Respiratory Syncytial Virus (RSV) continues to cause severe respiratory illness, and effective antiviral treatments are still lacking. While many viruses rely on host‑cell signaling pathways to replicate, the role of the MEK–ERK1/2 cascade in RSV’s replication cycle has been unclear, with studies in cell lines showing mixed results. To address this, we tracked ERK1/2 activation over time and space in primary normal human bronchial epithelial (NHBE) cells. We created NHBE cells expressing a genetically encoded ERK Kinase Translocation Reporter (KTR) and used live‑cell imaging to follow ERK activity. In infected cells, RSV triggered a steady increase in ERK1/2 activation beginning about 3–4 hours after infection and lasting over 20 hours, in contrast to earlier reports of two separate activation phases.

Next, we used specific inhibitors and a recombinant RSV‑based FRET assay to look at how the pathway works. Blocking ERK1/2 with Ulixertinib stopped the virus from entering cells, revealing a new role for ERK1/2 in the first stage of infection. After entry, inhibiting MEK1/2 (with U0126) or ERK1/2 sharply reduced viral mRNA transcription and the release of infectious particles, while viral genome replication was unaffected. Further experiments showed that MEK1/2 inhibition prevented the viral phosphoprotein (P) from becoming hyper‑phosphorylated, which is vital for the viral polymerase to work.

Overall, our findings show that the MEK–ERK1/2 pathway is a key host dependency factor for RSV at multiple stages, controlling both entry and transcription. This makes MEK–ERK1/2 a strong candidate for host‑directed antiviral strategies.