Publications by authors named "Andreas Pichlmair"

59 Publications

Disruption of disulfides within RBD of SARS-CoV-2 spike protein prevents fusion and represents a target for viral entry inhibition by registered drugs.

FASEB J 2021 06;35(6):e21651

Department of Synthetic Biology and Immunology, National Institute of Chemistry, Ljubljana, Slovenia.

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.
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http://dx.doi.org/10.1096/fj.202100560RDOI Listing
June 2021

Interferon-induced degradation of the persistent hepatitis B virus cccDNA form depends on ISG20.

EMBO Rep 2021 Jun 9;22(6):e49568. Epub 2021 May 9.

Institute of Virology, School of Medicine, Technical University of Munich / Helmholtz Zentrum München, Munich, Germany.

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.
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http://dx.doi.org/10.15252/embr.201949568DOI Listing
June 2021

The Zinc Finger Antiviral Protein ZAP Restricts Human Cytomegalovirus and Selectively Binds and Destabilizes Viral / Transcripts.

mBio 2021 05 4;12(3). Epub 2021 May 4.

Viral Immune Modulation Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany

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 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 locus may play an important role for HCMV replication. 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.
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http://dx.doi.org/10.1128/mBio.02683-20DOI Listing
May 2021

A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response.

Vaccines (Basel) 2021 Apr 27;9(5). Epub 2021 Apr 27.

EN-FIST, Centre of Excellence, 1000 Ljubljana, Slovenia.

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.
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http://dx.doi.org/10.3390/vaccines9050431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8146944PMC
April 2021

Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV.

Nature 2021 06 12;594(7862):246-252. Epub 2021 Apr 12.

Technical University of Munich, School of Medicine, Institute of Virology, Munich, Germany.

The emergence and global spread of SARS-CoV-2 has resulted in the urgent need for 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 pathophysiology. 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. Here we report a concurrent multi-omics study of SARS-CoV-2 and SARS-CoV. Using state-of-the-art proteomics, we profiled the interactomes of both viruses, as well as their influence on the transcriptome, proteome, ubiquitinome and phosphoproteome of a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed crosstalk between the perturbations taking place upon infection with SARS-CoV-2 and SARS-CoV at different levels and enabled identification of distinct 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 was specifically dysregulated by SARS-CoV-2 ORF3. The extensive dataset (available at https://covinet.innatelab.org ) highlights many hotspots that could be targeted by existing drugs and may be used to guide rational design of virus- and host-directed therapies, which we exemplify by identifying inhibitors of kinases and matrix metalloproteases with potent antiviral effects against SARS-CoV-2.
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http://dx.doi.org/10.1038/s41586-021-03493-4DOI Listing
June 2021

Human NLRP1 is a sensor for double-stranded RNA.

Science 2021 01 26;371(6528). Epub 2020 Nov 26.

Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany.

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 NLRP1B. 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 through its leucine-rich repeat domain, resulting in its NACHT domain gaining adenosine triphosphatase activity. Altogether, these results establish human NLRP1 as a direct sensor for dsRNA and thus RNA virus infection.
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http://dx.doi.org/10.1126/science.abd0811DOI Listing
January 2021

System-Based Approaches to Delineate the Antiviral Innate Immune Landscape.

Viruses 2020 10 21;12(10). Epub 2020 Oct 21.

School of Medicine, Institute of Virology, Technical University of Munich, 81675 Munich, Germany.

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.
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http://dx.doi.org/10.3390/v12101196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589202PMC
October 2020

Exploring the SARS-CoV-2 virus-host-drug interactome for drug repurposing.

Nat Commun 2020 07 14;11(1):3518. Epub 2020 Jul 14.

Chair of Experimental Bioinformatics, TUM School of Life Sciences, Technical University of Munich, München, Germany.

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/.
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http://dx.doi.org/10.1038/s41467-020-17189-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7360763PMC
July 2020

eIF2B as a Target for Viral Evasion of PKR-Mediated Translation Inhibition.

mBio 2020 07 14;11(4). Epub 2020 Jul 14.

