The 14-3-3 molecular scaffolds promote type I interferon (IFN) responses by stabilizing the interaction of RIG-I with the TRIM25 ligase. Viruses have evolved unique strategies to halt this cellular response to support their replication and spread. Here, we report that the ubiquitin deconjugase encoded in the N-terminus of the Epstein-Barr virus (EBV) large tegument protein BPLF1 harnesses 14-3-3 molecules to promote TRIM25 autoubiquitination and sequestration of the ligase into inactive protein aggregates. Catalytically inactive BPLF1 induced K48-linked autoubiquitination and degradation of TRIM25 while the ligase was mono- or di-ubiquitinated in the presence of the active viral enzyme and formed cytosolic aggregates decorated by the autophagy receptor p62/SQSTM1. Aggregate formation and the inhibition of IFN response were abolished by mutations of solvent exposed residues in helix-2 of BPLF1 that prevented binding to 14-3-3 while preserving both catalytic activity and binding to TRIM25. 14-3-3 interacted with the Coiled-Coil (CC) domain of TRIM25 in in vitro pulldown, while BPLF1 interacted with both the CC and B-box domains, suggesting that 14-3-3 positions BPLF1 at the ends of the CC dimer, close to known autoubiquitination sites. Our findings provide a molecular understanding of the mechanism by which a viral deubiquitinase inhibits the IFN response and emphasize the role of 14-3-3 proteins in modulating antiviral defenses. We have performed a molecular characterization of the mechanism by which the ubiquitin deconjugases encoded in the N-terminal domain of the herpesvirus large tegument proteins inhibit the type I IFN response. Starting from our previous finding that BPLF1, the Epstein-Barr virus (EBV) encoded member of the viral DUB family, induces the formation of a trimolecular complex including TRIM25 and 14-3-3 we now show that the complex promotes both the autoubiquitination and deubiquitination of TRIM25, which leads to sequestration of the ligase into protein aggregates decorated by the autophagy receptor p62/SQSTM1. Using mutants of a conserved putative protein-protein interaction motif in helix-2 of BPLF1 we show that binding to 14-3-3 is essential for this effect and for inhibition of the IFN response. Using 14-3-3 binding mutants in co-immunoprecipitation assays, we found that both BPLF1 and TRIM25 interact with the substrate binding groove of 14-3-3, suggesting that 14-3-3 serves as scaffold for the formation of the trimolecular complex. In vitro pulldown assays using TRIM25 subdomains and bacterially expressed BPLF1 and 14-3-3 suggest that 14-3-3 and BPLF1 interact with the tip of the coiled-coil domain, positioning the viral DUB close to a known autoubiquitination site in TRIM25. We used our findings to build a model of the trimeric complex based on available crystal structures and protein docking algorithms. The model provides a first characterization of the molecular interactions involved in the inhibition of TRIM25 by the viral DUB and has interesting implications for the regulation of TRIM25 activity.

Plant health and performance are highly dependent on the root microbiome. The impact of agricultural management on the soil microbiome has been studied extensively. However, a comprehensive understanding of how soil types and fertilization regimes affect both soil and root microbiome is still lacking, such as how fertilization regimes affect the root microbiome’s stability, and whether it follows the same patterns as the soil microbiome. In this study, we carried out a long-term experiment to see how different soil types, plant varieties, and fertilizer regimens affected the soil and root bacterial communities. Our results revealed higher stability of microbial networks under combined organic-inorganic fertilization than those relied solely on inorganic or organic fertilization. The root microbiome variation was predominantly caused by total nitrogen, while the soil microbiome variation was primarily caused by pH and soil organic matter. Bacteroidetes and Firmicutes were major drivers when the soil was amended with organic fertilizer, but Actinobacteria was found to be enriched in the soil when the soil was treated with inorganic fertilizer. Our findings demonstrate how the soil and root microbiome respond to diverse fertilizing regimes, and hence contribute to a better understanding of smart fertilizer as a strategy for sustainable agriculture.

