@article{Krasteva-Christ2022,

title = {Bitter Taste Signaling in Tracheal Epithelial Brush Cells Elicits Innate Immune Responses to Bacterial Infection},

author = {M.I. Hollenhorst, R. Nandigama, S.B. Evers, I. Gamayun, N.A. Wadood, A. Salah, M. Pieper, A. Wyatt, A. Stukalov, A. Gebhardt, W. Nadolni, W. Burow, C. Herr, C. Beisswenger, S. Kusumakshi, F. Ectors, T.I. Kichko, 

 L. Hübner, P. Reeh, A. Munder, S. Wienhold, M. Witzenrath, R. Bals, V. Flockerzi, T. Gudermann, M. Bischoff , P. Lipp, S. Zierler, V. Chubanov, A. Pichlmair, P. König, U. Boehm, G. Krasteva-Christ},

url = {https://doi.org/10.1172/JCI150951},

doi = {10.1172/JCI150951},

issn = {1558-8238},

year  = {2022},

date = {2022-07-01},

journal = {The Journal of Clinical Investigation},

volume = {132},

number = {13},

abstract = {Constant exposure of the airways to inhaled pathogens requires efficient early immune responses protecting against infections. How bacteria on the epithelial surface are detected and first-line protective mechanisms are initiated are not well understood. We have recently shown that tracheal brush cells (BCs) express functional taste receptors. Here we report that bitter taste signaling in murine BCs induces neurogenic inflammation. We demonstrate that BC signaling stimulates adjacent sensory nerve endings in the trachea to release the neuropeptides CGRP and substance P that mediate plasma extravasation, neutrophil recruitment, and diapedesis. Moreover, we show that bitter tasting quorum-sensing molecules from Pseudomonas aeruginosa activate tracheal BCs. BC signaling depends on the key taste transduction gene Trpm5, triggers secretion of immune mediators, among them the most abundant member of the complement system, and is needed to combat P. aeruginosa infections. Our data provide functional insight into first-line defense mechanisms against bacterial infections of the lung.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{10.7554/eLife.76804,

title = {The Interferon-inducible GTPase MxB Promotes Capsid Disassembly and Genome Release of Herpesviruses},

author = {M.C. Serrero and V. Girault and S. Weigang and T.M. Greco and A. Ramos-Nascimento and F. Anderson and A. Piras and A.H. Martinez and J. Hertzog and A. Binz and A. Pohlmann and U. Prank and J. Rehwinkel and R. Bauerfeind and I.M. Cristea and A. Pichlmair and G. Kochs and B. Sodeik},

editor = {Adam P Geballe and Päivi M Ojala},

url = {https://doi.org/10.7554/eLife.76804},

doi = {10.7554/eLife.76804},

issn = {2050-084X},

year  = {2022},

date = {2022-04-01},

journal = {eLife},

volume = {11},

pages = {e76804},

publisher = {eLife Sciences Publications, Ltd},

abstract = {Host proteins sense viral products and induce defence mechanisms, particularly in immune cells. Using cell-free assays and quantitative mass spectrometry, we determined the interactome of capsid-host protein complexes of herpes simplex virus and identified the large dynamin-like GTPase myxovirus resistance protein B (MxB) as an interferon-inducible protein interacting with capsids. Electron microscopy analyses showed that cytosols containing MxB had the remarkable capability to disassemble the icosahedral capsids of herpes simplex viruses and varicella zoster virus into flat sheets of connected triangular faces. In contrast, capsids remained intact in cytosols with MxB mutants unable to hydrolyse GTP or to dimerize. Our data suggest that MxB senses herpesviral capsids, mediates their disassembly, and thereby restricts the efficiency of nuclear targeting of incoming capsids and/or the assembly of progeny capsids. The resulting premature release of viral genomes from capsids may enhance the activation of DNA sensors, and thereby amplify the innate immune responses.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{10.1093/nar/gkab1248,

title = {Targeting Genomic SARS-CoV-2 RNA with siRNAs Allows Efficient Inhibition of Viral Replication and Spread},

author = {S. Ambike and C. Cheng and M. Feuerherd and S. Velkov and D. Baldassi and S.Q. Afridi and D. Porras-Gonzalez and X. Wei and P. Hagen and N. Kneidinger and M.G. Stoleriu and V. Grass and G. Burgstaller and A. Pichlmair and O.M. Merkel and C. Ko and T. Michler},

url = {https://doi.org/10.1093/nar/gkab1248},

doi = {10.1093/nar/gkab1248},

issn = {0305-1048},

year  = {2022},

date = {2022-01-11},

journal = {Nucleic Acids Research},

volume = {50},

number = {1},

pages = {333-349},

abstract = {A promising approach to tackle the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) could be small interfering (si)RNAs. So far it is unclear, which viral replication steps can be efficiently inhibited with siRNAs. Here, we report that siRNAs can target genomic RNA (gRNA) of SARS-CoV-2 after cell entry, and thereby terminate replication before start of transcription and prevent virus-induced cell death. Coronaviruses replicate via negative sense RNA intermediates using a unique discontinuous transcription process. As a result, each viral RNA contains identical sequences at the 5′ and 3′ end. Surprisingly, siRNAs were not active against intermediate negative sense transcripts. Targeting common sequences shared by all viral transcripts allowed simultaneous suppression of gRNA and subgenomic (sg)RNAs by a single siRNA. The most effective suppression of viral replication and spread, however, was achieved by siRNAs that targeted open reading frame 1 (ORF1) which only exists in gRNA. In contrast, siRNAs that targeted the common regions of transcripts were outcompeted by the highly abundant sgRNAs leading to an impaired antiviral efficacy. Verifying the translational relevance of these findings, we show that a chemically modified siRNA that targets a highly conserved region of ORF1, inhibited SARS-CoV-2 replication ex vivo in explants of the human lung. Our work encourages the development of siRNA-based therapies for COVID-19 and suggests that early therapy start, or prophylactic application, together with specifically targeting gRNA, might be key for high antiviral efficacy.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{EMSLANDER2022,

title = {Cell-free Production of Personalized Therapeutic Phages Targeting Multidrug-resistant Bacteria},

author = {Q. Emslander and K. Vogele and P. Braun and J. Stender and C. Willy and M. Joppich and J.A. Hammerl and M. Abele and C. Meng and A. Pichlmair and C. Ludwig and J.J. Bugert and F.C. Simmel and G.G. Westmeyer},

url = {https://www.sciencedirect.com/science/article/pii/S2451945622002355},

doi = {https://doi.org/10.1016/j.chembiol.2022.06.003},

issn = {2451-9456},

year  = {2022},

date = {2022-01-01},

journal = {Cell Chemical Biology},

abstract = {Bacteriophages are potent therapeutics against biohazardous bacteria, which rapidly develop multidrug resistance. However, routine administration of phage therapy is hampered by a lack of rapid production, safe bioengineering, and detailed characterization of phages. Thus, we demonstrate a comprehensive cell-free platform for personalized production, transient engineering, and proteomic characterization of a broad spectrum of phages. Using mass spectrometry, we validated hypothetical and non-structural proteins and could also monitor the protein expression during phage assembly. Notably, a few microliters of a one-pot reaction produced effective doses of phages against enteroaggregative Escherichia coli (EAEC), Yersinia pestis, and Klebsiella pneumoniae. By co-expressing suitable host factors, we could extend the range of cell-free production to phages targeting gram-positive bacteria. We further introduce a non-genomic phage engineering method, which adds functionalities for only one replication cycle. In summary, we expect this cell-free methodology to foster reverse and forward phage engineering and customized production of clinical-grade bacteriophages.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{https://doi.org/10.15252/embr.202154305,

title = {ADAM10 and ADAM17 Promote SARS-CoV-2 Cell Entry and Spike Protein-mediated Lung Cell Fusion},

author = {G. Jocher and V. Grass and S.K. Tschirner and L. Riepler and S. Breimann and T. Kaya and M. Oelsner and S.M. Hamad and L.I. Hofmann and C.P. Blobel and C.B. Schmidt-Weber and O. Gokce and C.A. Jakwerth and J. Trimpert and J. Kimpel and A. Pichlmair and S.F. Lichtenthaler},

url = {https://www.embopress.org/doi/abs/10.15252/embr.202154305},

doi = {https://doi.org/10.15252/embr.202154305},

year  = {2022},

date = {2022-01-01},

journal = {EMBO reports},

volume = {23},

number = {6},

pages = {e54305},

abstract = {Abstract The severe-acute-respiratory-syndrome-coronavirus-2 (SARS-CoV-2) is the causative agent of COVID-19, but host cell factors contributing to COVID-19 pathogenesis remain only partly understood. We identify the host metalloprotease ADAM17 as a facilitator of SARS-CoV-2 cell entry and the metalloprotease ADAM10 as a host factor required for lung cell syncytia formation, a hallmark of COVID-19 pathology. ADAM10 and ADAM17, which are broadly expressed in the human lung, cleave the SARS-CoV-2 spike protein (S) in vitro, indicating that ADAM10 and ADAM17 contribute to the priming of S, an essential step for viral entry and cell fusion. ADAM protease-targeted inhibitors severely impair lung cell infection by the SARS-CoV-2 variants of concern alpha, beta, delta, and omicron and also reduce SARS-CoV-2 infection of primary human lung cells in a TMPRSS2 protease-independent manner. Our study establishes ADAM10 and ADAM17 as host cell factors for viral entry and syncytia formation and defines both proteases as potential targets for antiviral drug development.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{WAGNER2022110214,

title = {Recruitment of Highly Cytotoxic CD8+ T Cell Receptors in Mild SARS-CoV-2 Infection},

author = {K.I. Wagner and L.M. Mateyka and S. Jarosch and V. Grass and S. Weber and K. Schober and M. Hammel and T. Burrell and B. Kalali and H. Poppert and H. Beyer and S. Schambeck and S. Holdenrieder and A. Strötges-Achatz and V. Haselmann and M. Neumaier and J. Erber and A. Priller and S. Yazici and H. Roggendorf and M. Odendahl and T. Tonn and A. Dick and K. Witter and H. Mijočević and U. Protzer and P.A. Knolle and A. Pichlmair and C.S. Crowell and M. Gerhard and E. D’Ippolito and D.H. Busch},

url = {https://www.sciencedirect.com/science/article/pii/S2211124721017186},

doi = {https://doi.org/10.1016/j.celrep.2021.110214},

issn = {2211-1247},

year  = {2022},

date = {2022-01-01},

journal = {Cell Reports},

volume = {38},

number = {2},

pages = {110214},

abstract = {T cell immunity is crucial for control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and has been studied widely on a quantitative level. However, the quality of responses, in particular of CD8+ T cells, has only been investigated marginally so far. Here, we isolate T cell receptor (TCR) repertoires specific for immunodominant SARS-CoV-2 epitopes restricted to common human Leukocyte antigen (HLA) class I molecules in convalescent individuals. SARS-CoV-2-specific CD8+ T cells are detected up to 12 months after infection. TCR repertoires are diverse, with heterogeneous functional avidity and cytotoxicity toward virus-infected cells, as demonstrated for TCR-engineered T cells. High TCR functionality correlates with gene signatures that, remarkably, could be retrieved for each epitope:HLA combination analyzed. Overall, our data demonstrate that polyclonal and highly functional CD8+ TCRs—classic features of protective immunity—are recruited upon mild SARS-CoV-2 infection, providing tools to assess the quality of and potentially restore functional CD8+ T cell immunity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Griffante2021,

