Circadian Clock Gene Modulation and Selective Reprogramming in Response to Copy-back Viral Genomes During Infection

The circadian clock regulates fundamental cellular processes that include the host response to infection. However, its intersection with the mechanisms driving antiviral immunity remains poorly explored. Copy-back viral genomes (cbVGs) generated during virus replication strongly stimulate immunity during infection with negative-sense RNA viruses. Here, we demonstrate that the model lung epithelial cells A549 are circadian-competent, and by tracking the circadian clock at single-cell resolution across an active infection timeline, we show that infection with Sendai virus progressively disrupts the molecular clock architecture, driven primarily by cbVGs. Furthermore, we show that MAVS signaling is essential for the induction of the circadian clock activating component ARNTL2, while PKR signaling is required for the cbVG-specific induction of the repressive components NR1D1, NR1D2, and PER1. A similar cbVG-specific signature was observed in cells infected with a cbVG-high respiratory syncytial virus stock, demonstrating that cbVG-driven clock reprogramming extends to phylogenetically distinct negative-sense RNA viruses. In addition, we established through gain- and loss-of-function experiments that ARNTL2 is functionally required for amplifying the transcriptional response to infection, with selective effects on the expression of specific antiviral genes including CCL5. Together, these findings demonstrate that the host clock is actively disrupted by a subset of viral genomes produced during virus replication and specific clock genes are repurposed to enhance the host response to infection.