Verger, A. | bioRxiv Science

bioRxiv2025

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

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. Author SummaryRespiratory syncytial virus (RSV) is a major pathogen for acute lower respiratory infections in infants and in the elderly. RSV elicits a remarkably weak immune response. It has developed a unique strategy to counteract the immune system, by encoding two small multifunctional proteins, RSV NS1 and NS2. NS1 is involved in interferon antagonism in the cytosol. Recently NS1 was shown to modulate host transcription in the nucleus. However, the mechanisms underpinning this function are not fully clear. Here we focus on the interplay between NS1 and the cellular MED25 coactivator protein, which can contribute to the antiviral response by activating several innate immune response genes. The MED25 C-terminal ACtivator Interacting Domain (ACID), a target of cellular transcription factors (TF), is a key feature for this function. To investigate the impact of MED25 hijacking by NS1, we combined in vitro biophysical experiments and cellular assays to probe the relationship between the stability of the NS1-MED25 ACID complex and RSV replication as well as antiviral responses. Our results suggest that this interaction is correlated with antiviral response antagonism, probably by hindering TFs to interact with MED25-ACID. This knowledge might pave the way for antiviral strategies aimed at stimulating appropriate immune responses.

pathology↗

bioRxiv2023

Assessment of machine-learning predictions for MED25 ACID domain interactions with transactivation domains

Human Mediator complex subunit MED25 binds transactivation domains (TADs) present in various cellular and viral proteins using two binding interfaces found on opposite sides of its ACID domain, and referenced as H1 and H2. Here, we use and compare deep learning methods to characterize Human MED25-TADs interfaces and assess the predicted models to published experimental data. For the H1 interface, AlphaFold produces predictions with high reliability scores that agree well with experimental data, while the H2 interface predictions appear inconsistent, preventing reliable binding modes. Despite these limitations, we experimentally assess the validity of Lana-1 and IE62 MED25 interface predictions. AlphaFold predictions also suggest the existence of a unique hydrophobic pocket for Arabidopsis MED25 ACID domain.

biochemistry↗

bioRxiv2020

Mediator complex subunit Med19 binds directly GATA DNA-binding zinc finger and functions with Med1 in GATA-driven gene regulation in vivo

The evolutionarily-conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Polymerase II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. However, binary MED subunit - TF partnerships are probably oversimplified models. Using Drosophila TFs of the GATA family - Pannier (Pnr) and Serpent (Srp) - as a model, we have previously established GATA cofactor evolutionarily-conserved function for the Med1 Mediator subunit. Here, we show that another subunit, Med19, is required for GATA-dependent gene expression and interacts physically with Pnr and Srp in cellulo, in vivo and in vitro through their conserved C-zinc finger (ZF), indicating general GATA co-activator functions. Interestingly, Med19 is critical for the regulation of all tested GATA target genes which is not the case for Med1, suggesting differential use of MED subunits by GATAs depending on the target gene. Lastly, despite their presumed distant position within the MED middle module, both subunits interact physically. In conclusion, our data shed new light first on the MED complex, engaging several subunits to mediate TF-driven transcriptional responses and second, on GATA TFs, showing that ZF DNA-binding domain also serves for transactivation.

developmental biology↗