Structure and mechanism of a novel cytomegaloviral DCAF mediating interferon antagonism

  1. Vu Thuy Khanh Le-Trilling
  2. Sofia Banchenko
  3. Darius Paydar
  4. Pia Madeleine Leipe
  5. Lukas Binting
  6. Simon Lauer
  7. Andrea Graziadei
  8. Christine Gotthold
  9. Jörg Bürger
  10. Thilo Bracht
  11. Barbara Sitek
  12. Robert Jan Lebbink
  13. Anna Malyshkina
  14. Thorsten Mielke
  15. Juri Rappsilber
  16. Christian M. T. Spahn
  17. Sebastian Voigt
  18. Mirko Trilling
  19. David Schwefel

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Abstract

Human cytomegalovirus (CMV) is a highly relevant and ubiquitously distributed human pathogen. Its rodent counterparts such as mouse and rat CMV serve as common infection models. Here, we conducted the first global proteome profiling of rat CMV-infected cells and 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. A cryo-electron microscopic structure determination revealed how E27 mimics molecular surface properties of cellular CRL4 substrate receptors called DDB1- and Cullin4-associated factors (DCAFs) to displace them from the catalytic core of CRL4. Moreover, structural analyses elucidated the mechanism of STAT2 recruitment and indicate that E27-binding additionally disturbs STAT2-dependent interferon signalling by occupying its 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.

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    Reply to the reviewers

    Manuscript number: RC-2022-01481R

    Corresponding author(s): Sebastian Voigt. Mirko Trilling, David Schwefel

    1. General Statements [optional]

    2. Description of the planned revisions

    Reviewer #1: Evidence, reproducibility and clarity

    Using proteome profiling of rat CMV infected cells, the authors of this study identify the E27 protein of rat cytomegalovirus as being crucial for proteasomal degradation of STAT2. Since E27 shares 56% sequence identity to the previously characterized STAT2 antagonist M27 of murine CMV the authors investigated association of E27 with the Cullin4-RING UbL CRL4. Using gel filtration chromatography they provide evidence that E27 forms a stable ternary complex with DDB1 and STAT2 suggesting that E27 bridges STAT2 to DDB1 which is further corroborated by data from cross-linking mass spectrometry. A cross-linked DDB1/DDA1/E27/STAT2 complex was then used for cryo-EM imaging experiments. The subsequent single particle analysis yielded a density map at 3.8 A resolution that was further used to generate an E27 molecular model. At this point it should be noted that resolution was not very high and data form AlphaFold2 prediction and CLMS experiments were necessary to build a model which was described as having "sufficient quality", however, no quality parameters are included for this model. In this model, a cryptic zinc-binding motif was identified that turned out to be well conserved in M27. At this point the study switches to a mutational analysis of M27: MCMV mutants either lacking M27 or bearing an AxAxxAA triple mutation were investigated both in cell culture and in animal models. Surprisingly, the M27-AxAxxA mutant while exhibiting attenuated IFN inhibition was still more active than an M27 deletion mutant. Later during the study it is postulated that this may be due to the fact that E27 binding to STAT2 abrogates the interaction with IRF9, however, this is only predicted from modeling and no experimental data are provided for this hypothesis. Furthermore, modeling approaches were used to predict how E27 replaces endogenous CRL4 substrate receptors and how E27 recruits STAT2 to mediate CRL4-catalysed ubiquitin transfer.

    Reviewer #1: Significance

    __Reviewer #1: __This is an interesting and well written paper describing for the first time in molecular detail how a cytomegalovirus-encoded interferon antagonist degrades STAT2 by mimicking the molecular surface properties of cellular CRL4 substrate receptors.

    This study should be of broad interest for both virologists and structural biologists.

    Authors Response: We thank the reviewer for the insightful and constructive evaluation. We are very grateful for highlighting the significance of our work.

    Reviewer #1: Major points

    __Reviewer #1: __To my opinion the authors should perform mutational analysis in the context of E27 and RCMV. I accept that switching to M27 may be easier due to established procedures for MCMV mutagenesis and analysis, however, since all structural work is primarily done on E27 it would be consequent to confirm these structural predictions in the context of E27 before switching to a related protein.

    Authors Response: As the Reviewer appreciated, there were multiple reasons for the switch from RCMV-E E27 to MCMV M27. Most importantly, the MCMV in vivo infection model in mice is very well-established. Please also note that MCMV is applied far more often by virologists and immunologist as a standard model. Thus, the extension of our findings from RCMV to MCMV increases the relevance and outreach of the study. By performing the experiments in the MCMV context, we also aimed to emphasise that the function of the zinc-binding motif, which structurally organises the DDB1-binding domain, is functionally conserved among E27/M27-like proteins. Obviously, Reviewer #1 could ask why we do not solve the structure of M27 parallel to E27. With the sole exception of E27, none of the rodent M27 homologues could be produced recombinantly in a soluble form, preventing the purification and structure analysis of M27.

    Since we agree with Reviewer #1 that the extension from E27 to M27 may read “a bit rough” without a mutational analysis in the E27 context, we will construct RCMV-E E27 mutants leading to Cys=>Ala exchanges in the Zn-binding motif. An analysis of the interaction between DDB1 and these E27 mutants will be included in the revised manuscript.

    __Reviewer #1: __Moreover, data on the replication of the generated E27 deletion RCMV should be included in the manuscript (i.e. growth curves).

    Authors Response: RCMV mutants lacking the E27 gene exhibit an impaired replication. According to the suggestion, the growth curves will be part of the revised manuscript.