title = {Human Cytomegalovirus-induced Host Protein Citrullination is Crucial for Viral Replication},

author = {G. Griffante and F. Gugliesi and S. Pasquero and V. DellÓste and M. Biolatti and A.J. Salinger and S. Mondal and P.R. Thompson and E. Weerapana and R.J. Lebbink and J.A. Soppe and T. Stamminger and V. Girault and A. Pichlmair and G. Oroszlán and D.M. Coen and M. De Andrea and S. Landolfo},

url = {http://www.nature.com/articles/s41467-021-24178-6},

doi = {10.1038/s41467-021-24178-6},

issn = {20411723},

year  = {2021},

date = {2021-12-01},

journal = {Nature Communications},

volume = {12},

number = {1},

pages = {3910},

abstract = {Citrullination is the conversion of arginine-to-citrulline by protein arginine deiminases (PADs), whose dysregulation is implicated in the pathogenesis of various types of cancers and autoimmune diseases. Consistent with the ability of human cytomegalovirus (HCMV) to induce post-translational modifications of cellular proteins to gain a survival advantage, we show that HCMV infection of primary human fibroblasts triggers PAD-mediated citrullination of several host proteins, and that this activity promotes viral fitness. Citrullinome analysis reveals significant changes in deimination levels of both cellular and viral proteins, with interferon (IFN)-inducible protein IFIT1 being among the most heavily deiminated one. As genetic depletion of IFIT1 strongly enhances HCMV growth, and in vitro IFIT1 citrullination impairs its ability to bind to 5'-ppp-RNA, we propose that viral-induced IFIT1 citrullination is a mechanism of HCMV evasion from host antiviral resistance. Overall, our findings point to a crucial role of citrullination in subverting cellular responses to viral infection.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Pennemann2021,

title = {Cross-species Analysis of Viral Nucleic Acid Interacting Proteins Identifies TAOKs as Innate Immune Regulators},

author = {F.L. Pennemann and A. Mussabekova and C. Urban and A. Stukalov and L.L. Andersen and V. Grass and T.M. Lavacca and C. Holze and L. Oubraham and Y. Benamrouche and E. Girardi and R.E. Boulos and R. Hartmann and G. Superti-Furga and M. Habjan and J. Imler and C. Meignin and A. Pichlmair},

url = {https://doi.org/10.1038/s41467-021-27192-w},

doi = {10.1038/s41467-021-27192-w},

issn = {2041-1723},

year  = {2021},

date = {2021-12-01},

journal = {Nature Communications},

volume = {12},

number = {1},

pages = {7009},

abstract = {The cell intrinsic antiviral response of multicellular organisms developed over millions of years and critically relies on the ability to sense and eliminate viral nucleic acids. Here we use an affinity proteomics approach in evolutionary distant species (human, mouse and fly) to identify proteins that are conserved in their ability to associate with diverse viral nucleic acids. This approach shows a core of orthologous proteins targeting viral genetic material and species-specific interactions. Functional characterization of the influence of 181 candidates on replication of 6 distinct viruses in human cells and flies identifies 128 nucleic acid binding proteins with an impact on virus growth. We identify the family of TAO kinases (TAOK1, −2 and −3) as dsRNA-interacting antiviral proteins and show their requirement for type-I interferon induction. Depletion of TAO kinases in mammals or flies leads to an impaired response to virus infection characterized by a reduced induction of interferon stimulated genes in mammals and impaired expression of srg1 and diedel in flies. Overall, our study shows a larger set of proteins able to mediate the interaction between viral genetic material and host factors than anticipated so far, attesting to the ancestral roots of innate immunity and to the lineage-specific pressures exerted by viruses.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Laudenbach2021,

title = {NUDT2 Initiates Viral RNA Degradation by Removal of 5'-phosphates},

author = {B.T. Laudenbach and K. Krey and Q. Emslander and L.L. Andersen and A. Reim and P. Scaturro and S. Mundigl and C. Dächert and K. Manske and M. Moser and J. Ludwig and D. Wohlleber and A. Kröger and M. Binder and A. Pichlmair},

url = {https://doi.org/10.1038/s41467-021-27239-y},

doi = {10.1038/s41467-021-27239-y},

issn = {2041-1723},

year  = {2021},

date = {2021-11-25},

journal = {Nature Communications},

volume = {12},

number = {1},

pages = {6918},

abstract = {While viral replication processes are largely understood, comparably little is known on cellular mechanisms degrading viral RNA. Some viral RNAs bear a 5'-triphosphate (PPP-) group that impairs degradation by the canonical 5textasciiacutex-3textasciiacutex degradation pathway. Here we show that the Nudix hydrolase 2 (NUDT2) trims viral PPP-RNA into monophosphorylated (P)-RNA, which serves as a substrate for the 5'-3' exonuclease XRN1. NUDT2 removes 5'-phosphates from PPP-RNA in an RNA sequence- and overhang-independent manner and its ablation in cells increases growth of PPP-RNA viruses, suggesting an involvement in antiviral immunity. NUDT2 is highly homologous to bacterial RNA pyrophosphatase H (RppH), a protein involved in the metabolism of bacterial mRNA, which is 5'-tri- or diphosphorylated. Our results show a conserved function between bacterial RppH and mammalian NUDT2, indicating that the function may have adapted from a protein responsible for RNA turnover in bacteria into a protein involved in the immune defence in mammals.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Fischer2021b,

title = {Single-cell RNA Sequencing Reveals ex vivo Signatures of SARS-CoV-2-reactive T Cells Through ‘Reverse Phenotyping'},

author = {D.S. Fischer and M. Ansari and K.I. Wagner and S. Jarosch and Y. Huang and C.H. Mayr and M. Strunz and N.J. Lang and E. D'Ippolito and M. Hammel and L. Mateyka and S. Weber and L.S. Wolff and K. Witter and I.E. Fernandez and G. Leuschner and K. Milger and M. Frankenberger and L. Nowak and K. Heinig-Menhard and I. Koch and M.G. Stoleriu and A. Hilgendorff and J. Behr and A. Pichlmair and B. Schubert and F.J. Theis and D.H. Busch and H.B. Schiller and K. Schober},

url = {http://www.nature.com/articles/s41467-021-24730-4},

doi = {10.1038/s41467-021-24730-4},

issn = {2041-1723},

year  = {2021},

date = {2021-07-26},

journal = {Nature Communications},

volume = {12},

number = {1},

pages = {4515},

abstract = {The in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping'. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Holleufer2021,

title = {Two cGAS-like Receptors Induce Antiviral Immunity in Drosophila},

author = {A. Holleufer and K.G. Winther and H.H. Gad and X. Ai and Y. Chen and L. Li and Z. Wei and H. Deng and J. Liu and N.A. Frederiksen and B. Simonsen and L.L. Andersen and K. Kleigrewe and L. Dalskov and A. Pichlmair and H. Cai and J. Imler and R. Hartmann},

url = {https://doi.org/10.1038/s41586-021-03800-z},

doi = {10.1038/s41586-021-03800-z},

issn = {1476-4687},

year  = {2021},

date = {2021-07-14},

journal = {Nature},

abstract = {In mammals, cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide (CDN) 2'3'-cGAMP in response to cytosolic DNA and this triggers an antiviral immune response. cGAS belongs to a large family of cGAS/DncV-like nucleotidyltransferases, present in both prokaryotes1 and eukaryotes2--5. In bacteria, these enzymes synthesize a range of cyclic oligonucleotide and have recently emerged as important regulators of phage infections6--8. Here, we identify two novel cGAS-like receptors (cGLRs) in the insect Drosophila melanogaster. We show that cGLR1 and cGLR2 activate Sting and NF-$kappa$B dependent antiviral immunity in response to infection with RNA or DNA viruses. cGLR1 is activated by dsRNA to produce the novel CDN 3'2'-cGAMP whereas cGLR2 produces a combination of 2'3'-cGAMP and 3'2' cGAMP in response to a yet unidentified stimulus. Our data establish cGAS as the founding member of a family of receptors sensing different types of nucleic acids and triggering immunity through production of CDNs beyond 2'3'-cGAMP.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Krivega2021,

title = {Genotoxic Stress in Constitutive Trisomies Induces Autophagy and the Innate Immune Response via the cGAS-STING Pathway},

author = {M. Krivega and C.M. Stiefel and S. Karbassi and L.L. Andersen and N.K. Chunduri and N. Donnelly and A. Pichlmair and Z. Storchová},

url = {https://doi.org/10.1038/s42003-021-02278-9},

doi = {10.1038/s42003-021-02278-9},

issn = {2399-3642},

year  = {2021},

date = {2021-07-02},

journal = {Communications Biology},

volume = {4},

number = {1},

pages = {831},

abstract = {Gain of even a single chromosome leads to changes in human cell physiology and uniform perturbations of specific cellular processes, including downregulation of DNA replication pathway, upregulation of autophagy and lysosomal degradation, and constitutive activation of the type I interferon response. Little is known about the molecular mechanisms underlying these changes. We show that the constitutive nuclear localization of TFEB, a transcription factor that activates the expression of autophagy and lysosomal genes, is characteristic of human trisomic cells. Constitutive nuclear localization of TFEB in trisomic cells is independent of mTORC1 signaling, but depends on the cGAS-STING activation. Trisomic cells accumulate cytoplasmic dsDNA, which activates the cGAS-STING signaling cascade, thereby triggering nuclear accumulation of the transcription factor IRF3 and, consequently, upregulation of interferon-stimulated genes. cGAS depletion interferes with TFEB-dependent upregulation of autophagy in model trisomic cells. Importantly, activation of both the innate immune response and autophagy occurs also in primary trisomic embryonic fibroblasts, independent of the identity of the additional chromosome. Our research identifies the cGAS-STING pathway as an upstream regulator responsible for activation of autophagy and inflammatory response in human cells with extra chromosomes, such as in Down syndrome or other aneuploidy-associated pathologies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Stukalov2021,

title = {Multilevel Proteomics Reveals Host Perturbations by SARS-CoV-2 and SARS-CoV},

author = {A. Stukalov and V. Girault and V. Grass and O. Karayel and V. Bergant and C. Urban and D.A. Haas and Y. Huang and L. Oubraham and A. Wang and M.S. Hamad and A. Piras and F.M. Hansen and M.C. Tanzer and I. Paron and L. Zinzula and T. Enghleitner and M. Reinecke and T.M. Lavacca and R. Ehmann and R. Wölfel and J. Jores and B. Kuster and U. Protzer and R. Rad and J. Ziebuhr and V. Thiel and P. Scaturro and M. Mann and A. Pichlmair},

