IFI16-mediated suppression of HSV-1 protein expression
- Organism: Homo sapiens, Human herpesvirus 1
- Instrument: Q Exactive HF
HSV-1, innate immunity, DNA sensing, IFI16
Lab head: Ileana Cristea
Submitter: Tim Howard
Human cells identify invading pathogens and activate immune signaling pathways through a wide array of pattern recognition receptors, such as 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 such as the prevalent human pathogen, herpes simplex virus 1 (HSV-1). Upon binding to the DNA genome of HSV-1, IFI16 both induces antiviral cytokine expression and suppresses virus gene expression. Here, we use a multi-omics 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 ChIP-seq, we find 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-US7. ATAC-seq analysis reveals 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-KO, indicating that expression of IFI16 globally induces chromatinization of viral DNA, regardless of IFI16 enrichment. Deletion of IFI16 also results in a global increase in the expression of HSV-1 proteins, as measured by parallel reaction monitoring-mass spectrometry. Altogether, we demonstrate that IFI16 interacts with the HSV-1 genome in a sequence-independent manner, and this interaction coordinates epigenetic silencing of the viral genome, resulting in decreased protein expression and virus replication.
Control and IFI16-KO HFF-1 cells were infected with HSV-1 and harvested at 1, 3 and 6 hours post-infection. A library of spectral peptides built using Proteome Discoverer 2.4 was used to measure the abundance of viral proteins via scheduled PRM analysis on a Thermo Q Exactive HF, with PRM data analysis performed in Skyline.
CRISPR/Cas9 was used to knock out IFI16 from human fibroblasts (HFF-1s) which were then infected with HSV-1 at an MOI of 5 PFU/cell. Samples were prepared using trypsin digestion on S-Traps following the manufacturer’s instructions. Elutions were diluted to 0.75 µg/µl for peptide LC-MS/MS analysis.
Created on 2/23/22, 5:07 PM