    Reviewer #1: The hypothesis that STAT2/E27 interaction is sterically incompatible with IRF9 binding is only based on structural prediction. It would help if the authors could present experimental evidence for such a mechanism.

    Authors Response: The hypothesis is based on three lines of argumentation: (i) structural data regarding the binding interface between STAT2 and E27 covering the known STAT2-IRF9 interface (Fig. 7F) (Rengachari et al., 2018). (ii) The finding that M27 mutants incapable to bind DDB1 and induce STAT2 degradation along the ubiquitin proteasome pathway retain a residual capacity to inhibit ISRE signaling, suggesting that the binding of M27 to STAT2 suffices to elicit some signaling inhibitory functions (Fig. 7G). (iii) To elicit their function, CRL4 substrate receptors such as E27 interact with two partners. As we discussed elsewhere (Le-Trilling and Trilling, 2020), a simultaneous development of two independent traits violates evolutionary and probability theories. Thus, these receptors must acquire their binding interfaces sequentially, and the first interaction must provide an evolutionary advantage allowing the fixation of the allele in the population. Afterwards, the second binding interface evolves. Thus, a hypothesis in which E27/M27 precursors evolved the capacity to bind STAT2, preventing its association with IRF9 thereby establishing relevant but incomplete IFN inhibition (before the DDB1 interface was invented leading to STAT2 degradation by the proteasome), provides a parsimonious explanation for all these findings without violating evolutionary constraints. To corroborate our argumentation, we will analyse if E27 indeed displaces IRF9 from STAT2 by analytical gel filtration and/or co-immunoprecipitation experiments.

    Reviewer #2: Evidence, reproducibility and clarity

    __Reviewer #2: __The manuscript entitled "Structure and mechanism of a novel cytomegaloviral DCAF mediating interferon antagonism" by Dr. Schwefel and colleagues cleverly combines biochemistry, mass-spectrometry, Cryo-EM and cell biology to dissect how RCMV-E hijacks its hosts ubiquitylation machinery to mediate proteasomal degradation of STAT2, a key player driving the antiviral IFN response. They identify E27 as DDB1-binding element, which is able promote CRL4-dependent ubiquitylation of STAT2, and demonstrate its effect on STAT2 levels by knockout RCMV-E strains. These findings are supported by in vitro reconstitution of the DDB1/E27/STAT2 complex and analyses via XL-MS and Cryo-EM. The obtained data are then powerfully validated and analysed in mutational strains via infection of homologue in vivo models. The results collectively explain how E27 mimics endogenous CRL4 substrate receptors, thereby recruiting STAT2 to be targeted by CLR4 for ubiquitylation in a NEDD8-dependent manner.

    Overall this is an important study that provides convincing insights on how rodent CMVs antagonize their host interferon response by exploiting its ubiquitin-proteasome system.

    The manuscript is well written and its introduction is extraordinarily comprehensive. There are a few minor points for the authors to consider below.

    Authors Response: We thank the reviewer for this very positive assessment.

    Reviewer #2: Significance

    Reviewer #2: The work of Schwefel and colleagues combines several powerful state-of-the art techniques to dissect the mechanism of the viral protein E27 and, for the first time, provides a rational for its ability to act as STAT2 antagonist. They performed outstanding structure-function analyses of the ubiquitin system, including the first global proteomic profiling of RCMV-infected cells, setting the standard for its human counterpart as rodent CMVs are commonly used as infection models. The manuscript is highly suitable for publication in any of the journals associated with the review commons platform.

    Authors Response: Again, we thank the reviewer for these kind words and the appreciation of our work.

    Reviewer #2: CROSS-CONSULTATION COMMENTS

    Reviewer #2: This reviewer agrees that at least testing mutants in the E27 in some assays would be appropriate.

    Authors Response: As detailed in the response to Reviewer #1, we will generate RCMV-E E27 mutants targeting the Zn-binding motif by site-directed mutagenesis. An analysis of the interaction between DDB1 and these E27 mutants will be included in the revised manuscript.

    Reviewer #3: Evidence, reproducibility and clarity

    __Reviewer #3: __Le-Trilling et al. present the first proteomic analysis of RCMV-infected cells, where they identified STAT2 as one of the most heavily downregulated (and degraded) proteins. This analysis showed that RCMV mediated degradation of STAT2 is conserved in closely related species used as animal models (rat and mouse) and human, despite the intra-host adaptation of each CMV. They also identify E27 as the RCMV factor that targets STAT2 for degradation, that exhibits ~50% homology with MCMV pM27. This study also identifies a Zinc binding motif in E27 using Cryo-EM which is conserved in other CMV species and is potentially involved in antagonising Type I and III responses.

    Reviewer #3: Significance

    __Reviewer #3: __The present work provides the first proteomics analysis of RCMV infection in rat cells, comparing infected vs non-infected rat fibroblasts to access potential RCMV targets. Then, it focuses on the characterisation of RCMV E27 and its role targeting and interacting with STAT2 (plus recruiting the Cul4 complex for STAT2 degradation). Finally, it provides the Cryo-EM structure of E27 and its CMV homologues, and the structure of the complex of E27 with elements of the CUL4 complex and STAT2. This is the first time that E27 function and structure are characterised. These are all novel findings - although the mouse homologue M27 has previously been found to interact with and degrade STAT2 (published by some of the same authors in Plos pathogens in 2011, (https://doi.org/10.1371/journal.ppat.1002069). Therefore the chief novel information is the structural studies.

    The manuscript will be of interest to researchers working with human and animal herpesviruses.