doi = {10.1038/s41586-021-03493-4},

issn = {0028-0836},

year  = {2021},

date = {2021-04-12},

journal = {Nature},

abstract = {The global emergence of SARS-CoV-2 urgently requires an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. Several individual omics studies have extended our knowledge of COVID-19 pathophysiology1–10. Integration of such datasets to obtain a holistic view of virus-host interactions and to define the pathogenic properties of SARS-CoV-2 is limited by the heterogeneity of the experimental systems. We therefore conducted a concurrent multi-omics study of SARS-CoV-2 and SARS-CoV. Using state-of-the-art proteomics, we profiled the interactome of both viruses, as well as their influence on transcriptome, proteome, ubiquitinome and phosphoproteome in a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed crosstalk between the perturbations taking place upon SARS-CoV-2 and SARS-CoV infections at different layers and identified unique and common molecular mechanisms of these closely related coronaviruses. The TGF-β pathway, known for its involvement in tissue fibrosis, was specifically dysregulated by SARS-CoV-2 ORF8 and autophagy by SARS-CoV-2 ORF3. The extensive dataset (available at https://covinet.innatelab.org) highlights many hotspots that can be targeted by existing drugs and it can guide rational design of virus- and host-directed therapies, which we exemplify by identifying kinase and MMPs inhibitors with potent antiviral effects against SARS-CoV-2.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{PEI2021109803b,

title = {Nuclear-localized Human Respiratory Syncytial Virus NS1 Protein Modulates Host Gene Transcription},

author = {J. Pei and N.R. Beri and A.J. Zou and P. Hubel and H.K. Dorando and V. Bergant and R.D. Andrews and J. Pan and J.M. Andrews and K.C.F. Sheehan and A. Pichlmair and G.K. Amarasinghe and S.L. Brody and J.E. Payton and D.W. Leung},

url = {https://www.sciencedirect.com/science/article/pii/S2211124721012638},

doi = {https://doi.org/10.1016/j.celrep.2021.109803},

issn = {2211-1247},

year  = {2021},

date = {2021-01-01},

journal = {Cell Reports},

volume = {37},

number = {2},

pages = {109803},

abstract = {Human respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in the pediatric, elderly, and immunocompromised individuals. RSV non-structural protein NS1 is a known cytosolic immune antagonist, but how NS1 modulates host responses remains poorly defined. Here, we observe NS1 partitioning into the nucleus of RSV-infected cells, including the human airway epithelium. Nuclear NS1 coimmunoprecipitates with Mediator complex and is chromatin associated. Chromatin-immunoprecipitation demonstrates enrichment of NS1 that overlaps Mediator and transcription factor binding within the promoters and enhancers of differentially expressed genes during RSV infection. Mutation of the NS1 C-terminal helix reduces NS1 impact on host gene expression. These data suggest that nuclear NS1 alters host responses to RSV infection by binding at regulatory elements of immune response genes and modulating host gene transcription. Our study identifies another layer of regulation by virally encoded proteins that shapes host response and impacts immunity to RSV.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{https://doi.org/10.1096/fj.202100560R,

title = {Disruption of Disulfides Within RBD of SARS-CoV-2 Spike Protein Prevents Fusion and Represents a Target for Viral Entry Inhibition by Registered Drugs},

author = {M. Manček-Keber and I. Hafner-Bratkovič and D. Lainšček and M. Benčina and T. Govednik and S. Orehek and T. Plaper and V. Jazbec and V. Bergant and V. Grass and A. Pichlmair and R. Jerala},

url = {https://faseb.onlinelibrary.wiley.com/doi/abs/10.1096/fj.202100560R},

doi = {https://doi.org/10.1096/fj.202100560R},

year  = {2021},

date = {2021-01-01},

journal = {The FASEB Journal},

volume = {35},

number = {6},

pages = {e21651},

abstract = {Abstract The SARS-CoV-2 pandemic imposed a large burden on health and society. Therapeutics targeting different components and processes of the viral infection replication cycle are being investigated, particularly to repurpose already approved drugs. Spike protein is an important target for both vaccines and therapeutics. Insights into the mechanisms of spike-ACE2 binding and cell fusion could support the identification of compounds with inhibitory effects. Here, we demonstrate that the integrity of disulfide bonds within the receptor-binding domain (RBD) plays an important role in the membrane fusion process although their disruption does not prevent binding of spike protein to ACE2. Several reducing agents and thiol-reactive compounds are able to inhibit viral entry. N-acetyl cysteine amide, L-ascorbic acid, JTT-705, and auranofin prevented syncytia formation, viral entry into cells, and infection in a mouse model, supporting disulfides of the RBD as a therapeutically relevant target.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{https://doi.org/10.15252/embr.201949568,

title = {Interferon-induced Degradation of the Persistent Hepatitis B Virus cccDNA Form Depends on ISG20},

author = {D. Stadler and M. Kächele and A.N. Jones and J. Hess and C. Urban and J. Schneider and Y. Xia and A. Oswald and F. Nebioglu and R. Bester and F. Lasitschka and M. Ringelhan and C. Ko and W. Chou and A. Geerlof and M.A. van de Klundert and J.M. Wettengel and P. Schirmacher and M. Heikenwälder and S. Schreiner and R. Bartenschlager and A. Pichlmair and M. Sattler and K. Unger and U. Protzer},

url = {https://www.embopress.org/doi/abs/10.15252/embr.201949568},

doi = {https://doi.org/10.15252/embr.201949568},

year  = {2021},

date = {2021-01-01},

journal = {EMBO reports},

volume = {22},

number = {6},

pages = {e49568},

abstract = {Abstract Hepatitis B virus (HBV) persists by depositing a covalently closed circular DNA (cccDNA) in the nucleus of infected cells that cannot be targeted by available antivirals. Interferons can diminish HBV cccDNA via APOBEC3-mediated deamination. Here, we show that overexpression of APOBEC3A alone is not sufficient to reduce HBV cccDNA that requires additional treatment of cells with interferon indicating involvement of an interferon-stimulated gene (ISG) in cccDNA degradation. Transcriptome analyses identify ISG20 as the only type I and II interferon-induced, nuclear protein with annotated nuclease activity. ISG20 localizes to nucleoli of interferon-stimulated hepatocytes and is enriched on deoxyuridine-containing single-stranded DNA that mimics transcriptionally active, APOBEC3A-deaminated HBV DNA. ISG20 expression is detected in human livers in acute, self-limiting but not in chronic hepatitis B. ISG20 depletion mitigates the interferon-induced loss of cccDNA, and co-expression with APOBEC3A is sufficient to diminish cccDNA. In conclusion, non-cytolytic HBV cccDNA decline requires the concerted action of a deaminase and a nuclease. Our findings highlight that ISGs may cooperate in their antiviral activity that may be explored for therapeutic targeting.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{doi:10.1128/mBio.02683-20,

title = {The Zinc Finger Antiviral Protein ZAP Restricts Human Cytomegalovirus and Selectively Binds and Destabilizes Viral \textit{UL4}/\textit{UL5} Transcripts},

author = {A.C. Gonzalez-Perez and M. Stempel and E. Wyler and C. Urban and A. Piras and T. Hennig and S. Ganskih and Y. Wei and A. Heim and M. Landthaler and A. Pichlmair and L. Dölken and M. Munschauer and F. Erhard and M.M. Brinkmann and S.M. Horner},

doi = {10.1128/mBio.02683-20  URL = https://journals.asm.org/doi/abs/10.1128/mBio.02683-20},

year  = {2021},

date = {2021-01-01},

journal = {mBio},

volume = {12},

number = {3},

pages = {e02683-20},

abstract = {Viral infections have a large impact on society, leading to major human and economic losses and even global instability. So far, many viral infections, including human cytomegalovirus (HCMV) infection, are treated with a small repertoire of drugs, often accompanied by the occurrence of resistant mutants. Interferon-stimulated gene products (ISGs) play a crucial role in early infection control. The ISG zinc finger CCCH-type antiviral protein 1 (ZAP/ZC3HAV1) antagonizes several RNA viruses by binding to CG-rich RNA sequences, whereas its effect on DNA viruses is less well understood. Here, we decipher the role of ZAP in the context of human cytomegalovirus (HCMV) infection, a β-herpesvirus that is associated with high morbidity in immunosuppressed individuals and newborns. We show that expression of the two major isoforms of ZAP, ZAP-S and ZAP-L, is induced during HCMV infection and that both negatively affect HCMV replication. Transcriptome and proteome analyses demonstrated that the expression of ZAP results in reduced viral mRNA and protein levels and decelerates the progression of HCMV infection. Metabolic RNA labeling combined with high-throughput sequencing (SLAM-seq) revealed that most of the gene expression changes late in infection result from the general attenuation of HCMV. Furthermore, at early stages of infection, ZAP restricts HCMV by destabilizing a distinct subset of viral mRNAs, particularly those from the previously uncharacterized UL4-UL6 HCMV gene locus. Through enhanced cross-linking immunoprecipitation and sequencing analysis (eCLIP-seq), we identified the transcripts expressed from this HCMV locus as the direct targets of ZAP. Moreover, our data show that ZAP preferentially recognizes not only CG, but also other cytosine-rich sequences, thereby expanding its target specificity. In summary, this report is the first to reveal direct targets of ZAP during HCMV infection, which strongly indicates that transcripts from the UL4-UL6 locus may play an important role for HCMV replication. IMPORTANCE Viral infections have a large impact on society, leading to major human and economic losses and even global instability. So far, many viral infections, including human cytomegalovirus (HCMV) infection, are treated with a small repertoire of drugs, often accompanied by the occurrence of resistant mutants. There is no licensed HCMV vaccine in sight to protect those most at risk, particularly immunocompromised individuals or pregnant women who might otherwise transmit the virus to the fetus. Thus, the identification of novel intervention strategies is urgently required. In this study, we show that ZAP decelerates the viral gene expression cascade, presumably by selectively handpicking a distinct set of viral transcripts for degradation. Our study illustrates the potent role of ZAP as an HCMV restriction factor and sheds light on a possible role for UL4 and/or UL5 early during infection, paving a new avenue for the exploration of potential targets for novel therapies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{vaccines9050431,

title = {A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response},

author = {D. Lainšček and T. Fink and V. Forstnerič and I. Hafner-Bratkovič and S. Orehek and Ž. Strmšek and M. Manček-Keber and P. Pečan and H. Esih and Š. Malenšek and J. Aupič and P. Dekleva and T. Plaper and S. Vidmar and L. Kadunc and M. Benčina and N. Omersa and G. Anderluh and F. Pojer and K. Lau and D. Hacker and B.E. Correia and D. Peterhoff and R. Wagner and V. Bergant and A. Herrmann and A. Pichlmair and R. Jerala},

url = {https://www.mdpi.com/2076-393X/9/5/431},

doi = {10.3390/vaccines9050431},

issn = {2076-393X},

year  = {2021},

date = {2021-01-01},

journal = {Vaccines},

volume = {9},

number = {5},

abstract = {The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and β-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small β-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The β-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the β-annulus peptide for vaccine design.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{molecules26020287,

title = {The UFM1 Pathway Impacts HCMV US2-Mediated Degradation of HLA Class I},

author = {A.B.C. Schuren and I.G.J. Boer and E.M. Bouma and M.L. Van de Weijer and A.I. Costa and P. Hubel and A. Pichlmair and R.J. Lebbink and E.J.H.J. Wiertz},

