The Nuclear DNA Sensor IFI16 Indiscriminately Binds to and Diminishes Accessibility of the HSV-1 Genome to Suppress Infection

Abstract

Human cells identify invading pathogens and activate immune signaling pathways through a wide array of pattern recognition receptors, including DNA sensors. The interferon-inducible protein 16 (IFI16) is a nuclear DNA sensor that recognizes double-stranded DNA from a number of viral sources, including genomes of nuclear-replicating viruses. Among these is the prevalent human pathogen herpes simplex virus 1 (HSV-1). Upon binding to the HSV-1 DNA genome, IFI16 both induces antiviral cytokine expression and suppresses virus gene expression. Here, we used a multiomics approach of DNA sequencing techniques paired with targeted mass spectrometry to obtain an extensive view of the interaction between IFI16 and the HSV-1 genome and how this binding affects the viral DNA structure and protein expression. Through chromatin immunoaffinity purification coupled with next-generation DNA sequencing (ChIP-seq), we found that IFI16 binds to the HSV-1 genome in a sequence-independent manner while simultaneously exhibiting broad enrichment at two loci: UL30, the viral DNA polymerase gene, and US1 to US7. The assay for transposase-accessible chromatin with sequencing (ATAC-seq) revealed that these two regions are among the most accessible stretches of DNA on the genome, thereby facilitating IFI16 binding. Accessibility of the entire HSV-1 genome is elevated upon IFI16 knockout, indicating that expression of IFI16 globally induces chromatinization of viral DNA. Deletion of IFI16 also results in a global increase in the expression of HSV-1 proteins, as measured by parallel reaction monitoring-mass spectrometry of viral proteins representing 80% of the HSV-1 genome. Altogether, we demonstrate that IFI16 interacts with the HSV-1 genome in a sequence-independent manner, coordinating epigenetic silencing of the viral genome and decreasing protein expression and virus replication. IMPORTANCE Mammalian host defense against viral infection includes broad-acting cellular restriction factors, as well as effectors of intrinsic and innate immunity. IFI16 is a critical nuclear host defense factor and intrinsic immune protein involved in binding viral DNA genomes, thereby repressing the replication of nucleus-replicating viruses, including the human herpes simplex virus 1. What has remained unclear is where on the viral genome IFI16 binds and how binding affects both viral DNA structural accessibility and viral protein expression. Our study provides a global view of where and how a nuclear restriction factor of DNA viruses associates with viral genomes to exert antiviral functions during early stages of an acute virus infection. Our study can additionally serve as a systems-level model to evaluate nuclear DNA sensor interactions with viral genomes, as well as the antiviral outcomes of transcriptionally silencing pathogen-derived DNA.

Keywords: ATAC-seq; ChIP-seq; DNA sensor; HSV-1; IFI16; PRM; proteomics; targeted mass spectrometry; virus infection; virus-host interactions.

FIG 1

Multiomics platform to define IFI16 binding and suppression of herpesviral genomes. Omics techniques were integrated to facilitate investigation into IFI16 binding to HSV-1 DNA and IFI16-mediated suppression of viral genes. Immunoaffinity purification of IFI16-DNA complexes followed by Illumina sequencing (IFI16 ChIP-seq) was used to map interactions between IFI16 and HSV-1 DNA. Dynamics of the HSV-1 genome accessibility during infection were investigated using ATAC-seq in WT and IFI16-KO cells. A targeted MS (parallel reaction monitoring [PRM]-MS) assay monitoring HSV-1 protein abundances was used to measure differences in viral protein expression in WT and IFI16-KO cells.