    My field of expertise is in Virology, Innate Immunity and host-virus interactions from an evolutionary perspective. I do not have expertise in Cryo-EM, so I could not evaluate the methods used in the section.

    __Authors Response: __We thank the reviewer for the positive evaluation of our work and its significance.

    Reviewer #3: Major points

    __Reviewer #3: __1. The authors claim the identification of a Zinc-binding motif in the protein E27 (RCMV) using Cryo-EM, then validation of the phenotype with MCMV WT, delM27 and M27 AxAxxA. To justify the change to MCMV to perform the functional validation, they stated "MCMV M27, the closest E27 homologue, exhibits 56% and 76% amino acid sequence identity and similarity, respectively (Fig. S4B). E27 and M27 AlphaFold2 structure predictions are almost indistinguishable (RMSD of 1.195 Å, 6652 aligned atoms) (Figs. 3B, S4A), and structural alignment of these predictions demonstrated conservation of side chain positions involved in zinc-binding (Fig. 3C). Thus, M27 represents a valid model to study functional consequences of interference with the zinc coordination motif through site-directed mutagenesis, and to test the predictive power of our E27/M27 model". Although they rationalise the change to MCMV to validate the functional outcomes of the newly identified zinc binding motif with alignments and Cryo-EM data, it falls within the DDB1 binding region that is less conserved (Fig S4B). The addition of a mouse model here provides a solid result but given the aim of the paper is to provide a proper characterisation of RCMV and elucidate some inter-species adaptations, I strongly recommend the validation with E27 here given the potential impact of this motif. Rather than having to repeat this in a rat model (which would clearly be a large amount of work), this could simply be achieved by constructing the relevant deletion / mutant viruses and assessing in vitro in a relevant cell line (readout - either virus titre or luciferase assay as shown in Figure 3G/H).

    __Authors Response: __Please also see our responses to the other reviewers. Briefly, we will apply side-directed mutagenesis to alter the CxCxxC motif in E27 that binds the zinc ion, and analyse the interaction of these E27 mutants with DDB1. In this context, we would like to add that almost two thirds of E27 residues in direct contact with DDB1 are at least type-conserved in M27, and the zinc-coordinating side chains are totally conserved (Fig. 3C). Together with a predicted similar structural organization of the respective binding regions (Fig. S11), and in light of our MCMV mutagenesis results (Fig. 7), it is highly likely that the DDB1-binding mode is conserved between E27 and M27. As mentioned above, we will put this assumption to the test in the revision process.

    __Reviewer #3: __Furthermore, in Figure 2, the GF assay was performed using full-length DDB1, however CLMS was performed using DDB1 delBPB (interchange between these two proteins continues in the remainder of the paper). This should be at least justified, and preferably one or other of wt DDB1 and DDB1 delBPB used in the GF or CLMS assay where this has not yet been performed. Later on in the results section (Fig 5E), the authors use wt DDB1 while in fig 4 they used the delBPB to describe the interaction with E27 - would be relevant to have consistency across the paper and some supplementary data that could support using one or the other in each assay.

    __Authors Response: __Protein complex preparations including full length DDB1 did not yield cryo-EM reconstructions at appropriate resolution for model building, almost certainly due to the known flexibility of the DDB1 BPB, impeding proper alignment of the cryo-EM particle images. This is why we switched to DDB1ΔBPB. Importantly, the structure model including full length DDB1 (Fig. S12B) clearly demonstrates that the BPB is located on the opposite side of the E27 binding interface on DDB1 (where it is situated to flexibly connect to the CUL4 scaffold to create the ubiquitination zone around immobilised substrates [Fig. 6]). This rules out an involvement of DDB1 BPB in E27- and/or STAT2-binding processes. Several previous studies have employed DDB1ΔBPB to facilitate structure determination, and have successfully applied the resulting structural models for functional follow-up experiments in the context of complete CRL4 assemblies (Bussiere et al., 2020; Petzold et al., 2016; Slabicki et al., 2020). Nevertheless, we will repeat GF experiments with DDB1ΔBPB for consistency and include these data in the revised manuscript.

    Reviewer #3: Minor points

    __Reviewer #3: __2. Although they present sufficient detail in the methods, further details in the text should be given as to the number of repeats performed in each case, and whether the data shown is representative or based on an average of repeats (preferably the latter; if representative, the data for other repeats should be shown in supplementary information).

    Authors Response: We will add this information in the revised version of the manuscript.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Reviewer #1: Major points

    __Reviewer #1: __Resolution of the cryoEM structure is rather low and many predictions of the manuscript are based on modeling using AlphaFold2 prediction. The authors describe their model as of "sufficient quality", however, no quality measures are included in the manuscript. At least the discussion should address limitations of the used approach.

    Authors Response: While we apologize for not sufficiently describing our quality measures, we respectfully disagree regarding the conclusion. Our resolution (3.8 Å, map 1) lies well within the 3–4 Å resolution range of the vast majority of structures deposited to the Electron Microscopy Data Bank during the last five years (https://www.emdataresource.org/statistics.html). Nevertheless, de novo modelling in this resolution regime is challenging. This is why we sought additional guidance through cross-linking mass spectrometry (XL-MS) restraints and AlphaFold2. Please also note that modelling of E27 was not based solely on the AlphaFold2 prediction. Instead, a partial model corresponding to the α-domain was manually built in map 1, guided by XL-MS information (see Methods - “Model building and refinement” and Fig. S5B, grey cartoon). This partial model proved to be in very good agreement with AlphaFold2 predictions (RMSD of 1.489 Å, 2764 aligned atoms). Only after this initial sanity check, the computational prediction was used for model completion, adjustment, and refinement.

