Charge-Mediated Pyrin Oligomerization Nucleates Antiviral IFI16 Sensing of Herpesvirus DNA

Oligomerization is required for IFI16 association with HSV-1 genomes and downstream antiviral functions. (A) Immunofluorescence microscopy of full-length IFI16-GFP, bearing mutations at R23, and ICP4. HEK293 FlpIn cells inducibly expressing the indicated IFI16-GFP fusions were infected with ICP0-RF HSV-1 for 24 h (multiplicity of infection [MOI] of 0.5). Representative cells are shown at the edge of a developing plaque. Bars, 5 μm. (B) Western blots of HEK293 FlpIn cells expressing the indicated IFI16-GFP fusions that affect its oligomerization state were infected with HSV-1 for 12 h (MOI of 1). Densitometry analyses of ICP8 and ICP27 are indicated relative to the GFP control and normalized to tubulin levels. (C) As in panel B with cells expressing IFI16-GFP bearing HIN mutations. (D) As in panel B with cells expressing IFI16-GFP bearing mutations at R23. (E) Progeny ICP0-RF HSV-1 titers (MOI of 0.2) from infected HEK293 FlpIn cells expressing IFI16-GFP R23 mutants (n = 3). (F) Cytokine mRNA levels in HEK293T cells cotransfected with STING and IFI16-GFP R23 mutants for 24 h. Abundances were normalized to β-actin. Values are means ± standard errors of the means (SEM) (error bars) (n = 3). Values that are significantly different (P ≤ 0.05) from those of the control by Student’s t test are indicated by a bar and asterisk. See also Fig. S2.

Alternative PYDs can functionally replace that of IFI16 to engage IFI16-mediated antiviral activity during HSV-1 infection. (A) Schematics of the PYHIN proteins IFI16, AIM2, IFIX, and MNDA and the generated chimera fusions to GFP. The PYD of IFI16 was swapped with that of the other PYHIN proteins. (B) Immunofluorescence microscopy of GFP and ICP4 in HEK293T cells transfected with the indicated chimera-GFP constructs. Cells were transfected and subsequently infected with ICP0-RF HSV-1 for 24 h (MOI of 0.5). Representative cells are shown at the edge of a developing plaque. Bars, 5 μm. (C) Western blots of HEK293Ts transfected with the indicated chimera-GFP constructs. Separate cell samples were both cross-linked and kept non-cross-linked for analysis. (D) Western blots of cells as in panel B infected with ICP0-RF HSV-1 for 12 h (MOI of 1). Densitometry analyses of phospho-TBK-1 and phospho-IRF3 are indicated relative to the HIN-GFP control and normalized to tubulin levels.

IFI16 oligomerization state dictates its interactions with transcription regulatory proteins during HSV-1 infection. (A) IP-MS/MS workflow to characterize IFI16 oligomerization-dependent protein interactions in HEK293Ts with or without ICP0-RF HSV-1 infection (6 hpi, MOI of 10, n = 2) (orange circles in cells represent viral capsids docked at the nucleus, green circles represent oligomerized IFI16 puncta at the nuclear periphery). Cells were transfected with either GFP or IFI16-GFP constructs (WT, oligomerization-competent R23K, oligomerization-deficient R23Q). Validation experiments were performed using targeted MS/MS (PRM) in CRISPR-HFFs that lacked endogenous IFI16 and stably expressed CRISPR-resistant IFI16 constructs (WT, R23K, and R23Q). In the HFF validations, the same ICP0-RF infection conditions were maintained. (B) Specificity-filtered IFI16 interactions were assembled into an interaction network via Cytoscape, categorized by Gene Ontology (GO) analysis. Gray lines indicate known interactions with other proteins as assessed in the Reactome database. Node colors represent relative MS¹ protein abundances enriched with either R23K IFI16 (yellow) or R23Q IFI16 (blue). (C) Targeted PRM analysis of IFI16 oligomerization-dependent interactions with UBTF in HFFs stably expressing IFI16-GFP constructs as in panel A. Representative peptide values for each protein are relative to those of the WT IFI16 IP and normalized by average IFI16 abundance during ICP0-RF HSV-1 infection (6 hpi, MOI of 10). Values are means ± SEM (error bars) across peptides. Statistically significant differences were determined by one-tailed, paired Student’s t test (*, P < 0.05). (D) Immunofluorescence microscopy of IFI16 and UBTF in HFFs either mock infected or infected with WT HSV-1 at 1 hpi (MOI of 10). Bars, 5 μm. (E) Western blot of ICP8 and ICP27 in CRISPR HFFs (Scrambled and UBTF) infected with WT HSV-1 at 8 hpi (MOI of 1). (F) Progeny virus titers from CRISPR HFFs (Scrambled and UBTF) infected with WT HSV-1 at 0, 6, 9, 24, and 48 hpi (MOI of 0.2; n = 3). Statistically significant differences were determined by unpaired Student’s t test (*, P < 0.05). (G) As in panel C, upon assessment of representative peptides for ND10 body components (PML and DAXX). See also Fig. S3 and Tables S1 to S3.