Journal Article

A dual interaction between RSV NS1 and MED25 ACID domain reshapes antiviral responses

Verger, Alexis, Ait-Mouhoub, Celia, Dong, Jiawei, Bajorek, Monika, Descamps, Delphyne, Eleouet, Jean-Francois, Fourati, Slim, Noiray, Magali, Nedvedova, Stepanka, Fix, Jenna, Sizun, Christina

2025

Overview

Respiratory syncytial virus (RSV), the most common cause of bronchiolitis and pneumonia in infants, elicits a remarkably weak innate immune response. This is partly due to type I interferon (IFN) antagonism by the non-structural RSV NS1 protein. It was recently suggested that NS1 could modulate host transcription via an interaction with the MED25 subunit of the Mediator complex. Previous work emphasized the role of the NS1 C-terminal helix α3 for recruitment of the MED25 ACID domain, a target of transcription factors (TFs). Here we show that the NS1 α/β core domain binds to MED25 ACID and acts cooperatively with NS1 α3 to achieve nanomolar affinity. The strong interaction is rationalized by the dual NS1 binding site on MED25 ACID predicted by AlphaFold and confirmed by NMR, which overlaps with the two canonical binding interfaces of TF transactivation domains. Single amino acid substitutions in the NS1 α/β domain, notably NS1 E110A, significantly reduced the affinity of NS1 for MED25 ACID, both in vitro and in cellula. These mutations resulted in attenuated replication of recombinant RSV (rRSV-mCherry). They did not significantly upregulate type I or III IFN levels in IFN-competent BEAS-2B cells, contrary to the NS1 α3 deletion. However, in line with attenuated replication, the NS1 E110A mutation enhanced expression of the antiviral interferon-stimulated gene ISG15, and NS1 I54A upregulated ISG15, OAS1A and IFIT1 in IFN-competent cells. In MED25-knockdown cells, rRSV-mCherry replication was further attenuated at a late post-infection timepoint. The difference between WT and NS1 mutant rRSV-mCherry was partially lost, suggesting that the NS1–MED25 ACID complex contributes to controlling antiviral responses at this timepoint. The strong interaction and the extended binding interface between NS1 and MED25 ACID provide evidence for a mechanism, where NS1 blocks access of transcription factors to MED25, and thereby MED25-mediated transcription activation.

• Publisher: Public Library of Science,PLOS,Public Library of Science (PLoS)
• Subject: Affinity / Amino acids / Analysis / Antiviral Agents / Apoptosis / Attenuation / Binding sites / Biology and Life Sciences / Bronchopneumonia / DNA binding proteins / Gene deletion / Gene expression / Grants / Health aspects / Herpes viruses / Humans / Immune response / Immunity, Innate / Innate immunity / Interfaces / Interferon / Life Sciences / Mass spectrometry / Mediator Complex - genetics / Mediator Complex - immunology / Mediator Complex - metabolism / Medicine and Health Sciences / Mutation / NMR / NS1 protein / Nuclear magnetic resonance / Peptides / Protein Domains / Proteins / Replication / Research and Analysis Methods / Respiratory syncytial virus / Respiratory Syncytial Virus Infections - immunology / Respiratory Syncytial Virus Infections - metabolism / Respiratory Syncytial Virus Infections - virology / Respiratory Syncytial Virus, Human - immunology / Respiratory Syncytial Virus, Human - metabolism / RNA polymerase / Transcription activation / Transcription factors / Viral Nonstructural Proteins - genetics / Viral Nonstructural Proteins - immunology / Viral Nonstructural Proteins - metabolism / Virus Replication