    We now added graphical overviews of model fits in Figs. S5 and S10. Furthermore, we included detailed views of the fit of relevant side chains involved in intermolecular interaction to the experimental density (Fig. S7, S9). We also calculated and listed quality indicators of the model-to-map fit in Table S1 (correlation coefficients and model resolution based upon model-map FSC). To ensure the validity of our atomic model using an alternative method besides cryo-EM and XL-MS, we have performed site-directed mutagenesis of critical binding regions in E27, followed by in vitro reconstitution and analytical GF (Fig. S7B, C, S9B, C). The text was revised accordingly (see p10 [ll22] and p14 [ll26]).

    __Reviewer #1: __The authors identify a cryptic zinc-binding motif in E27 that is conserved in homologous proteins. For this reviewer it is not clear: is there experimental evidence for zinc binding of E27 or can the presence of zinc reliably be detected in their structural data? If not, it would be worth to confirm zinc binding.

    Authors Response: Our structural data show a tetragonal metal coordination geometry, involving three cysteine side chains and one histidine side chain, with coordination bond lengths of 2.2 Å between the histidine nitrogen and the metal ion, and of 2.4 Å between the cysteine sulfurs and the metal ion. The density feature cannot be explained by another type of side chain interaction, e.g. a disulfide bond, because this would lead to a steric clash with the remaining adjacent side chains. Based on the knowledge on metal-binding sites in proteins and metal-coordination chemistry, these characteristics indicate the presence of a structural zinc-binding site for the following reasons: (i) after magnesium, zinc is the second most prevalent metal in the Protein Data Bank (https://metalpdb.cerm.unifi.it/getSummary), however, magnesium is coordinated octahedrally by oxygen ligands (Tang and Yang, 2013); (ii) the most abundant zinc ligands are cysteine and histidine; (iii) the most abundant zinc coordination number is four ligands; (iv) the average coordination bond lengths are 2.12±0.19 Å and 2.33±0.12Å for nitrogen-zinc and sulfur-zinc interactions, respectively (Ireland and Martin, 2019; Laitaoja et al., 2013), which is in very good agreement with our structural observations. We included this argumentation in the revised manuscript (see p9 [ll21]), and added Fig. S5C for visualization.

    Reviewer #2: Minor points

    Reviewer #2: Page 2, line 3. "Here," should be inserted before "Global proteome profiling..." to highlight the work of this manuscript.

    Authors Response: We changed the text accordingly.

    Reviewer #2: Page 3, line 21. "IFNs" instead of "IFN"

    Authors Response: We changed the text accordingly.

    Reviewer #2: Page 4, lines 9,15,27. "Ubiquitin Ligases (UbL)" is not a common abbreviation and could be mistaken for Ubl (Ubiquitin-like proteins). Possible abbreviation is "E3s" for Ubiquitin E3 ligases

    Authors Response: We have amended the respective abbreviations accordingly.

    Reviewer #2: Page 4 line 25. "RBX1" is the more common term for "ROC1"

    Authors Response: This has been corrected throughout the manuscript.

    Reviewer #2: Page 5 lines 1-9. Citing of the first structure of DDB1 in complex with a viral protein is recommended. (Ti Li et al. Cell 2006)

    Authors Response: We thank the reviewer for this important suggestions and cited this landmark publication.

    Reviewer #2: Figure 1 a) STAT2 dot is cut off in second panel. I recommend highlighting STAT2 in both panels.

    We amended the figure accordingly. We furthermore additionally highlighted the “STAT2” text in both panels by increasing the font size and putting it in bold type.

    Reviewer #2: Page 7 line 17. "Cross-linking MS (CLMS)" is commonly abbreviated as (XL-MS)

    Authors Response: We changed the text accordingly.

    Reviewer #2: Figure 2 a-c) These panels could benefit from thinner lines in order to increase visibility of chromatograms and cross-links.

    Authors Response: The panels were changed accordingly.

    Reviewer #2: Figure 2 a-b) Could the authors elaborate on why STAT2 is stoichiometrically

    underrepresented in the SDS-PAGE of the E27/DDB1/STAT2 complex?

    Authors Response: We applaud Reviewer #2 for their in-depth examination. Honestly, we were also puzzled by this. Based on the cryo-EM single particle analysis, we found an explanation: We separated a major contamination in silico during 2D classification (~12% of all particles). Out of curiosity, we reconstructed a density map from these particles (now shown in Fig. S3). The map was identical to a previous cryo-EM structure of the E. coli protein ArnA (Yang et al., 2019), a notorious contaminant in E. coli Ni-NTA protein purifications (Andersen et al., 2013). ArnA migrates similar to E27 on the SDS-PAGE, the band runs just a little bit faster (compare fraction 6 [ArnA] and fractions 8/9 [E27] from the SDS-PAGE of the analytical GF run of E27 in isolation, Fig. 2A, green trace). However, in analytical GF, ArnA elutes at higher molecular weight fractions, since it forms a hexamers (Ve~10.2 ml). Incidentally, this elution volume of the ArnA hexamer almost equals the one of DDB1 or DDB1ΔBPB/DDA1/E27/STAT2 complexes. This leads to a superposition of ArnA and E27 bands in the respective SDS-PAGE lanes corresponding to GF fraction 6. Accordingly, we conclude that it is actually not STAT2 that is underrepresented, but rather E27 seems overrepresented due to SDS-PAGE band overlap with the ArnA contaminant. We have now indicated the contaminant in Fig. 2A, amended the legend, and extended Fig. S3 to indicate at which point of the cryo-EM analysis the contaminating ArnA particles were separated, and to show the ArnA model to map fit.

