Effect of ILF3 on translation during homeostasis and the antiviral response

Upon detection of viral infections, cells activate the expression of type I interferons (IFNs) and pro-inflammatory cytokines to control viral dissemination. As part of their antiviral response, cells also trigger the translational shutoff response which prevents translation of viral mRNAs and cellular mRNAs in a non-selective manner. Intriguingly, mRNAs encoding for antiviral factors bypass this translational shutoff, suggesting the presence of additional regulatory mechanisms enabling expression of the self-defence genes. Here, we identified the dsRNA binding protein ILF3 as an essential host factor required for efficient translation of the central antiviral cytokine, IFNB1, and a subset of interferon-stimulated genes. By combining polysome profiling and next-generation sequencing, ILF3 was also found to be necessary to establish the dsRNA-induced transcriptional and translational programs. We propose a central role for the host factor ILF3 in enhancing expression of the antiviral defence mRNAs in cellular conditions where cap-dependent translation is compromised. In this dataset we use polysome profiling in combination with RNA-seq to investigate the effect of ILF3 on the translation of mRNAs in HeLa cells in homeostasis and the antiviral response. To study the effect of ILF3 on translation, HeLa cells were first depleted of ILF3 by transfection with either a pool of siRNAs that target all splice isoforms of ILF3 (siILF3) or a pool of non-targeting control (Mock) siRNAs for 72h. Following the depletion, the antiviral response was activated by transfection with poly(I:C) for 4 hours. 30min prior to harvest, the cells were treated with 50ng/mL Cycloheximide to inhibit protein synthesis. Cytoplasmic lysates of identical numbers of cells were then subject to separation by velocity sedimentation through 10-60% sucrose gradients. This separated the subpolysomal fractions (which include free mRNA complexed to RNPs and the 40S, 60S and 80S ribosomal subunits) with the polysomal fractions (>2 polysomes) which were collected in a fraction collector. RNA was extracted from the subpolysomal and polysomal pools by precipitation with ethanol followed by Trizol LS RNA purification. The purified RNA was then enriched for poly(A) tailed mRNAs and subject to Illumina based RNA sequencing.