Institute for Virology, FB10-Veterinary Medicine, Justus-Liebig University, Giessen, Germany

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. 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.
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http://dx.doi.org/10.1128/mBio.00976-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7360930PMC
July 2020

Reduced mitochondrial resilience enables non-canonical induction of apoptosis after TNF receptor signaling in virus-infected hepatocytes.

J Hepatol 2020 12 26;73(6):1347-1359. Epub 2020 Jun 26.

Institute of Pathology, Technical University of Munich, Munich, Germany.

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.
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http://dx.doi.org/10.1016/j.jhep.2020.06.026DOI Listing
December 2020

Data, Reagents, Assays and Merits of Proteomics for SARS-CoV-2 Research and Testing.

Mol Cell Proteomics 2020 09 26;19(9):1503-1522. Epub 2020 Jun 26.

Chair of Proteomics and Bioanalytics, Technical University of Munich, Freising, Germany; Bavarian Center for Biomolecular Mass Spectrometry (BayBioMS), Technical University of Munich, Freising, Germany. Electronic address:

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.
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http://dx.doi.org/10.1074/mcp.RA120.002164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7780043PMC
September 2020

Persistent Innate Immune Stimulation Results in IRF3-Mediated but Caspase-Independent Cytostasis.

Viruses 2020 06 11;12(6). Epub 2020 Jun 11.

Research Group "Dynamics of Early Viral Infection and the Innate Antiviral Response", Division Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.

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.
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http://dx.doi.org/10.3390/v12060635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7354422PMC
June 2020

The Cytomegalovirus Tegument Protein UL35 Antagonizes Pattern Recognition Receptor-Mediated Type I IFN Transcription.

Microorganisms 2020 May 26;8(6). Epub 2020 May 26.

Institute of Genetics, Technische Universität Braunschweig, 38106 Braunschweig, Germany.

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.
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http://dx.doi.org/10.3390/microorganisms8060790DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7356634PMC
May 2020

The alternative cap-binding complex is required for antiviral defense in vivo.

PLoS Pathog 2019 12 19;15(12):e1008155. Epub 2019 Dec 19.

Institute of Virology, Technical University of Munich, School of Medicine, Munich, Germany.

Cellular response to environmental challenges requires immediate and precise regulation of transcriptional programs. During viral infections, this includes the expression of antiviral genes that are essential to combat the pathogen. Transcribed mRNAs are bound and escorted to the cytoplasm by the cap-binding complex (CBC). We recently identified a protein complex consisting of NCBP1 and NCBP3 that, under physiological conditions, has redundant function to the canonical CBC, consisting of NCBP1 and NCBP2. Here, we provide evidence that NCBP3 is essential to mount a precise and appropriate antiviral response. Ncbp3-deficient cells allow higher virus growth and elicit a reduced antiviral response, a defect happening on post-transcriptional level. Ncbp3-deficient mice suffered from severe lung pathology and increased morbidity after influenza A virus challenge. While NCBP3 appeared to be particularly important during viral infections, it may be more broadly involved to ensure proper protein expression.
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http://dx.doi.org/10.1371/journal.ppat.1008155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6946169PMC
December 2019

ER-shaping atlastin proteins act as central hubs to promote flavivirus replication and virion assembly.

Nat Microbiol 2019 12 21;4(12):2416-2429. Epub 2019 Oct 21.

Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.

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.
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http://dx.doi.org/10.1038/s41564-019-0586-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6881184PMC
December 2019

Chasing Intracellular Zika Virus Using Proteomics.

Viruses 2019 09 19;11(9). Epub 2019 Sep 19.

School of Medicine, Institute of Virology, Technical University of Munich, Schneckenburgerstr. 8, 81675 Munich, Germany.

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.
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http://dx.doi.org/10.3390/v11090878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6783930PMC
September 2019

Chemoenzymatic Total Synthesis of Sorbicatechol Structural Analogues and Evaluation of Their Antiviral Potential.

Chembiochem 2020 02 19;21(4):492-495. Epub 2019 Nov 19.

Biosystems Chemistry, Department of Chemistry and, Center for Integrated Protein Science Munich (CIPSM), Technical University of Munich, Lichtenbergstrasse 4, 85748, Garching, Germany.