    In addition to this, it might be that potential STAT2 degradation products (marked by ** in Fig. 2), which seem to co-migrate with STAT2/E27 complexes, occupy FL STAT2 binding sites on E27.

    Reviewer #2: Paragraph "The E27 structure.." page 9. Placing this paragraph after the overall

    structure is recommended.

    Authors Response: Accordingly, we have now moved this section to the end of the results section.

    Reviewer #2: Figure 3 a) The grey mesh being laid over the ribbon structures is not contributing to the overall visibility. Adding a panel of the cryo-EM structure alone in cost of alphafold models is recommended.

    Figure 4a) same issue with grey mesh

    Authors Response: Thank you very much for the very good suggestions. We have removed the mesh representation, and included panels just showing the segmented cryo-EM map in the new Fig. 3A.

    Reviewer #2: c) panels could benefit from fewer amino acids being labeled/shown

    Authors Response: We understand the motives of the Reviewer. However, we would prefer to depict all relevant side chain interactions in these panels. The rearrangement of the figure, i.e. showing the overview of the interacting regions before the detailed panels, should make them more accessible (new Fig. 3B).

    __Reviewer #2: __d) may want to avoid red-green coloring to improve for colorblindness

    Authors Response: We are deeply sorry for our ignorance in this regard. We changed the colors accordingly (see new Fig. 3B, C).

    __Reviewer #2: __Figure 6a) s.a grey mesh

    Authors Response: We removed the mesh representations and included panels just showing the segmented cryo-EM density in the new Fig. 5C.

    Reviewer #2: CROSS-CONSULTATION COMMENTS

    __Reviewer #2: __A 3.8 A overall resolution map and the approach to fitting may be suitable, but it is unclear from the authors' figures whether the side-chains shown in the figures are clearly visible in the map or if they are modeled by some other approach. Side chains should ideally be visible in the maps if shown in figures, and if not, close-ups of the corresponding regions of the maps should be shown with sufficient depthcue to allow the reader to gauge how the map corresponds to the model.

    Authors Response: This is a crucial point. As mentioned in the response to Reviewer #1, major point 2, we have now included very detailed views of the fit of relevant side chains involved in intermolecular interaction to the experimental density (Fig. S7, S9).

    __Reviewer #2: __Along these lines, the figures with the mesh maps do not clearly show how well the model fits the map. This needs to be clearly visible in figures, and ideally maps and models provided to reviewers in order for the reviewers to gauge the level of accuracy of the fit.

    Authors Response: Please see our response to Reviewer #1, major point 2. Briefly, we have now included graphical overviews of model fits in Figs. S5 and S10. We also calculated and listed quality indicators of the model-to-map fit in Table S1 (correlation coefficients and model resolution based upon model-map FSC). To ensure the validity of our atomic model using an alternative method besides cryo-EM and XL-MS, we have performed site-directed mutagenesis of critical binding regions in E27, followed by in vitro reconstitution and analytical GF (Fig. S7B, C, S9B, C). The text was extended accordingly (see p10 [ll22] and p14 [ll26]).

    __Reviewer #2: __At minimum, the authors have nicely assembled proteomics and cell biological data indicating that E27 hijacks CRL4 to turn over Stat2 in rat cells in a manner paralagous to M27 hijacking in mouse cells, biophysical/structural data for a model of a CUL4-DDB1-E27-Stat2 complex, and mutagenesis of a putative zinc binding site in M27.

    I feel most of the issues raised by all 3 reviewers could be addressed in the text, with more clarity about the structural models, and better explanation for why the construct with proteins from various organisms were used for structural studies (the authors had made human DDB1 before, and it expressed well, and perhaps didn't consider to make from rat? Or this mixture expressed, purified best? Gave best quality EM data?).

    Authors Response: We thank Reviewer #2 for her/his overall assessment. As mentioned in the two cross-consultation comments before, and in the response to Reviewer #1, major point 2, we strived to provide adequate measures allowing to judge the quality of our structural models in the present updated version of the manuscript. In addition, as indicated in the response to reviewer #3, major point 2, we have now added Fig. S12 and extended the Discussion to explain and justify the use of different protein constructs.

    __Reviewer #2: __Also, the presentation of the zinc binding site should come after the overall structure. As for the use of MCMV to assess the role of the zinc binding site, placing this last in the text might allow this to flow better.

    Authors Response: Thank you very much for this suggestion. The manuscript has been restructured as recommended: details of the zinc-binding motif and the MCMV assays are now shown in Fig. 7 and described in the text just before the Discussion.

    Reviewer #3: Major points

    __Reviewer #3: __2. Given that previous data in mice showed that the E27 homologue pM27 binds a component of host Cullin4-RING UbLs (CRL4), to induce the poly-ubiquitination of STAT2, the current study also addressed if this mechanism was preserved in RCMV. Yet, they seemed to do this with E27, rnSTAT2 and hsDDB1 - Page 7 lines 1 to 3: "These results prompted us to explore the association of E27 with Rattus norvegicus (rn) STAT2 and Homo sapiens (hs) DDB1 in vitro. Importantly, 1128 of 1140 amino acids are identical between hsDDB1 and rnDDB1 (...)". They identify the residues and regions where the DDB1 is different between both species, but should provide a structure/alignment with this highlighted. In addition, DDB1 is a DNA damage protein that is annotated in the Rattus norvegicus genome. The authors should justify the assays between rnSTAT2-hsDDB1 instead of using the both proteins from rn, and present the equivalent data for rnDDB1 in the paper.