url = {https://www.mdpi.com/1420-3049/26/2/287},

doi = {10.3390/molecules26020287},

issn = {1420-3049},

year  = {2021},

date = {2021-01-01},

journal = {Molecules},

volume = {26},

number = {2},

abstract = {To prevent accumulation of misfolded proteins in the endoplasmic reticulum, chaperones perform quality control on newly translated proteins and redirect misfolded proteins to the cytosol for degradation by the ubiquitin-proteasome system. This pathway is called ER-associated protein degradation (ERAD). The human cytomegalovirus protein US2 induces accelerated ERAD of HLA class I molecules to prevent immune recognition of infected cells by CD8+ T cells. Using US2-mediated HLA-I degradation as a model for ERAD, we performed a genome-wide CRISPR/Cas9 library screen to identify novel cellular factors associated with ERAD. Besides the identification of known players such as TRC8, p97, and UBE2G2, the ubiquitin-fold modifier1 (UFM1) pathway was found to affect degradation of HLA-I. UFMylation is a post-translational modification resembling ubiquitination. Whereas we observe ubiquitination of HLA-I, no UFMylation was detected on HLA-I or several other proteins involved in degradation of HLA-I, suggesting that the UFM1 pathway impacts ERAD in a different manner than ubiquitin. Interference with the UFM1 pathway seems to specifically inhibit the ER-to-cytosol dislocation of HLA-I. In the absence of detectable UFMylation of HLA-I, UFM1 may contribute to US2-mediated HLA-I degradation by misdirecting protein sorting indirectly. Mass spectrometry analysis of US2-expressing cells showed that ribosomal proteins are a major class of proteins undergoing extensive UFMylation; the role of these changes in protein degradation may be indirect and remains to be established.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{KreyPichlmair2020,

title = {System-based Approaches to Delineate the Antiviral Innate Immune Landscape},

author = {K. Krey and A. Babnis and A. Pichlmair},

url = {https://pubmed.ncbi.nlm.nih.gov/33096788/

https://innatelab.virologie.med.tum.de/archives/1071},

doi = {10.3390/v12101196},

issn = {19994915},

year  = {2020},

date = {2020-10-01},

booktitle = {Viruses},

journal = {Viruses},

volume = {12},

number = {10},

publisher = {MDPI AG},

abstract = {Viruses pose substantial challenges for society, economy, healthcare systems, and research. Their distinctive pathologies are based on specific interactions with cellular factors. In order to develop new antiviral treatments, it is of central importance to understand how viruses interact with their host and how infected cells react to the virus on a molecular level. Invading viruses are commonly sensed by components of the innate immune system, which is composed of a highly effective yet complex network of proteins that, in most cases, mediate efficient virus inhibition. Central to this process is the activity of interferons and other cytokines that coordinate the antiviral response. So far, numerous methods have been used to identify how viruses interact with cellular processes and revealed that the innate immune response is highly complex and involves interferon-stimulated genes and their binding partners as functional factors. Novel approaches and careful experimental design, combined with large-scale, high-throughput methods and cutting-edge analysis pipelines, have to be utilized to delineate the antiviral innate immune landscape at a global level. In this review, we describe different currently used screening approaches, how they contributed to our knowledge on virus–host interactions, and essential considerations that have to be taken into account when planning such experiments.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{UrbanPichlmair2020_V,

title = {Persistent Innate Immune Stimulation Results in IRF3-Mediated but Caspase-Independent Cytostasis},

author = { C. Urban and H. Welsch and K. Heine and S. Wüst and D.A. Haas and C. Dächert and A. Pandey and A. Pichlmair and M. Binder},

url = {https://www.mdpi.com/1999-4915/12/6/635

https://innatelab.virologie.med.tum.de/archives/1005},

doi = {10.3390/v12060635},

issn = {1999-4915},

year  = {2020},

date = {2020-06-11},

journal = {Viruses},

volume = {12},

number = {6},

abstract = {Persistent virus infection continuously produces non-self nucleic acids that activate cell-intrinsic immune responses. However, the antiviral defense evolved as a transient, acute phase response and the effects of persistently ongoing stimulation onto cellular homeostasis are not well understood. To study the consequences of long-term innate immune activation, we expressed the NS5B polymerase of Hepatitis C virus (HCV), which in absence of viral genomes continuously produces immune-stimulatory RNAs. Surprisingly, within 3 weeks, NS5B expression declined and the innate immune response ceased. Proteomics and functional analyses indicated a reduced proliferation of those cells most strongly stimulated, which was independent of interferon signaling but required mitochondrial antiviral signaling protein (MAVS) and interferon regulatory factor 3 (IRF3). Depletion of MAVS or IRF3, or overexpression of the MAVS-inactivating HCV NS3/4A protease not only blocked interferon responses but also restored cell growth in NS5B expressing cells. However, pan-caspase inhibition could not rescue the NS5B-induced cytostasis. Our results underline an active counter selection of cells with prolonged innate immune activation, which likely constitutes a cellular strategy to prevent persistent virus infections.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{FabitsBrinkmann2020_V,

title = {The Cytomegalovirus Tegument Protein UL35 Antagonizes Pattern Recognition Receptor-Mediated Type I IFN Transcription},

author = { M. Fabits and V.G. Magalhães and B. Chan and V. Girault and E. Elbasani and E. Rossetti and E. Saeland and M. Messerle and A. Pichlmair and V.J. Lisnić and M.M. Brinkmann},

url = {https://www.mdpi.com/2076-2607/8/6/790

https://innatelab.virologie.med.tum.de/archives/1010},

doi = {10.3390/microorganisms8060790},

issn = {2076-2607},

year  = {2020},

date = {2020-05-26},

journal = {Microorganisms},

volume = {8},

number = {6},

abstract = {The rapid activation of pattern recognition receptor (PRR)-mediated type I interferon (IFN) signaling is crucial for the host response to infection. In turn, human cytomegalovirus (HCMV) must evade this potent response to establish life-long infection. Here, we reveal that the HCMV tegument protein UL35 antagonizes the activation of type I IFN transcription downstream of the DNA and RNA sensors cGAS and RIG-I, respectively. We show that ectopic expression of UL35 diminishes the type I IFN response, while infection with a recombinant HCMV lacking UL35 induces an elevated type I IFN response compared to wildtype HCMV. With a series of luciferase reporter assays and the analysis of signaling kinetics upon HCMV infection, we observed that UL35 downmodulates PRR signaling at the level of the key signaling factor TANK-binding kinase 1 (TBK1). Finally, we demonstrate that UL35 and TBK1 co-immunoprecipitate when co-expressed in HEK293T cells. In addition, we show that a previously reported cellular binding partner of UL35, O-GlcNAc transferase (OGT), post-translationally GlcNAcylates UL35, but that this modification is not required for the antagonizing effect of UL35 on PRR signaling. In summary, we have identified UL35 as the first HCMV protein to antagonize the type I IFN response at the level of TBK1, thereby enriching our understanding of how this important herpesvirus escapes host immune responses.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Zecha1503,

title = {Data, Reagents, Assays and Merits of Proteomics for SARS-CoV-2 Research and Testing},

author = {J. Zecha, C.Y. Lee, F.P. Bayer, C. Meng, V. Grass, J. Zerweck, K. Schnatbaum, T. Michler, A. Pichlmair, C. Ludwig and B. Kuster},

url = {https://www.mcponline.org/content/19/9/1503},

doi = {10.1074/mcp.RA120.002164},

issn = {1535-9476},

year  = {2020},

date = {2020-01-01},

journal = {Molecular & Cellular Proteomics},

volume = {19},

number = {9},

pages = {1503--1522},

publisher = {American Society for Biochemistry and Molecular Biology},

abstract = {As the COVID-19 pandemic continues to spread, thousands of scientists around the globe have changed research direction to understand better how the virus works and to find out how it may be tackled. The number of manuscripts on preprint servers is soaring and peer-reviewed publications using MS-based proteomics are beginning to emerge. To facilitate proteomic research on SARS-CoV-2, the virus that causes COVID-19, this report presents deep-scale proteomes (10,000 proteins; >130,000 peptides) of common cell line models, notably Vero E6, Calu-3, Caco-2, and ACE2-A549 that characterize their protein expression profiles including viral entry factors such as ACE2 or TMPRSS2. Using the 9 kDa protein SRP9 and the breast cancer oncogene BRCA1 as examples, we show how the proteome expression data can be used to refine the annotation of protein-coding regions of the African green monkey and the Vero cell line genomes. Monitoring changes of the proteome on viral infection revealed widespread expression changes including transcriptional regulators, protease inhibitors, and proteins involved in innate immunity. Based on a library of 98 stable-isotope labeled synthetic peptides representing 11 SARS-CoV-2 proteins, we developed PRM (parallel reaction monitoring) assays for nano-flow and micro-flow LC–MS/MS. We assessed the merits of these PRM assays using supernatants of virus-infected Vero E6 cells and challenged the assays by analyzing two diagnostic cohorts of 24 (+30) SARS-CoV-2 positive and 28 (+9) negative cases. In light of the results obtained and including recent publications or manuscripts on preprint servers, we critically discuss the merits of MS-based proteomics for SARS-CoV-2 research and testing.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{doi:10.1002/cbic.201900472,

title = {Chemoenzymatic Total Synthesis of Sorbicatechol Structural Analogues and Evaluation of Their Antiviral Potential},

author = {A. Sib and T.M. Milzarek and A. Herrmann and L. Oubraham and J.I. Müller and A. Pichlmair and R. Brack-Werner and T.A.M. Gulder},

url = {https://chemistry-europe.onlinelibrary.wiley.com/doi/abs/10.1002/cbic.201900472},

doi = {10.1002/cbic.201900472},

year  = {2020},

date = {2020-01-01},

journal = {ChemBioChem},

volume = {21},

number = {4},

pages = {492-495},

abstract = {Abstract Sorbicillinoids are fungal polyketides characterized by highly complex and diverse molecular structures, with considerable stereochemical intricacy combined with a high degree of oxygenation. Many sorbicillinoids possess promising biological activities. An interesting member of this natural product family is sorbicatechol A, which is reported to have antiviral activity, particularly against influenza A virus (H1N1). Through a straightforward, one-pot chemoenzymatic approach with recently developed oxidoreductase SorbC, the characteristic bicyclo[2.2.2]octane core of sorbicatechol is structurally diversified by variation of its natural 2-methoxyphenol substituent. This facilitates the preparation of a focused library of structural analogues bearing substituted aromatic systems, alkanes, heterocycles, and ethers. Fast access to this structural diversity provides an opportunity to explore the antiviral potential of the sorbicatechol family.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Bauernfriedeabd0811,

title = {Human NLRP1 is a Sensor for Double-stranded RNA},

author = {S. Bauernfried and M.J. Scherr and A. Pichlmair and K.E. Duderstadt and V. Hornung},

url = {https://science.sciencemag.org/content/early/2020/11/24/science.abd0811},

doi = {10.1126/science.abd0811},

issn = {0036-8075},

year  = {2020},

date = {2020-01-01},

journal = {Science},

publisher = {American Association for the Advancement of Science},

abstract = {Inflammasomes function as intracellular sensors of pathogen infection or cellular perturbation and thereby play a central role in numerous diseases. Given the high abundance of NLRP1 in epithelial barrier tissues, we screened a diverse panel of viruses for inflammasome activation in keratinocytes. We identified Semliki Forest virus (SFV), a positive-strand RNA virus, as a potent activator of human, but not murine NLRP1. SFV replication and the associated formation of double-stranded (ds) RNA was required to engage the NLRP1 inflammasome. Moreover, delivery of long dsRNA was sufficient to trigger activation. Biochemical studies revealed that NLRP1 binds dsRNA via its LRR, resulting in its NACHT domain gaining ATPase activity. Altogether, these results establish human NLRP1 as a direct sensor for dsRNA and thus RNA virus infection.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Wuerthe00976-20,

