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

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

Authors

Ait-Mouhoub, C.; Dong, J.; Noiray, M.; Fix, J.; Nedvedova, S.; Verger, A.; Eleouet, J.-F.; Descamps, D.; Bajorek, M.; Sizun, C.

Abstract

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 the type I interferon (IFN-I) antagonism of 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 3 for recruitment of the MED25 ACID domain, a target of transcription factors (TFs). Here we show that the NS1 /{beta} core domain binds to MED25 ACID, and acts cooperatively with NS1 3 to achieve nanomolar affinity. This strong interaction is rationalized by the dual NS1 binding site on MED25 ACID predicted by AlphaFold3, which overlaps with the two canonical binding interfaces of TF transactivation domains (TADs), H1 and H2. By NMR, we confirmed that the NS1 /{beta} core domain targets the H2 interface. Several single amino acid NS1 /{beta} core domain mutations displayed reduced affinity for MED25 ACID, both in vitro and in cellula, at a comparable extent to the deletion of NS1 3. These mutations resulted in attenuated replication of recombinant RSV (rRSV) and increased expression of several antiviral interferon-stimulated genes (ISG) in interferon-competent cells. In MED25 knockdown cells, rRSV-mCherry replication was further attenuated, in line with the upregulation of IFT1 and ISG15 ISGs. The difference between WT and NS1 mutant rRSV-mCherry was partially lost, suggesting that RSV uses MED25 to control antiviral responses, by a mechanism involving the NS1-MED25 ACID complex. The strong interaction and the extended binding surface of NS1 on MED25 ACID provide evidence for a mechanism, where NS1 blocks access of transcription factors to MED25 ACID, and thereby MED25 mediated transcription activation.

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