    Authors Response: Among the 12 alterations between human and rat DDB1, 4 are type-conserved (Fig. S12A). Thus, >99% of amino acids are identical or similar. We mapped all exchanges on a model of full length human DDB1 bound to E27 and the rat STAT2 CCD. None are involved in intermolecular interactions (Fig. S12B, C). Please note that due to the high conservation of DDB1 across eukaryotes, this inter-species approach has been used by us and others to study DDB1-containing complexes (e.g., the SV5V, WHX, SIV Vpx and Vpr, zebrafish DDB2, and chicken CRBN proteins have been in vitro reconstituted with human DDB1 for structural characterisation) and valid biological conclusions have been drawn from these studies (Angers et al., 2006; Banchenko et al., 2021; Fischer et al., 2014; Fischer et al., 2011; Li et al., 2006; Li et al., 2010; Schwefel et al., 2015; Schwefel et al., 2014; Wu et al., 2015).

    Reviewer #3: Minor points

    __Reviewer #3: __1. In fig 5D, the authors present the H-box alignment, where it is clear that this motif is not conserved. The lack of H-box conservation should be discussed in the results and discussion, to provide an explanation for the competition/binding observed.

    Authors Response: We respectfully disagree. There is conservation of amino acid side chains, regarding their physicochemical properties, observable in the H-box motif. Furthermore, the secondary structure is conserved. Please note, that the H-box is not our invention but rather represented a well-accepted motif known in the field, see e.g., (Li et al., 2010). We extended the discussion to cover this point (p21 [ll15]).

    __Reviewer #3: __3. The authors commence their abstract justifying the study on the grounds of the usefulness of rodent HCMV counterparts as common infection models for HCMV. They should return to this theme in the discussion - what is the usefulness of their findings with regards to HCMV (particularly given the relatively low homology between E27 and HCMV pUL27, and the alternative mechanism for STAT2 antagonism encoded by HCMV UL145)?

    Authors Response: We extended the discussion in this regard. Briefly, our data, to our knowledge for the first time, reveal that RCMV (like MCMV) exploits CRL4 to induce proteasomal degradation of STAT2. With pUL145, HCMV relies on an analogous protein. In clear contrast to HCMV, RMCV and MCMV are both amenable to in vivo experiments in small animal models. Over 40 years ago, HCMV has been called the troll of transplantation due to its grim impact on immunosuppressed individuals after transplantation surgery (Balfour, 1979). Despite tremendous efforts, HCMV still harms and kills graft recipients. While MCMV allows various experiments regarding general principles of cytomegaloviral pathogenesis and antiviral immunity, one shortcoming is that the mouse obviously is a rather small animal, preventing various chirurgical and solid organ transplantation (SOT) procedures. In clear contrast, SOT procedures that are indispensable for human medicine can be recapitulated in rat models. Thus, according to our opinion, our work lays the molecular foundation for future studies addressing the relevance of STAT2 and CMV-induced STAT2 degradation in rat SOT models.

    4. Description of analyses that authors prefer not to carry out

    -

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  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Le-Trilling et al. present the first proteomic analysis of RCMV-infected cells, where they identified STAT2 as one of the most heavily downregulated (and degraded) proteins. This analysis showed that RCMV mediated degradation of STAT2 is conserved in closely related species used as animal models (rat and mouse) and human, despite the intra-host adaptation of each CMV. They also identify E27 as the RCMV factor that targets STAT2 for degradation, that exhibits ~50% homology with MCMV pM27. This study also identifies a Zinc binding motif in E27 using Cryo-EM which is conserved in other CMV species and is potentially involved in antagonising Type I and III responses.

    Major and minor concerns to be addressed:

    Major points

    1. The authors claim the identification of a Zinc-binding motif in the protein E27 (RCMV) using Cryo-EM, then validation of the phenotype with MCMV WT, delM27 and M27 AxAxxA. To justify the change to MCMV to perform the functional validation, they stated "MCMV M27, the closest E27 homologue, exhibits 56% and 76% amino acid sequence identity and similarity, respectively (Fig. S4B). E27 and M27 AlphaFold2 structure predictions are almost indistinguishable (RMSD of 1.195 Å, 6652 aligned atoms) (Figs. 3B, S4A), and structural alignment of these predictions demonstrated conservation of side chain positions involved in zinc-binding (Fig. 3C). Thus, M27 represents a valid model to study functional consequences of interference with the zinc coordination motif through site-directed mutagenesis, and to test the predictive power of our E27/M27 model". Although they rationalise the change to MCMV to validate the functional outcomes of the newly identified zinc binding motif with alignments and Cryo-EM data, it falls within the DDB1 binding region that is less conserved (Fig S4B). The addition of a mouse model here provides a solid result but given the aim of the paper is to provide a proper characterisation of RCMV and elucidate some inter-species adaptations, I strongly recommend the validation with E27 here given the potential impact of this motif. Rather than having to repeat this in a rat model (which would clearly be a large amount of work), this could simply be achieved by constructing the relevant deletion / mutant viruses and assessing in vitro in a relevant cell line (readout - either virus titre or luciferase assay as shown in Figure 3G/H).
    2. Given that previous data in mice showed that the E27 homologue pM27 binds a component of host Cullin4-RING UbLs (CRL4), to induce the poly-ubiquitination of STAT2, the current study also addressed if this mechanism was preserved in RCMV. Yet, they seemed to do this with E27, rnSTAT2 and hsDDB1 - Page 7 lines 1 to 3: "These results prompted us to explore the association of E27 with Rattus norvegicus (rn) STAT2 and Homo sapiens (hs) DDB1 in vitro. Importantly, 1128 of 1140 amino acids are identical between hsDDB1 and rnDDB1 (...)". They identify the residues and regions where the DDB1 is different between both species, but should provide a structure/alignment with this highlighted. In addition, DDB1 is a DNA damage protein that is annotated in the Rattus norvegicus genome. The authors should justify the assays between rnSTAT2-hsDDB1 instead of using the both proteins from rn, and present the equivalent data for rnDDB1 in the paper. Furthermore, in Figure 2, the GF assay was performed using full-length DDB1, however CLMS was performed using DDB1 delBPB (interchange between these two proteins continues in the remainder of the paper). This should be at least justified, and preferably one or other of wt DDB1 and DDB1 delBPB used in the GF or CLMS assay where this has not yet been performed. Later on in the results section (Fig 5E), the authors use wt DDB1 while in fig 4 they used the delBPB to describe the interaction with E27 - would be relevant to have consistency across the paper and some supplementary data that could support using one or the other in each assay.

