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Structure and mechanism of a novel cytomegaloviral DCAF mediating interferon antagonism

ABSTRACT: Human cytomegalovirus (CMV) is a highly relevant pathogen, and its rodent counterparts serve as common infection models. Global proteome profiling of rat CMV (RCMV)-infected cells uncovered a pronounced loss of the transcription factor STAT2, which is crucial for interferon signalling. Deletion mutagenesis documented that STAT2 is targeted by the viral protein E27. Cellular analyses and in vitro reconstitution showed that E27 exploits host-derived Cullin4-RING ubiquitin ligases (CRL4) to induce poly-ubiquitylation and proteasomal degradation of STAT2. Cryo-electron microscopic structure determination revealed how E27 mimics molecular surface properties of cellular CRL4 substrate receptors to displace them from DDB1. Moreover, structure analyses solved the mechanism of STAT2 recruitment, and indicate that E27-binding disturbs the formation of active STAT2 complexes by occupying the IRF9-binding interface. Our results provide first structural insights into the cytomegalovirus-encoded interferon antagonism and establish an atomic model for STAT2 counteraction by CRL4 misappropriation, with important implications for viral immune evasion.

ORGANISM(S): Cellular Organisms

SUBMITTER: Juri Rappsilber

PROVIDER: PXD033366 | JPOST Repository | Thu Dec 15 00:00:00 GMT 2022

REPOSITORIES: jPOST

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Structure and mechanism of a novel cytomegaloviral DCAF mediating interferon antagonism

Project description:Human cytomegalovirus (CMV) is a highly relevant pathogen, and its rodent counterparts serve as common infection models. Global proteome profiling of rat CMV-infected cells uncovered a pronounced loss of the transcription factor STAT2, which is crucial for interferon signalling. Deletion mutagenesis documented that STAT2 is targeted by the viral protein E27. Cellular and in vitro analyses showed that E27 exploits host-derived Cullin4-RING ubiquitin ligases (CRL4) to induce poly-ubiquitylation and proteasomal degradation of STAT2. Cryo-electron microscopic structure determination revealed how E27 mimics molecular surface properties of cellular CRL4 substrate receptors to displace them from DDB1. Moreover, structural analyses elucidated the mechanism of STAT2 recruitment and indicate that E27-binding additionally disturbs STAT2 activation by occupying the IRF9 binding interface. For the first time, these data provide structural insights into cytomegalovirus-encoded interferon antagonism and establish an atomic model for STAT2 counteraction by CRL4 misappropriation with important implications for viral immune evasion.

2022-12-09 | PXD033639 | Pride

Lavigne2021 - Non-spatial model of viral infection dynamics and interferon response of well-mixed viral infection

Project description:This ordinary differential equation model is described in the following article: "Autocrine and paracrine interferon signalling as ‘ring vaccination’ and ‘contact tracing’ strategies to suppress virus infection in a host" G. Michael Lavigne, Hayley Russell, Barbara Sherry and Ruian Ke DOI: 10.1098/rspb.2020.3002 Comment: This model is based on the ordinary differential equations of the non-spatial model of well-mixed viral infection stated in the manuscript (Eq. 2.1 in the article). Abstract: The innate immune response, particularly the interferon response, represents a first line of defence against viral infections. The interferon molecules produced from infected cells act through autocrine and paracrine signalling to turn host cells into an antiviral state. Although the molecular mechanisms of IFN signalling have been well characterized, how the interferon response collectively contribute to the regulation of host cells to stop or suppress viral infection during early infection remain unclear. Here, we use mathematical models to delineate the roles of the autocrine and the paracrine signalling, and show that their impacts on viral spread are dependent on how infection proceeds. In particular, we found that when infection is well-mixed, the paracrine signalling is not as effective; by contrast, when infection spreads in a spatial manner, a likely scenario during initial infection in tissue, the paracrine signalling can impede the spread of infection by decreasing the number of susceptible cells close to the site of infection. Furthermore, we argue that the interferon response can be seen as a parallel to population-level epidemic prevention strategies such as ‘contact tracing’ or ‘ring vaccination’. Thus, our results here may have implications for the outbreak control at the population scale more broadly.

2021-08-11 | BIOMD0000001021 | BioModels

Doa10/MARCH6 architecture interconnects E3 ligase activity with lipid-binding transmembrane channel to regulate SQLE

Project description:Transmembrane E3 ligases play crucial roles in homeostasis. Much protein and organelle quality control, and metabolic regulation, are determined by ER-resident MARCH6 E3 ligases, including Doa10 in yeast. Here, we present Doa10/MARCH6 structural analysis by cryo-EM and AlphaFold predictions, and a structure-based mutagenesis campaign. The majority of MARCH6/Doa10 adopts a unique circular structure within the membrane. This channel is established by a lipid-binding scaffold, gated by a flexible helical bundle. The ubiquitylation active site is positioned over the channel by connections between the cytosolic E3 ligase RING domain and the membrane-spanning scaffold and gate. Assaying 95 MARCH6 variants for effects on stability of the well-characterized substrate SQLE, which regulates cholesterol levels, revealed crucial roles of the gated channel and RING domain consistent with AlphaFold-models of substrate-engaged and ubiquitylation complexes. SQLE degradation further depends on connections between the channel and RING domain, and lipid binding sites, revealing how interconnected MARCH6/Doa10 elements could orchestrate metabolic signals, substrate binding, and E3 ligase activity.

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Free SepF blocks recruitment of late cell division proteins

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KSHV chromatin looping facilitates effective gene expression: high-resolution mapping of K-Rta binding sites in the KSHV genome [ChIP-seq]

Project description:The three-dimensional structure of chromatin via formation of genomic loops allows cellular RNA polymerase II to access distal promoters, thus it has a significant impact on the outcome of gene expression. The Kaposi’s sarcoma-associated herpesvirus (KSHV) entire lytic transcriptional program of 80 plus genes is initiated by a single viral transactivator, K-Rta. Here we examined the relationship between the KSHV genomic structure and K-Rta recruitment sites with unbiased Capture Hi-C analyses. The studies showed that KSHV forms a unique genomic structure, which coordinates a domain-specific viral gene expression program via K-Rta recruitment, and identified a number of direct physical, long-range, and dynamic genomic interactions. Chromatin immunoprecipitation-sequencing analyses identified a limited number of K-Rta recruitment sites in the KSHV genome, including the K12 and PAN RNA promoters. KSHV engineered to harbor point mutations in the K-Rta-responsive elements (RE) at these promoters significantly attenuated not only the direct downstream gene, but also distal viral gene expression in a domain-specific manner. Despite the long distance between two RE sites in the linear orientation, efficient recruitment of K-Rta to either promoter required intact K-Rta REs at both promoters. Finally, inducible genomic loops generated during KSHV reactivation occurred preferentially at K-Rta recruitment sites. Mutation of a K-Rta RE inhibited formation of inducible genomic loops, gene expression of its destination of looping genes, and KSHV replication in de novo infected human gingival epithelial cells. These results suggest that the KSHV genome is programed to form both stable and inducible chromatin hubs for effective KSHV gene expression, and partly explains why K-Rta has such a dominant effect on KSHV lytic gene transcription.

2018-01-22 | GSE99943 | GEO

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2017-03-12 | GSE95464 | GEO

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