title = {eIF2B as a Target for Viral Evasion of PKR-Mediated Translation Inhibition},

author = {J.D. Wuerth, M. Habjan, M. Kainulainen, B. Berisha, D. Bertheloot, G. Superti-Furga, A. Pichlmair and F. Weber},

editor = {Tetsuro Ikegami and Thomas E Morrison},

url = {https://mbio.asm.org/content/11/4/e00976-20},

doi = {10.1128/mBio.00976-20},

year  = {2020},

date = {2020-01-01},

journal = {mBio},

volume = {11},

number = {4},

publisher = {American Society for Microbiology},

abstract = {RNA-activated protein kinase (PKR) is a major innate immune factor that senses viral double-stranded RNA (dsRNA) and phosphorylates eukaryotic initiation factor (eIF) 2α. Phosphorylation of the α subunit converts the eIF2αβγ complex into a stoichiometric inhibitor of eukaryotic initiation factor eIF2B, thus halting mRNA translation. To escape this protein synthesis shutoff, viruses have evolved countermechanisms such as dsRNA sequestration, eIF-independent translation by an internal ribosome binding site, degradation of PKR, or dephosphorylation of PKR or of phospho-eIF2α. Here, we report that sandfly fever Sicilian phlebovirus (SFSV) confers such a resistance without interfering with PKR activation or eIF2α phosphorylation. Rather, SFSV expresses a nonstructural protein termed NSs that strongly binds to eIF2B. Although NSs still allows phospho-eIF2α binding to eIF2B, protein synthesis and virus replication are unhindered. Hence, SFSV encodes a unique PKR antagonist that acts by rendering eIF2B resistant to the inhibitory action of bound phospho-eIF2α.IMPORTANCE RNA-activated protein kinase (PKR) is one of the most powerful antiviral defense factors of the mammalian host. PKR acts by phosphorylating mRNA translation initiation factor eIF2α, thereby converting it from a cofactor to an inhibitor of mRNA translation that strongly binds to initiation factor eIF2B. To sustain synthesis of their proteins, viruses are known to counteract this on the level of PKR or eIF2α or by circumventing initiation factor-dependent translation altogether. Here, we report a different PKR escape strategy executed by sandfly fever Sicilian virus (SFSV), a member of the increasingly important group of phleboviruses. We found that the nonstructural protein NSs of SFSV binds to eIF2B and protects it from inactivation by PKR-generated phospho-eIF2α. Protein synthesis is hence maintained and the virus can replicate despite ongoing full-fledged PKR signaling in the infected cells. Thus, SFSV has evolved a unique strategy to escape the powerful antiviral PKR.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{LAMPL20201347,

title = {Reduced Mitochondrial Resilience Enables Non-canonical Induction of Apoptosis after TNF Receptor Signaling in Virus-infected Hepatocytes},

author = {S. Lampl and M.K. Janas and S. Donakonda and M. Brugger and K. Lohr and A. Schneider and K. Manske and L.E. Sperl and S. Kläger and B. Küster and J. Wettmarshausen and C. Müller and M. Laschinger and D. Hartmann and N. Hüser and F. Perocchi and P. Schmitt-Kopplin and F. Hagn and L. Zender and V. Hornung and C. Borner and A. Pichlmair and H. Kashkar and M. Klingenspor and M. Prinz and S. Schreiner and M. Conrad and P.J. Jost and H. Zischka and K. Steiger and M. Krönke and D. Zehn and U. Protzer and M. Heikenwälder and P.A. Knolle and D. Wohlleber},

url = {http://www.sciencedirect.com/science/article/pii/S0168827820303986},

doi = {https://doi.org/10.1016/j.jhep.2020.06.026},

issn = {0168-8278},

year  = {2020},

date = {2020-01-01},

journal = {Journal of Hepatology},

volume = {73},

number = {6},

pages = {1347 - 1359},

abstract = {Background & Aims 

Selective elimination of virus-infected hepatocytes occurs through virus-specific CD8 T cells recognizing peptide-loaded MHC molecules. Herein, we report that virus-infected hepatocytes are also selectively eliminated through a cell-autonomous mechanism. 

Methods 

We generated recombinant adenoviruses and genetically modified mouse models to identify the molecular mechanisms determining TNF-induced hepatocyte apoptosis in vivo and used in vivo bioluminescence imaging, immunohistochemistry, immunoblot analysis, RNAseq/proteome/phosphoproteome analyses, bioinformatic analyses, mitochondrial function tests. 

Results 

We found that TNF precisely eliminated only virus-infected hepatocytes independently of local inflammation and activation of immune sensory receptors. TNF receptor I was equally relevant for NF-kB activation in healthy and infected hepatocytes, but selectively mediated apoptosis in infected hepatocytes. Caspase 8 activation downstream of TNF receptor signaling was dispensable for apoptosis in virus-infected hepatocytes, indicating an unknown non-canonical cell-intrinsic pathway promoting apoptosis in hepatocytes. We identified a unique state of mitochondrial vulnerability in virus-infected hepatocytes as the cause for this non-canonical induction of apoptosis through TNF. Mitochondria from virus-infected hepatocytes showed normal biophysical and bioenergetic functions but were characterized by reduced resilience to calcium challenge. In the presence of unchanged TNF-induced signaling, reactive oxygen species-mediated calcium release from the endoplasmic reticulum caused mitochondrial permeability transition and apoptosis, which identified a link between extrinsic death receptor signaling and cell-intrinsic mitochondrial-mediated caspase activation. 

Conclusion 

Our findings reveal a novel concept in immune surveillance by identifying a cell-autonomous defense mechanism that selectively eliminates virus-infected hepatocytes through mitochondrial permeability transition. 

Lay summary 

The liver is known for its unique immune functions. Herein, we identify a novel mechanism by which virus-infected hepatocytes can selectively eliminate themselves through reduced mitochondrial resilience to calcium challenge.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{38ecc3ec211f42b29472cbcd2d8bf8e8,

title = {Exploring the SARS-CoV-2 Virus-host-drug Interactome for Drug Repurposing},

author = {S. Sadegh, J. Matschinske, D.B. Blumenthal, G. Galindez, T. Kacprowski, M. List, R. Nasirigerdeh, M. Oubounyt, A. Pichlmair, T.D. Rose, M. Salgado-Albarrán, J. Späth, A. Stukalov, N.K. Wenke, K. Yuan, J.K. Pauling and J. Baumbach},

doi = {10.1038/s41467-020-17189-2},

year  = {2020},

date = {2020-01-01},

journal = {Nature Communications},

volume = {11},

number = {1},

abstract = {textcopyright 2020, The Author(s). Coronavirus Disease-2019 (COVID-19) is an infectious disease caused by the SARS-CoV-2 virus. Various studies exist about the molecular mechanisms of viral infection. However, such information is spread across many publications and it is very time-consuming to integrate, and exploit. We develop CoVex, an interactive online platform for SARS-CoV-2 host interactome exploration and drug (target) identification. CoVex integrates virus-human protein interactions, human protein-protein interactions, and drug-target interactions. It allows visual exploration of the virus-host interactome and implements systems medicine algorithms for network-based prediction of drug candidates. Thus, CoVex is a resource to understand molecular mechanisms of pathogenicity and to prioritize candidate therapeutics. We investigate recent hypotheses on a systems biology level to explore mechanistic virus life cycle drivers, and to extract drug repurposing candidates. CoVex renders COVID-19 drug research systems-medicine-ready by giving the scientific community direct access to network medicine algorithms. It is available at https://exbio.wzw.tum.de/covex/.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{GebhardtPichlmair2019_PP,

title = {The Alternative Cap-binding Complex is Required for Antiviral Defense in vivo},

author = {A. Gebhardt and V. Bergant and D. Schnepf and M. Moser and A. Meiler and D. Togbe and C. Mackowiak and L.S. Reinert and S.R. Paludan and B. Ryffel and A. Stukalov and P. Staeheli and A. Pichlmair},

url = {https://doi.org/10.1371/journal.ppat.1008155},

doi = {10.1371/journal.ppat.1008155},

year  = {2019},

date = {2019-12-19},

journal = {PLOS Pathogens},

volume = {15},

number = {12},

pages = {1-22},

publisher = {Public Library of Science},

abstract = {Infection with viruses and other pathogens requires appropriate cellular countermeasures, which involve swift and accurate adaptation of gene expression profiles. mRNAs encoding for immune-regulatory and effector proteins need to be transported into the cytoplasm in order to generate proteins necessary to fight the pathogen. Here we show that this process requires proper functionality of the Nuclear cap protein 3 (NCBP3), a protein recently identified to contribute to an alternative mRNA cap-binding complex. An Ncbp3-deficient mouse model allowed higher virus growth in vitro and showed high susceptibility to influenza A virus challenge in vivo. While NCBP3-deficient cells were able to transcriptionally upregulate cytokine mRNAs, generation of cytokines was significantly reduced in the absence of NCBP3. Our data shows a non-redundant function of NCBP3 and the alternative cap-binding complex in antiviral responses. More broadly, this work demonstrates a yet unappreciated aspect of post-transcriptional gene regulation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{ScaturroPichlmair2019_V,

title = {Chasing Intracellular Zika Virus Using Proteomics},

author = {P. Scaturro and A.L. Kastner and A. Pichlmair},

url = {https://www.mdpi.com/1999-4915/11/9/878},

doi = {10.3390/v11090878},

year  = {2019},

date = {2019-09-19},

journal = {Viruses},

volume = {11},

number = {9},

pages = {878},

abstract = {Flaviviruses are the most medically relevant group of arboviruses causing a wide range of diseases in humans and are associated with high mortality and morbidity, as such posing a major health concern. Viruses belonging to this family can be endemic (e.g., dengue virus), but can also cause fulminant outbreaks (e.g., West Nile virus, Japanese encephalitis virus and Zika virus). Intense research efforts in the past decades uncovered shared fundamental strategies used by flaviviruses to successfully replicate in their respective hosts. However, the distinct features contributing to the specific host and tissue tropism as well as the pathological outcomes unique to each individual flavivirus are still largely elusive. The profound footprint of individual viruses on their respective hosts can be investigated using novel technologies in the field of proteomics that have rapidly developed over the last decade. An unprecedented sensitivity and throughput of mass spectrometers, combined with the development of new sample preparation and bioinformatics analysis methods, have made the systematic investigation of virus–host interactions possible. Furthermore, the ability to assess dynamic alterations in protein abundances, protein turnover rates and post-translational modifications occurring in infected cells now offer the unique possibility to unravel complex viral perturbations induced in the infected host. In this review, we discuss the most recent contributions of mass spectrometry–based proteomic approaches in flavivirus biology with a special focus on Zika virus, and their basic and translational potential and implications in understanding and characterizing host responses to arboviral infections.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{PlaszczycaBartenschlager2019_Pp,