    Minor points:

    1. In fig 5D, the authors present the H-box alignment, where it is clear that this motif is not conserved. The lack of H-box conservation should be discussed in the results and discussion, to provide an explanation for the competition/binding observed.
    2. Although they present sufficient detail in the methods, further details in the text should be given as to the number of repeats performed in each case, and whether the data shown is representative or based on an average of repeats (preferably the latter; if representative, the data for other repeats should be shown in supplementary information).
    3. The authors commence their abstract justifying the study on the grounds of the usefulness of rodent HCMV counterparts as common infection models for HCMV. They should return to this theme in the discussion - what is the usefulness of their findings with regards to HCMV (particularly given the relatively low homology between E27 and HCMV pUL27, and the alternative mechanism for STAT2 antagonism encoded by HCMV UL145)?

    Significance

    The present work provides the first proteomics analysis of RCMV infection in rat cells, comparing infected vs non-infected rat fibroblasts to access potential RCMV targets. Then, it focuses on the characterisation of RCMV E27 and its role targeting and interacting with STAT2 (plus recruiting the Cul4 complex for STAT2 degradation). Finally, it provides the Cryo-EM structure of E27 and its CMV homologues, and the structure of the complex of E27 with elements of the CUL4 complex and STAT2. This is the first time that E27 function and structure are characterised. These are all novel findings - although the mouse homologue M27 has previously been found to interact with and degrade STAT2 (published by some of the same authors in Plos pathogens in 2011, (https://doi.org/10.1371/journal.ppat.1002069). Therefore the chief novel information is the structural studies.

    The manuscript will be of interest to researchers working with human and animal herpesviruses.

    My field of expertise is in Virology, Innate Immunity and host-virus interactions from an evolutionary perspective. I do not have expertise in Cryo-EM, so I could not evaluate the methods used in the section.

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript entitled "Structure and mechanism of a novel cytomegaloviral DCAF mediating interferon antagonism" by Dr. Schwefel and colleagues cleverly combines biochemistry, mass-spectrometry, Cryo-EM and cell biology to dissect how RCMV-E hijacks its hosts ubiquitylation machinery to mediate proteasomal degradation of STAT2, a key player driving the antiviral IFN response. They identify E27 as DDB1-binding element, which is able promote CRL4-dependent ubiquitylation of STAT2, and demonstrate its effect on STAT2 levels by knockout RCMV-E strains. These findings are supported by in vitro reconstitution of the DDB1/E27/STAT2 complex and analyses via XL-MS and Cryo-EM. The obtained data are then powerfully validated and analysed in mutational strains via infection of homologue in vivo models. The results collectively explain how E27 mimics endogenous CRL4 substrate receptors, thereby recruiting STAT2 to be targeted by CLR4 for ubiquitylation in a NEDD8-dependent manner.

    Overall this is an important study that provides convincing insights on how rodent CMVs antagonize their host interferon response by exploiting its ubiquitin-proteasome system. The manuscript is well written and its introduction is extraordinarily comprehensive. There are a few minor points for the authors to consider below.

    Minor points:

    Page 2, line 3. "Here," should be inserted before "Global proteome profiling..." to highlight the work of this manuscript.

    Page 3, line 21. "IFNs" instead of "IFN"

    Page 4, lines 9,15,27. "Ubiquitin Ligases (UbL)" is not a common abbreviation and could be mistaken for Ubl (Ubiquitin-like proteins). Possible abbreviation is "E3s" for Ubiquitin E3 ligases

    Page 4 line 25. "RBX1" is the more common term for "ROC1"

    Page 5 lines 1-9. Citing of the first structure of DDB1 in complex with a viral protein
    is recommended. (Ti Li et al. Cell 2006)

    Figure 1 a) STAT2 dot is cut off in second panel. I recommend highlighting STAT2
    in both panels.

    Page 7 line 17. "Cross-linking MS (CLMS)" is commonly abbreviated as (XL-MS)

    Figure 2 a-c) These panels could benefit from thinner lines in order to increase visibility of chromatograms and cross-links.

    Figure 2 a-b) Could the authors elaborate on why STAT2 is stoichiometrically underrepresented in the SDS-PAGE of the E27/DDB1/STAT2 complex?

    Paragraph "The E27 structure.." page 9. Placing this paragraph after the overall structure is recommended.

    Figure 3 a) The grey mesh being laid over the ribbon structures is not contributing to the overall visibility. Adding a panel of the cryo-EM structure alone in cost of alphafold models is recommended.

