Webminar: A dual binding mode between RSV NS1 and MED25 contributes to reshaping of antiviral responses.

Abstract:

Respiratory syncytial virus (RSV), responsible for pneumonia in infants, elicits a weak innate immune response. This is partially mediated by the non-structural NS1 protein of RSV. We and others found that NS1 interacts with the MED25 subunit of the Mediator complex. NS1 binds to the MED25-ACID domain, targeted by transcription activators. This suggests that NS1 could modulate host transcription, by an unknown mechanism, as NS1 lacks a DNA binding domain. NS1 consists of a globular core domain and a C-terminal helix α3. α3 was reported to be involved in modulation of host gene expression. The α3 peptide binds to the “H2” transactivation domain (TAD)-binding face of MED25-ACID, with micromolar affinity. However, the affinity of full-length NS1 is 15 nM. Structural predictions for a heterodimer by AlphaFold2 hint at a dual binding mode, involving both TAD-binding faces of MED25-ACID, “H1” and “H2”, as well as α3 and the core domain of NS1. This rationalizes binding observed for the NS1Δα3 deletion mutant to MED25-ACID. Structural analysis of NS1 by NMR revealed significant conformational plasticity that could promote interactions with partner proteins versus self-assembly. To investigate the individual roles of NS1 subdomains, we designed mutations in the NS1 core domain. These NS1 mutants displayed significantly weaker interaction with MED25-ACID than WT NS1. Using recombinant RSV, we observed that rRSV mutants were attenuated and induced increased production of several interferon-stimulated genes, as compared to wild-type rRSV. Taken together, our results suggest that a strong interaction with MED25-ACID, achieved by cooperative binding of α3 and the core domain, is correlated to antiviral response antagonism.