title = {A Novel Interaction Between Dengue Virus Nonstructural Protein 1 and the NS4A-2K-4B Precursor is Required for Viral RNA Replication but not for Formation of the Membranous Replication Organelle},

author = {A. Płaszczyca and P. Scaturro and C.J. Neufeldt and M. Cortese and B. Cerikan and S. Ferla and A. Brancale and A. Pichlmair and R. Bartenschlager},

doi = {10.1371/journal.ppat.1007736},

issn = {1553-7374},

year  = {2019},

date = {2019-05-09},

journal = {PLoS Pathogens},

volume = {15},

pages = {e1007736},

abstract = {Dengue virus (DENV) has emerged as major human pathogen. Despite the serious socio-economic impact of DENV-associated diseases, antiviral therapy is missing. DENV replicates in the cytoplasm of infected cells and induces a membranous replication organelle, formed by invaginations of the endoplasmic reticulum membrane and designated vesicle packets (VPs). Nonstructural protein 1 (NS1) of DENV is a multifunctional protein. It is secreted from cells to counteract antiviral immune responses, but also critically contributes to the severe clinical manifestations of dengue. In addition, NS1 is indispensable for viral RNA replication, but the underlying molecular mechanism remains elusive. In this study, we employed a combination of genetic, biochemical and imaging approaches to dissect the determinants in NS1 contributing to its various functions in the viral replication cycle. Several important observations were made. First, we identified a cluster of amino acid residues in the exposed region of the β-ladder domain of NS1 that are essential for NS1 secretion. Second, we revealed a novel interaction of NS1 with the NS4A-2K-4B cleavage intermediate, but not with mature NS4A or NS4B. This interaction is required for RNA replication, with two residues within the connector region of the NS1 "Wing" domain being crucial for binding of the NS4A-2K-4B precursor. By using a polyprotein expression system allowing the formation of VPs in the absence of viral RNA replication, we show that the NS1 -NS4A-2K-4B interaction is not required for VP formation, arguing that the association between these two proteins plays a more direct role in the RNA amplification process. Third, through analysis of polyproteins containing deletions in NS1, and employing a trans-complementation assay, we show that both cis and trans acting elements within NS1 contribute to VP formation, with the capability of NS1 mutants to form VPs correlating with their capability to support RNA replication. In conclusion, these results reveal a direct role of NS1 in VP formation that is independent from RNA replication, and argue for a critical function of a previously unrecognized NS4A-2K-NS4B precursor specifically interacting with NS1 and promoting viral RNA replication.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{HubelPichlmair2019_NI,

title = {A Protein-interaction Network of Interferon-stimulated Genes Extends the Innate Immune System Landscape},

author = { P. Hubel and C. Urban and V. Bergant and W.M. Schneider and B. Knauer and A. Stukalov and P. Scaturro and A. Mann and L. Brunotte and H.H. Hoffmann and J.W. Schoggins and M. Schwemmle and M. Mann and C.M. Rice and A. Pichlmair},

url = {https://www.nature.com/articles/s41590-019-0323-3

https://innatelab.virologie.med.tum.de/archives/664},

doi = {10.1038/s41590-019-0323-3},

issn = {1529-2916},

year  = {2019},

date = {2019-03-04},

journal = {Nat. Immunol.},

abstract = {Interferon-stimulated genes (ISGs) form the backbone of the innate immune system and are important for limiting intra- and intercellular viral replication and spread. We conducted a mass-spectrometry-based survey to understand the fundamental organization of the innate immune system and to explore the molecular functions of individual ISGs. We identified interactions between 104 ISGs and 1,401 cellular binding partners engaging in 2,734 high-confidence interactions. 90% of these interactions are unreported so far, and our survey therefore illuminates a far wider activity spectrum of ISGs than is currently known. Integration of the resulting ISG-interaction network with published datasets and functional studies allowed us to identify regulators of immunity and processes related to the immune system. Given the extraordinary robustness of the innate immune system, this ISG network may serve as a blueprint for therapeutic targeting of cellular systems to efficiently fight viral infections.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{neufeldt_er-shaping_2019,

title = {ER-shaping Atlastin Proteins Act as Central Hubs to Promote Flavivirus Replication and Virion Assembly},

author = {C.J. Neufeldt and M. Cortese and P. Scaturro and B. Cerikan and J.G. Wideman and K. Tabata and T. Moraes and O. Oleksiuk and A. Pichlmair and R. Bartenschlager},

url = {http://www.nature.com/articles/s41564-019-0586-3},

doi = {10.1038/s41564-019-0586-3},

issn = {2058-5276},

year  = {2019},

date = {2019-01-01},

urldate = {2021-07-01},

journal = {Nature Microbiology},

volume = {4},

number = {12},

pages = {2416--2429},

abstract = {Flaviviruses, including dengue virus and Zika virus, extensively remodel the cellular endomembrane network to generate replication organelles that promote viral genome replication and virus production. However, it remains unclear how these membranes and associated cellular proteins act during the virus cycle. Here, we show that atlastins (ATLs), a subset of ER-resident proteins involved in neurodegenerative diseases, have dichotomous effects on flaviviruses—with ATL2 depletion leading to replication organelle defects, and ATL3 depletion to changes in virus production pathways. We characterized non-conserved functional domains in ATL paralogues and show that the ATL interactome is profoundly reprogrammed following dengue virus infection. Screen analysis confirmed non-redundant ATL functions and identified a specific role for ATL3, and its interactor ARF4, in vesicle trafficking and virion maturation. Our data identify ATLs as central hubs targeted by flaviviruses to establish their replication organelle and to achieve efficient virion maturation and secretion.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Scaturro2018zika,

title = {An Orthogonal Proteomic Survey Uncovers Novel Zika Virus Host Factors},

author = {P. Scaturro and A. Stukalov and D.A. Haas and M. Cortese and K. Draganova and A. Płaszczyca and R. Bartenschlager and M. Götz and A. Pichlmair},

url = {https://www.nature.com/articles/s41586-018-0484-5

https://innatelab.virologie.med.tum.de/archives/458},

doi = {10.1038/s41586-018-0484-5},

issn = {1476-4687},

year  = {2018},

date = {2018-09-03},

journal = {Nature},

abstract = {Zika virus (ZIKV) has recently emerged as a global health concern owing to its widespread diffusion and its association with severe neurological symptoms and microcephaly in newborns1. However, the molecular mechanisms that are responsible for the pathogenicity of ZIKV remain largely unknown. Here we use human neural progenitor cells and the neuronal cell line SK-N-BE2 in an integrated proteomics approach to characterize the cellular responses to viral infection at the proteome and phosphoproteome level, and use affinity proteomics to identify cellular targets of ZIKV proteins. Using this approach, we identify 386 ZIKV-interacting proteins, ZIKV-specific and pan-flaviviral activities as well as host factors with known functions in neuronal development, retinal defects and infertility. Moreover, our analysis identified 1,216 phosphorylation sites that are specifically up- or downregulated after ZIKV infection, indicating profound modulation of fundamental signalling pathways such as AKT, MAPK–ERK and ATM–ATR and thereby providing mechanistic insights into the proliferation arrest elicited by ZIKV infection. Functionally, our integrative study identifies ZIKV host-dependency factors and provides a comprehensive framework for a system-level understanding of ZIKV-induced perturbations at the levels of proteins and cellular pathways.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Scaturro2018oxe,

title = {Oxeiptosis — a Cell Death Pathway to Mitigate Damage Caused by Radicals},

author = {P. Scaturro and A. Pichlmair},

doi = {10.1038/s41418-018-0134-3},

year  = {2018},

date = {2018-05-29},

journal = {Cell Death Diff.},

volume = {25},

number = {7},

pages = {1191-1193},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Haas2018,

title = {Viral Targeting of TFIIB Impairs de novo Polymerase II Recruitment and Affects Antiviral Immunity},

author = {D.A. Haas and A. Meiler and K. Geiger and C. Vogt and E. Preuss and G. Kochs and A. Pichlmair},

url = {http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1006980},

doi = {10.1371/journal.ppat.1006980},

issn = {1553-7374},

year  = {2018},

date = {2018-04-30},

journal = {PLOS Pathogens},

volume = {14},

number = {4},

pages = {e1006980},

abstract = {Viruses have evolved a plethora of mechanisms to target host antiviral responses. Here, we propose a yet uncharacterized mechanism of immune regulation by the orthomyxovirus Thogoto virus (THOV) ML protein through engaging general transcription factor TFIIB. ML generates a TFIIB depleted nuclear environment by re-localizing it into the cytoplasm. Although a broad effect on gene expression would be anticipated, ML expression, delivery of an ML-derived functional domain or experimental depletion of TFIIB only leads to altered expression of a limited number of genes. Our data indicate that TFIIB is critically important for the de novo recruitment of Pol II to promoter start sites and that TFIIB may not be required for regulated gene expression from paused promoters. Since many immune genes require de novo recruitment of Pol II, targeting of TFIIB by THOV represents a neat mechanism to affect immune responses while keeping other cellular transcriptional activities intact. Thus, interference with TFIIB activity may be a favourable site for therapeutic intervention to control undesirable inflammation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Holze2017,

title = {Oxeiptosis, a ROS-induced Caspase-independent Apoptosis-like Cell-death Pathway},

author = {C. Holze and C. Michaudel and C. Mackowiak and D.A. Haas and C. Benda and P. Hubel and F.L. Pennemann and D. Schnepf and J. Wettmarshausen and M. Braun and D.W. Leung and G.K. Amarasinghe and F. Perocchi and P. Staeheli and B. Ryffel and A. Pichlmair},

url = {https://www.nature.com/articles/s41590-017-0013-y},

doi = {10.1038/s41590-017-0013-y},

year  = {2017},

date = {2017-12-18},

journal = {Nat. Immunol.},

volume = {19},

number = {2},

pages = {130-140},

abstract = {Reactive oxygen species (ROS) are generated by virus-infected cells; however, the physiological importance of ROS generated under these conditions is unclear. Here we found that the inflammation and cell death induced by exposure of mice or cells to sources of ROS were not altered in the absence of canonical ROS-sensing pathways or known cell-death pathways. ROS-induced cell-death signaling involved interactions among the cellular ROS sensor and antioxidant factor KEAP1, the phosphatase PGAM5 and the proapoptotic factor AIFM1. Pgam5 –/– mice showed exacerbated lung inflammation and proinflammatory cytokines in an ozone-exposure model. Similarly, challenge with influenza A virus led to increased infiltration of the virus, lymphocytic bronchiolitis and reduced survival of Pgam5 –/– mice. This pathway, which we have called ‘oxeiptosis’, was a ROS-sensitive, caspase independent, non-inflammatory cell-death pathway and was important for protection against inflammation induced by ROS or ROS-generating agents such as viral pathogens.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Vonderstein2017,

title = {Viperin Targets Flavivirus Virulence by Inducing Assembly of Non-infectious Capsid Particles},

author = {K. Vonderstein and E. Nilsson and P. Hubel and L. Nygård Skalman and A. Upadhyay and J. Pasto and A. Pichlmair and R. Lundmark and A.K. Överby},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5730767/},