    Figure 4a) same issue with grey mesh c) panels could benefit from fewer amino acids being labeled/shown d) may want to avoid red-green coloring to improve for colorblindness

    Figure 6a) s.a grey mesh

    Referees cross-commenting

    A 3.8 A overall resolution map and the approach to fitting may be suitable, but it is unclear from the authors' figures whether the side-chains shown in the figures are clearly visible in the map or if they are modeled by some other approach. Side chains should ideally be visible in the maps if shown in figures, and if not, close-ups of the corresponding regions of the maps should be shown with sufficient depthcue to allow the reader to gauge how the map corresponds to the model.

    Along these lines, the figures with the mesh maps do not clearly show how well the model fits the map. This needs to be clearly visible in figures, and ideally maps and models provided to reviewers in order for the reviewers to gauge the level of accuracy of the fit.

    At minimum, the authors have nicely assembled proteomics and cell biological data indicating that E27 hijacks CRL4 to turn over Stat2 in rat cells in a manner paralagous to M27 hijacking in mouse cells, biophysical/structural data for a model of a CUL4-DDB1-E27-Stat2 complex, and mutagenesis of a putative zinc binding site in M27.

    I feel most of the issues raised by all 3 reviewers could be addressed in the text, with more clarity about the structural models, and better explanation for why the construct with proteins from various organisms were used for structural studies (the authors had made human DDB1 before, and it expressed well, and perhaps didn't consider to make from rat? Or this mixture expressed, purified best? Gave best quality EM data?). Also, the presentation of the zinc binding site should come after the overall structure.

    As for the use of MCMV to assess the role of the zinc binding site, placing this last in the text might allow this to flow better. This reviewer agrees that at least testing mutants in the E27 in some assays would be appropriate.

    Significance

    The work of Schwefel and colleagues combines several powerful state-of-the art techniques to dissect the mechanism of the viral protein E27 and, for the first time, provides a rational for its ability to act as STAT2 antagonist. They performed outstanding structure-function analyses of the ubiquitin system, including the first global proteomic profiling of RCMV-infected cells, setting the standard for its human counterpart as rodent CMVs are commonly used as infection models. The manuscript is highly suitable for publication in any of the journals associated with the review commons platform.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Using proteome profiling of rat CMV infected cells, the authors of this study identify the E27 protein of rat cytomegalovirus as being crucial for proteasomal degradation of STAT2. Since E27 shares 56% sequence identity to the previously characterized STAT2 antagonist M27 of murine CMV the authors investigated association of E27 with the Cullin4-RING UbL CRL4. Using gel filtration chromatography they provide evidence that E27 forms a stable ternary complex with DDB1 and STAT2 suggesting that E27 bridges STAT2 to DDB1 which is further corroborated by data from cross-linking mass spectrometry. A cross-linked DDB1/DDA1/E27/STAT2 complex was then used for cryo-EM imaging experiments. The subsequent single particle analysis yielded a density map at 3.8 A resolution that was further used to generate an E27 molecular model. At this point it should be noted that resolution was not very high and data form AlphaFold2 prediction and CLMS experiments were necessary to build a model which was described as having "sufficient quality", however, no quality parameters are included for this model. In this model, a cryptic zinc-binding motif was identified that turned out to be well conserved in M27. At this point the study switches to a mutational analysis of M27: MCMV mutants either lacking M27 or bearing an AxAxxAA triple mutation were investigated both in cell culture and in animal models. Surprisingly, the M27-AxAxxA mutant while exhibiting attenuated IFN inhibition was still more active than an M27 deletion mutant. Later during the study it is postulated that this may be due to the fact that E27 binding to STAT2 abrogates the interaction with IRF9, however, this is only predicted from modeling and no experimental data are provided for this hypothesis. Furthermore, modeling approaches were used to predict how E27 replaces endogenous CRL4 substrate receptors and how E27 recruits STAT2 to mediate CRL4-catalysed ubiquitin transfer.

    Major comments:

    1. To my opinion the authors should perform mutational analysis in the context of E27 and RCMV. I accept that switching to M27 may be easier due to established procedures for MCMV mutagenesis and analysis, however, since all structural work is primarily done on E27 it would be consequent to confirm these structural predictions in the context of E27 before switching to a related protein. Moreover, data on the replication of the generated E27 deletion RCMV should be included in the manuscript (i.e. growth curves).
    2. Resolution of the cryoEM structure is rather low and many predictions of the manuscript are based on modeling using AlphaFold2 prediction. The authors describe their model as of "sufficient quality", however, no quality measures are included in the manuscript. At least the discussion should address limitations of the used approach.
    3. The authors identify a cryptic zinc-binding motif in E27 that is conserved in homologous proteins. For this reviewer it is not clear: is there experimental evidence for zinc binding of E27 or can the presence of zinc reliably be detected in their structural data? If not, it would be worth to confirm zinc binding.
    4. The hypothesis that STAT2/E27 interaction is sterically incompatible with IRF9 binding is only based on structural prediction. It would help if the authors could present experimental evidence for such a mechanism.

    Significance

    This is an interesting and well written paper describing for the first time in molecular detail how a cytomegalovirus-encoded interferon antagonist degrades STAT2 by mimicking the molecular surface properties of cellular CRL4 substrate receptors.

    This study should be of broad interest for both virologists and structural biologists.

  5. Version published to 10.1101/2022.05.05.490734v2 on bioRxiv
  6. Version published to 10.1101/2022.05.05.490734v1 on bioRxiv