FIG 2

IFI16 indiscriminately binds to the HSV-1 genome and is enriched at the genes UL30 and US1–US7. (A) Schematic of the HSV-1 genome displaying the unique long (U_L) and unique short (U_S) regions, terminal repeats (TR_L and TR_S), and inverted repeats (IR_L and IR_S). Genes are shown as boxes, with inner white arrows indicating gene orientation. Genes that are highlighted in teal and gray indicate regions of interest that are discussed below (teal, ChIP-seq; gray, ATAC-seq). Red triangles mark locations of viral origins of replication. (B to D) IFI16 ChIP-seq was performed in HFF-1 cells following infection with ICP0-RF HSV-1, and samples were harvested at the indicated times postinfection (MOI = 10). Data were first normalized for sequencing depth and viral genome number using input sample ChIP-seq reads. Coverage tracks are displayed as the difference in average reads recovered from IFI16 and IgG immunoaffinity purifications (IFI16 − IgG). Scales representing normalized read counts are in brackets. GC percentage in panel B was measured as the average in 50-bp bins, and the mean GC content of the genome was 68%. Vertical red lines in panels C and D indicate viral origins of replication. Two biological replications were used for 1 hpi, and three biological replications were used for 3 and 6 hpi (50-bp bins).

FIG 3

HSV-1 genome transposase accessibility correlates with IFI16 binding. (A) ATAC-seq was performed following infection with ICP0-RF HSV-1 and Tn5 transposase treatment at the indicated times postinfection in control HFF-1 cells (MOI = 10). Data were normalized by sequencing depth and viral genome numbers (quantified via qPCR). Coverage tracks represent the average of three biological replicates at 1 and 3 hpi and two biological replicates for 6 hpi (50-bp bins). Scales representing normalized read counts are in brackets. (B) Scatterplots showing correlation between average ATAC-seq and average IFI16 ChIP-seq signal within each 50-bp bin. Left column, no filter; center column, bins within the IFI16 ChIP-seq-enriched genes UL30 and US1–US7 (including intergenic regions) are highlighted in light gray; right column, bins within the ATAC-seq-enriched genes are highlighted and baseline data points are excluded. Regions were determined using sequencing data at 6 hpi. UL30, 62,100 to 66,000 bp; RS1 (left), 127,100 to 131,500 bp; US1–US7, 132,000 to 141,100 bp; US8–US12, 141,100 to 146,700 bp; RS1 (right), 146,700 to 151,100 bp.

FIG 4

IFI16 reduces transposase accessibility of the HSV-1 genome throughout infection. (A) ATAC-seq data from ICP0-RF-infected IFI16-KO HFF-1 cells (1, 3, and 6 hpi; MOI of 10) were normalized by sequencing depth and viral genome numbers (quantified via qPCR). Coverage tracks at each time postinfection represent the average of biological replicates from IFI16-KO cells, the difference between IFI16-KO and Ctrl cells (data shown in Fig. 2; Δ, IFI16-KO minus control), and the ratio between IFI16-KO and control cells (IFI16-KO/Ctrl). The baseline for the ratio coverage tracks was set at 1.0 in each graph, and all values below 1.0 are red. Three biological replicates were used for 1 and 3 hpi, and two biological replicates were used for 6 hpi (50-bp bins). Scales representing normalized read counts are in brackets. (B) Line graphs representing the average ATAC-seq signal from IFI16-KO (black) and control (teal) cells in all HSV-1 open reading frames, scaled to 2 kbp.

FIG 5

Expression of IFI16 globally suppresses HSV-1 protein levels. Targeted PRM-MS was used to quantify HSV-1 protein levels in Ctrl and IFI16-KO cells following infection with ICP0-RF HSV-1 at 1, 3, and 6 hpi (MOI = 5). Three biological replicates. (A) Heat maps represent the normalized abundance of HSV-1 proteins in Ctrl and IFI16-KO cells, quantified from two or more precursor ions. (B) Log₂ fold change between IFI16-KO and Ctrl cells. ND, not detected. Horizontal red lines in panels A and B indicate regions of IFI16 enrichment. (C) Bar graphs displaying the protein abundances of the genes surrounding UL30 and in the U_S region. Error bars represent the standard errors of the means. Values that are significantly different between IFI16-KO and Ctrl samples (Student's t test, P < 0.05) are indicated by an asterisk. (D) Key insights into the IFI16 antiviral mechanism revealed by multiomics approach of ChIP-seq (left), ATAC-seq (center), and targeted mass spectrometry (right).