MHC-I epitope presentation to CD8 ⁺ T cells is directly dependent on peptide loading and selection during antigen processing. However, the exact molecular bases underlying peptide selection and binding by MHC-I remain largely unknown. Within the peptide-loading complex, the peptide editor tapasin is key to the selection of MHC-I–bound peptides. Here, we have determined an ensemble of crystal structures of MHC-I in complex with the peptide exchange-associated dipeptide GL, as well as the tapasin-associated scoop loop, alone or in combination with candidate epitopes. These results combined with mutation analyses allow us to propose a molecular model underlying MHC-I peptide selection by tapasin. The N termini of bound peptides most probably bind first in the N-terminal and middle region of the MHC-I peptide binding cleft, upon which the peptide C termini are tested for their capacity to dislodge the tapasin scoop loop from the F pocket of the MHC-I cleft. Our results also indicate important differences in peptide selection between different MHC-I alleles.

Eukaryotic organelles have developed elaborate protein quality control systems to ensure their normal activity, among which Deg/HtrA proteases play an essential role. Plant Deg2 protease is a homologue of prokaryotic DegQ/DegP proteases and is located in the chloroplast stroma, where its proteolytic activity is required to maintain the efficiency of photosynthetic machinery during stress. Here, we demonstrate that Deg2 exhibits dual protease-chaperone activities, and we present the hexameric structure of Deg2 complexed with co-purified peptides. The structure shows that Deg2 contains a unique second PDZ domain (PDZ2) following a conventional PDZ domain (PDZ1), with PDZ2 orchestrating the cage assembly of Deg2. We discovered a conserved internal ligand for PDZ2 that mediates hexamer formation and thus locks the protease in the resting state. These findings provide insight into the diverse modes of PDZ domain-mediated regulation of Deg proteases.

Ras-GTPase-activating protein SH3-domain-binding proteins (G3BP) are critical for the formation of stress granules (SGs) through their RNA- and ribosome-binding properties. SARS-CoV-2 nucleocapsid (N) protein exhibits strong binding affinity for G3BP and inhibits infection-induced SG formation soon after infection. To study the impact of the G3BP-N interaction on viral replication and pathogenesis in detail, we generated a mutant SARS-CoV-2 (RATA) that specifically lacks the G3BP-binding motif in the N protein. RATA triggers a stronger and more persistent SG response in infected cells, showing reduced replication across various cell lines, and greatly reduced pathogenesis in K18-hACE2 transgenic mice. At early times of infection, G3BP and WT N protein strongly colocalise with dsRNA and with non-structural protein 3 (nsp3), a component of the pore complex in double membrane vesicles (DMVs) from which nascent viral RNA emerges. Furthermore, G3BP-N complexes promote highly localized translation of viral mRNAs in the immediate vicinity of the DMVs and thus contribute to efficient viral gene expression and replication. In contrast, G3BP is absent from the DMVs in cells infected with RATA and translation of viral mRNAs is less efficient. This work provides a fuller understanding of the multifunctional roles of G3BP in SARS-CoV-2 infection.

Viral escape from CD8⁺ cytotoxic T lymphocyte responses correlates with disease progression and represents a significant challenge for vaccination. Here, we demonstrate that CD8⁺ T cell recognition of the naturally occurring MHC-I-restricted LCMV-associated immune escape variant Y4F is restored following vaccination with a proline-altered peptide ligand (APL). The APL increases MHC/peptide (pMHC) complex stability, rigidifies the peptide and facilitates T cell receptor (TCR) recognition through reduced entropy costs. Structural analyses of pMHC complexes before and after TCR binding, combined with biophysical analyses, revealed that although the TCR binds similarly to all complexes, the p3P modification alters the conformations of a very limited amount of specific MHC and peptide residues, facilitating efficient TCR recognition. This approach can be easily introduced in peptides restricted to other MHC alleles, and can be combined with currently available and future vaccination protocols in order to prevent viral immune escape. Viral escape mutagenesis correlates often with disease progression and represents a major hurdle for vaccination-based therapies. Here, we have designed and developed a novel generation of altered epitopes that re-establish and enhance significantly CD8⁺ T cell recognition of a naturally occurring viral immune escape variant. Biophysical and structural analyses provide a clear understanding of the molecular mechanisms underlying this reestablished recognition. We believe that this approach can be implemented to currently available or novel vaccination approaches to efficiently restore T cell recognition of virus escape variants to control disease progression.