doi = {10.1128/JVI.01751-17},

issn = {1098-5514},

year  = {2017},

date = {2017-10-18},

journal = {J Virol},

volume = {92},

number = {1},

pages = {e01751-17},

abstract = {Efficient antiviral immunity requires interference with virus replication at multiple layers targeting diverse steps in the viral life cycle. Here we describe a novel flavivirus inhibition mechanism that results in interferon-mediated obstruction of tick-borne encephalitis virus particle assembly, and involves release of malfunctional membrane associated capsid (C) particles. This mechanism is controlled by the activity of the interferon-induced protein viperin, a broad spectrum antiviral interferon stimulated gene. Through analysis of the viperin-interactome, we identified the Golgi Brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1), as the cellular protein targeted by viperin. Viperin-induced antiviral activity as well as C-particle release was stimulated by GBF1 inhibition and knock down, and reduced by elevated levels of GBF1. Our results suggest that viperin targets flavivirus virulence by inducing the secretion of unproductive non-infectious virus particles, by a GBF1-dependent mechanism. This yet undescribed antiviral mechanism allows potential therapeutic intervention. The interferon response can target viral infection on almost every level, however, very little is known about interference of flavivirus assembly. Here we show that interferon, through the action of viperin, can disturb assembly of tick-borne encephalitis virus. The viperin protein is highly induced after viral infection and exhibit broad-spectrum antiviral activity. However, the mechanism of action is still elusive and appear to vary between the different viruses, indicating that cellular targets utilized by several viruses might be involved. In this study we show that viperin induce capsid particle release by interacting and inhibiting the function of the cellular protein Golgi Brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1). GBF1 is a key protein in the cellular secretory pathway and essential in the life cycle of many viruses, also targeted by viperin, implicating GBF1 as a novel putative drug target.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{GebhardtLaudenbach2017,

title = {Discrimination of Self and Non-Self Ribonucleic Acids},

author = {A. Gebhardt and B. Laudenbach and A. Pichlmair},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5439445/},

doi = {10.1089/jir.2016.0092},

year  = {2017},

date = {2017-05-01},

journal = {J Interferon Cytokine Res.},

volume = {37},

number = {5},

pages = {184-197},

abstract = {Most virus infections are controlled through the innate and adaptive immune system. A surprisingly limited number of so-called pattern recognition receptors (PRRs) have the ability to sense a large variety of virus infections. The reason for the broad activity of PRRs lies in the ability to recognize viral nucleic acids. These nucleic acids lack signatures that are present in cytoplasmic cellular nucleic acids and thereby marking them as pathogen-derived. Accumulating evidence suggests that these signatures, which are predominantly sensed by a class of PRRs called retinoic acid-inducible gene I (RIG-I)-like receptors and other proteins, are not unique to viruses but rather resemble immature forms of cellular ribonucleic acids generated by cellular polymerases. RIG-I-like receptors, and other cellular antiviral proteins, may therefore have mainly evolved to sense nonprocessed nucleic acids typically generated by primitive organisms and pathogens. This capability has not only implications on induction of antiviral immunity but also on the function of cellular proteins to handle self-derived RNA with stimulatory potential.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{WillemsenBinder2017_MC,

title = {Phosphorylation-Dependent Feedback Inhibition of RIG-I by DAPK1 Identified by Kinome-wide siRNA Screening.},

author = {J. Willemsen and O. Wicht and J.C. Wolanski and N. Baur and S. Bastian and D.A. Haas and P. Matula and B. Knapp and L. Meyniel-Schicklin and C. Wang and R. Bartenschlager and V. Lohmann and K. Rohr and H. Erfle and L. Kaderali and J. Marcotrigiano and A. Pichlmair and M. Binder},

url = {https://doi.org/10.1016/j.molcel.2016.12.021},

doi = {10.1016/j.molcel.2016.12.021},

issn = {1097-4164},

year  = {2017},

date = {2017-02-02},

journal = {Mol Cell},

volume = {65},

pages = {403--415.e8},

abstract = {Cell-autonomous induction of type I interferon must be stringently regulated. Rapid induction is key to control virus infection, whereas proper limitation of signaling is essential to prevent immunopathology and autoimmune disease. Using unbiased kinome-wide RNAi screening followed by thorough validation, we identified 22 factors that regulate RIG-I/IRF3 signaling activity. We describe a negative-feedback mechanism targeting RIG-I activity, which is mediated by death associated protein kinase 1 (DAPK1). RIG-I signaling triggers DAPK1 kinase activation, and active DAPK1 potently inhibits RIG-I stimulated IRF3 activity and interferon-beta production. DAPK1 phosphorylates RIG-I in vitro at previously reported as well as other sites that limit 5'ppp-dsRNA sensing and virtually abrogate RIG-I activation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Abbas2017,

title = {Structure of Human IFIT1 with Capped RNA Reveals Adaptable mRNA Binding and Mechanisms for Sensing N1 and N2 Ribose 2'-O methylations},

author = {Y.M. Abbas and B. Laudenbach and S. Martínez-Montero and R. Cencic and M. Habjan and A. Pichlmair and M.J. Damha and J. Pelletier and B. Nagar},

doi = {10.1073/pnas.1612444114},

issn = {1091-6490},

year  = {2017},

date = {2017-01-01},

journal = {Proc Natl Acad Sci U S A},

volume = {114},

pages = {E2106--E2115},

abstract = {IFIT1 (IFN-induced protein with tetratricopeptide repeats-1) is an effector of the host innate immune antiviral response that prevents propagation of virus infection by selectively inhibiting translation of viral mRNA. It relies on its ability to compete with the translation initiation factor eIF4F to specifically recognize foreign capped mRNAs, while remaining inactive against host mRNAs marked by ribose 2'-O methylation at the first cap-proximal nucleotide (N1). We report here several crystal structures of RNA-bound human IFIT1, including a 1.6-Å complex with capped RNA. IFIT1 forms a water-filled, positively charged RNA-binding tunnel with a separate hydrophobic extension that unexpectedly engages the cap in multiple conformations ( and ) giving rise to a relatively plastic and nonspecific mode of binding, in stark contrast to eIF4E. Cap-proximal nucleotides encircled by the tunnel provide affinity to compete with eIF4F while allowing IFIT1 to select against N1 methylated mRNA. Gel-shift binding assays confirm that N1 methylation interferes with IFIT1 binding, but in an RNA-dependent manner, whereas translation assays reveal that N1 methylation alone is not sufficient to prevent mRNA recognition at high IFIT1 concentrations. Structural and functional analysis show that 2'-O methylation at N2, another abundant mRNA modification, is also detrimental for RNA binding, thus revealing a potentially synergistic role for it in self- versus nonself-mRNA discernment. Finally, structure-guided mutational analysis confirms the importance of RNA binding for IFIT1 restriction of a human coronavirus mutant lacking viral N1 methylation. Our structural and biochemical analysis sheds new light on the molecular basis for IFIT1 translational inhibition of capped viral RNA.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Brady2017,

title = {Molluscum Contagiosum Virus Protein MC005 Inhibits NF-κB Activation by Targeting NEMO-Regulated IκB Kinase Activation.},

author = {G. Brady and D.A. Haas and P.J. Farrell and A. Pichlmair and A.G. Bowie},

doi = {10.1128/JVI.00545-17},

issn = {1098-5514},

year  = {2017},

date = {2017-01-01},

journal = {J Virol},

volume = {91},

abstract = {Molluscum contagiosum virus (MCV), the only known extant human-adapted poxvirus, causes a long-duration infection characterized by skin lesions that typically display an absence of inflammation despite containing high titers of live virus. Despite this curious presentation, MCV is very poorly characterized in terms of host-pathogen interactions. The absence of inflammation around MCV lesions suggests the presence of potent inhibitors of human antiviral immunity and inflammation. However, only a small number of MCV immunomodulatory genes have been characterized in detail. It is likely that many more remain to be discovered, given the density of such sequences in other poxvirus genomes. NF-κB activation occurs in response to both virus-induced pattern recognition receptor (PRR) signaling and cellular activation by virus-induced proinflammatory cytokines like tumor necrosis factor and interleukin-1. Activated NF-κB drives cytokine and interferon gene expression, leading to inflammation and virus clearance. We report that MC005, which has no orthologs in other poxvirus genomes, is a novel inhibitor of PRR- and cytokine-stimulated NF-κB activation. MC005 inhibited NF-κB proximal to the IκB kinase (IKK) complex, and unbiased affinity purification revealed that MC005 interacts with the IKK subunit NEMO (NF-κB essential modulator). MC005 binding to NEMO prevents the conformational priming of the IKK complex that occurs when NEMO binds to ubiquitin chains during pathway activation. These data reveal a novel mechanism of poxvirus inhibition of human innate immunity, validate current dynamic models of NEMO-dependent IKK complex activation, and further clarify how the human-adapted poxvirus MCV can so effectively evade antiviral immunity and suppress inflammation to persist in human skin lesions. Poxviruses adapt to specific hosts over time, evolving and tailoring elegantly precise inhibitors of the rate-limiting steps within the signaling pathways that control innate immunity and inflammation. These inhibitors reveal new features of the antiviral response, clarify existing models of signaling regulation while offering potent new tools for approaching therapeutic intervention in autoimmunity and inflammatory disease. Molluscum contagiosum virus (MCV) is the only known extant poxvirus specifically adapted to human infection and appears adept at evading normal human antiviral responses, yet it remains poorly characterized. We report the identification of MCV protein MC005 as an inhibitor of the pathways leading to the activation of NF-κB, an essential regulator of innate immunity. Further, identification of the mechanism of inhibition of NF-κB by MC005 confirms current models of the complex way in which NF-κB is regulated and greatly expands our understanding of how MCV so effectively evades human immunity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Goonawardane2017a,

title = {Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions.},

author = {N. Goonawardane and A. Gebhardt and C. Bartlett and A. Pichlmair and M. Harris},

doi = {10.1128/JVI.00805-17},

issn = {1098-5514},

year  = {2017},

date = {2017-01-01},

journal = {J Virol},

volume = {91},

abstract = {Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a phosphoprotein that plays key, yet poorly defined, roles in both virus genome replication and virion assembly/release. It has been proposed that differential phosphorylation could act as a switch to regulate the various functions of NS5A; however, the mechanistic details of the role of this posttranslational modification in the virus life cycle remain obscure. We previously reported (D. Ross-Thriepland, J. Mankouri, and M. Harris, J Virol 89:3123-3135, 2015, doi:10.1128/JVI.02995-14) a role for phosphorylation at serine 225 (S225) of NS5A in the regulation of JFH-1 (genotype 2a) genome replication. A phosphoablatant (S225A) mutation resulted in a 10-fold reduction in replication and a perinuclear restricted distribution of NS5A, whereas the corresponding phosphomimetic mutation (S225D) had no phenotype. To determine the molecular mechanisms underpinning this phenotype we conducted a label-free proteomics approach to identify cellular NS5A interaction partners. This analysis revealed that the S225A mutation disrupted the interactions of NS5A with a number of cellular proteins, in particular the nucleosome assembly protein 1-like protein 1 (NAP1L1), bridging integrator 1 (Bin1, also known as amphiphysin II), and vesicle-associated membrane protein-associated protein A (VAP-A). These interactions were validated by immunoprecipitation/Western blotting, immunofluorescence, and proximity ligation assay. Importantly, small interfering RNA (siRNA)-mediated knockdown of NAP1L1, Bin1 or VAP-A impaired viral genome replication and recapitulated the perinuclear redistribution of NS5A seen in the S225A mutant. These results demonstrate that S225 phosphorylation regulates the interactions of NS5A with a defined subset of cellular proteins. Furthermore, these interactions regulate both HCV genome replication and the subcellular localization of replication complexes. Hepatitis C virus is an important human pathogen. The viral nonstructural 5A protein (NS5A) is the target for new antiviral drugs. NS5A has multiple functions during the virus life cycle, but the biochemical details of these roles remain obscure. NS5A is known to be phosphorylated by cellular protein kinases, and in this study, we set out to determine whether this modification is required for the binding of NS5A to other cellular proteins. We identified 3 such proteins and show that they interacted only with NS5A that was phosphorylated on a specific residue. Furthermore, these proteins were required for efficient virus replication and the ability of NS5A to spread throughout the cytoplasm of the cell. Our results help to define the function of NS5A and may contribute to an understanding of the mode of action of the highly potent antiviral drugs that are targeted to NS5A.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Mazouzi2016,

