% FA, 1% ACN. Peptides were analyzed by nano-liquid chromatography coupled to tandem mass spectrometry with a Q Exactive HF Hybrid Quadrupole-Orbitrap intrument (Thermo Scientific) using parallel-reaction monitoring (PRM) mode. 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. The instrument was set to 15,000 resolution, AGC target was set to 1E5, MIT of 100 ms, 0.8 m/z isolation window, and NCE set to 27. An MS1 scan was acquired after every 20 PRM scans. MS1 acquisition was performed at 15,000 resolution, 25 ms MIT, and full scan range of 350-1800 m/z.

CRISPR/Cas9 was used to knock out IFI16 from human fibroblasts (HFF-1s) which were then transduced to express IFI16 mutants tagged with GFP or GFP alone.