title = {A Comprehensive Analysis of the Dynamic Response to Aphidicolin-Mediated Replication Stress Uncovers Targets for ATM and ATMIN.},

author = {A. Mazouzi and A. Stukalov and A.C. Müller and D. Chen and M. Wiedner and J. Prochazkova and S. Chiang and M. Schuster and F.P. Breitwieser and A. Pichlmair and S.F. El-Khamisy and C. Bock and R. Kralovics and J. Colinge and K.L. Bennett and J.I. Loizou},

doi = {10.1016/j.celrep.2016.03.077},

issn = {2211-1247},

year  = {2016},

date = {2016-01-01},

journal = {Cell reports},

volume = {15},

pages = {893--908},

abstract = {The cellular response to replication stress requires the DNA-damage-responsive kinase ATM and its cofactor ATMIN; however, the roles of this signaling pathway following replication stress are unclear. To identify the functions of ATM and ATMIN in response to replication stress, we utilized both transcriptomics and quantitative mass-spectrometry-based phosphoproteomics. We found that replication stress induced by aphidicolin triggered widespread changes in both gene expression and protein phosphorylation patterns. These changes gave rise to distinct early and late replication stress responses. Furthermore, our analysis revealed previously unknown targets of ATM and ATMIN downstream of replication stress. We demonstrate ATMIN-dependent phosphorylation of H2AX and of CRMP2, a protein previously implicated in Alzheimer's disease but not in the DNA damage response. Overall, our dataset provides a comprehensive resource for discovering the cellular responses to replication stress and, potentially, associated pathologies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Stukalov2015,

title = {Gambling with Flu: "All in" to Maximize Reward},

author = {A. Stukalov and A. Pichlmair},

url = {https://www.sciencedirect.com/science/article/pii/S1931312815004643},

doi = {10.1016/j.chom.2015.11.010},

issn = {1934-6069},

year  = {2015},

date = {2015-12-09},

journal = {Cell host microbe},

volume = {18},

number = {6},

pages = {643--645},

abstract = {In this issue of Cell Host & Microbe, Tripathi et al. (2015) report an in-depth meta-analysis of eight influenza virus siRNA screens combined with viral-host protein interactome data. The integration of the different omics datasets highlights candidate genes and pathways for further investigation and potential therapeutic targeting in the future.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Gebhardt2015,

title = {mRNA Export Through an Additional Cap-binding Complex Consisting of NCBP1 and NCBP3},

author = {A. Gebhardt and M. Habjan and C. Benda and A. Meiler and D.A. Haas and M.Y. Hein and A. Mann and M. Mann and B. Habermann and A. Pichlmair},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4595607/},

doi = {10.1038/ncomms9192},

issn = {2041-1723},

year  = {2015},

date = {2015-09-18},

journal = {Nat commun},

volume = {6},

pages = {8192},

abstract = {The flow of genetic information from DNA to protein requires polymerase-II-transcribed RNA characterized by the presence of a 5'-cap. The cap-binding complex (CBC), consisting of the nuclear cap-binding protein (NCBP) 2 and its adaptor NCBP1, is believed to bind all capped RNA and to be necessary for its processing and intracellular localization. Here we show that NCBP1, but not NCBP2, is required for cell viability and poly(A) RNA export. We identify C17orf85 (here named NCBP3) as a cap-binding protein that together with NCBP1 forms an alternative CBC in higher eukaryotes. NCBP3 binds mRNA, associates with components of the mRNA processing machinery and contributes to poly(A) RNA export. Loss of NCBP3 can be compensated by NCBP2 under steady-state conditions. However, NCBP3 becomes pivotal under stress conditions, such as virus infection. We propose the existence of an alternative CBC involving NCBP1 and NCBP3 that plays a key role in mRNA biogenesis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Heinz2015,

title = {The Lipid-Modifying Enzyme SMPDL3B Negatively Regulates Innate Immunity.},

author = {L.X. Heinz and C.L. Baumann and M.S. Köberlin and B. Snijder and R. Gawish and G. Shui and O. Sharif and I.M. Aspalter and A.C. Müller and R.K. Kandasamy and F.P. Breitwieser and A. Pichlmair and M. Bruckner and M. Rebsamen and S. Blüml and T. Karonitsch and A. Fauster and J. Colinge and K.L. Bennett and S. Knapp and M.R Wenk and G. Superti-Furga},

doi = {10.1016/j.celrep.2015.05.006},

issn = {2211-1247},

year  = {2015},

date = {2015-01-01},

journal = {Cell reports},

volume = {11},

pages = {1919--1928},

abstract = {Lipid metabolism and receptor-mediated signaling are highly intertwined processes that cooperate to fulfill cellular functions and safeguard cellular homeostasis. Activation of Toll-like receptors (TLRs) leads to a complex cellular response, orchestrating a diverse range of inflammatory events that need to be tightly controlled. Here, we identified the GPI-anchored Sphingomyelin Phosphodiesterase, Acid-Like 3B (SMPDL3B) in a mass spectrometry screening campaign for membrane proteins co-purifying with TLRs. Deficiency of Smpdl3b in macrophages enhanced responsiveness to TLR stimulation and profoundly changed the cellular lipid composition and membrane fluidity. Increased cellular responses could be reverted by re-introducing affected ceramides, functionally linking membrane lipid composition and innate immune signaling. Finally, Smpdl3b-deficient mice displayed an intensified inflammatory response in TLR-dependent peritonitis models, establishing its negative regulatory role in vivo. Taken together, our results identify the membrane-modulating enzyme SMPDL3B as a negative regulator of TLR signaling that functions at the interface of membrane biology and innate immunity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Brady2015,

title = {Poxvirus Protein MC132 from Molluscum Contagiosum Virus Inhibits NF-B Activation by Targeting p65 for Degradation.},

author = {G. Brady and D.A. Haas and P.J. Farrell and A. Pichlmair and A.G. Bowie},

doi = {10.1128/JVI.00799-15},

issn = {1098-5514},

year  = {2015},

date = {2015-01-01},

journal = {J Virol},

volume = {89},

pages = {8406--8415},

abstract = {Molluscum contagiosum virus (MCV) is unique in being the only known extant, human-adapted poxvirus, yet to date, it is very poorly characterized in terms of host-pathogen interactions. MCV causes persistent skin lesions filled with live virus, but these are generally immunologically silent, suggesting the presence of potent inhibitors of human antiviral immunity and inflammation. Fewer than five MCV immunomodulatory genes have been characterized in detail, but it is likely that many more remain to be discovered given the density of such sequences in all well-characterized poxviruses. Following virus infection, NF-B activation occurs in response to both pattern recognition receptor (PRR) signaling and cellular activation by virus-elicited proinflammatory cytokines, such as tumor necrosis factor (TNF). As such, NF-B activation is required for virus detection, antiviral signaling, inflammation, and clearance of viral infection. Hence, we screened a library of MCV genes for effects on TNF-stimulated NF-B activation. This revealed MC132, a unique protein with no orthologs in other poxviral genomes, as a novel inhibitor of NF-B. Interestingly, MC132 also inhibited PRR- and virus-activated NF-B, since MC132 interacted with the NF-B subunit p65 and caused p65 degradation. Unbiased affinity purification to identify host targets of MC132 revealed that MC132 acted by targeting NF-B p65 for ubiquitin-dependent proteasomal degradation by recruiting p65 to a host Cullin-5/Elongin B/Elongin C complex. These data reveal a novel mechanism for poxviral inhibition of human innate immunity and further clarify how the human-adapted poxvirus MCV can so effectively evade antiviral immunity to persist in skin lesions.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Habjan2015,

title = {Cytoplasmic Sensing of Viral Nucleic Acids},

author = {M. Habjan and A. Pichlmair},

doi = {10.1016/j.coviro.2015.01.012},

issn = {1879-6265},

year  = {2015},

date = {2015-01-01},

journal = {Current opinion in virology},

volume = {11},

pages = {31--37},

abstract = {Viruses are the most abundant pathogens on earth. A fine-tuned framework of intervening pathways is in place in mammalian cells to orchestrate the cellular defence against these pathogens. Key for this system is sensor proteins that recognise specific features associated with nucleic acids of incoming viruses. Here we review the current knowledge on cytoplasmic sensors for viral nucleic acids. These sensors induce expression of cytokines, affect cellular functions required for virus replication and directly target viral nucleic acids through degradation or sequestration. Their ability to respond to a given nucleic acid is based on both the differential specificity of the individual proteins and the downstream signalling or adaptor proteins. The cooperation of these multiple proteins and pathways plays a key role in inducing successful immunity against virus infections.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Eberle2014,

title = {The Lysine Methyltransferase SMYD3 Interacts with Hepatitis C Virus NS5A and is a Negative Regulator of Viral Particle Production},

author = {C. Eberle and M. Zayas and A. Stukalov and A. Pichlmair and G. Alvisi and A.C. Müller and K.L. Bennett and R. Bartenschlager and G. Superti-Furga},

doi = {10.1016/j.virol.2014.05.016},

issn = {1096-0341},

year  = {2014},

date = {2014-01-01},

journal = {Virology},

volume = {462-463},

pages = {34--41},

abstract = {Hepatitis C virus (HCV) is a considerable global health and economic burden. The HCV nonstructural protein (NS) 5A is essential for the viral life cycle. The ability of NS5A to interact with different host and viral proteins allow it to manipulate cellular pathways and regulate viral processes, including RNA replication and virus particle assembly. As part of a proteomic screen, we identified several NS5A-binding proteins, including the lysine methyltransferase SET and MYND domain containing protein 3 (SMYD3). We confirmed the interaction in the context of viral replication by co-immunoprecipitation and co-localization studies. Mutational analyses revealed that the MYND-domain of SMYD3 and domain III of NS5A are required for the interaction. Overexpression of SMYD3 resulted in decreased intracellular and extracellular virus titers, whilst viral RNA replication remained unchanged, suggesting that SMYD3 negatively affects HCV particle production in a NS5A-dependent manner.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}