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.
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http://dx.doi.org/10.1002/cbic.201900472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065055PMC
February 2020

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.

PLoS Pathog 2019 05 9;15(5):e1007736. Epub 2019 May 9.

Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany.

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.
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http://dx.doi.org/10.1371/journal.ppat.1007736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6508626PMC
May 2019

A protein-interaction network of interferon-stimulated genes extends the innate immune system landscape.

Nat Immunol 2019 04 4;20(4):493-502. Epub 2019 Mar 4.

Innate Immunity Laboratory, Max-Planck Institute of Biochemistry, Munich, Germany.

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.
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http://dx.doi.org/10.1038/s41590-019-0323-3DOI Listing
April 2019

Oxeiptosis: a discreet way to respond to radicals.

Curr Opin Immunol 2019 02 18;56:37-43. Epub 2018 Oct 18.

Immunopathology of Virus Infections, Institute of Virology, Technical University of Munich, Schneckenburger Str. 8, 81675 Munich, Germany; German Center for Infection Research (DZIF), Munich Partner Site, Germany. Electronic address:

One of the best-studied cellular responses to toxic signals and pathogens is programmed cell death. Over the past years, it became apparent that the specific mechanisms of cell death have tremendous influence at both cellular and organismal level, highlighting the importance of sensors and pathways involved in this decision-making process. Central signalling molecules involved in a variety of cell death pathways are reactive oxygen species (ROS). However, the molecular mechanisms regulating differential responses and cellular fates to distinct ROS levels remain incompletely understood. Recently, we uncovered a caspase-independent cell-death pathway named 'oxeiptosis', which links the ROS sensing capacity of KEAP1 to a cell death pathway involving PGAM5 and AIFM1. Alike apoptosis, oxeiptosis is anti-inflammatory when activated by increased intracellular ROS levels and upon pathogens encounter. Here we discuss the potential impact of oxeiptosis in pathogens clearance and teratogenic cells.
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http://dx.doi.org/10.1016/j.coi.2018.10.006DOI Listing
February 2019

NSs Protein of Sandfly Fever Sicilian Phlebovirus Counteracts Interferon (IFN) Induction by Masking the DNA-Binding Domain of IFN Regulatory Factor 3.

J Virol 2018 12 12;92(23). Epub 2018 Nov 12.

Institute for Virology, Philipps-University Marburg, Marburg, Germany

Sandfly fever Sicilian virus (SFSV) is one of the most widespread and frequently identified members of the genus (order , family ) infecting humans. Being transmitted by sandflies, SFSV causes a self-limiting, acute, often incapacitating febrile disease ("sandfly fever," "Pappataci fever," or "dog disease") that has been known since at least the beginning of the 20th century. We show that, similarly to other pathogenic phleboviruses, SFSV suppresses the induction of the antiviral type I interferon (IFN) system in an NSs-dependent manner. SFSV NSs interfered with the TBK1-interferon regulatory factor 3 (IRF3) branch of the RIG-I signaling pathway but not with NF-κB activation. Consistently, we identified IRF3 as a host interactor of SFSV NSs. In contrast to IRF3, neither the IFN master regulator IRF7 nor any of the related transcription factors IRF2, IRF5, and IRF9 were bound by SFSV NSs. In spite of this specificity for IRF3, NSs did not inhibit its phosphorylation, dimerization, or nuclear accumulation, and the interaction was independent of the IRF3 activation or multimerization state. In further studies, we identified the DNA-binding domain of IRF3 (amino acids 1 to 113) as sufficient for NSs binding and found that SFSV NSs prevented the association of activated IRF3 with the IFN-β promoter. Thus, unlike highly virulent phleboviruses, which either destroy antiviral host factors or sequester whole signaling chains into inactive aggregates, SFSV modulates type I IFN induction by directly masking the DNA-binding domain of IRF3. Phleboviruses are receiving increased attention due to the constant discovery of new species and the ongoing spread of long-known members of the genus. Outbreaks of sandfly fever were reported in the 19th century, during World War I, and during World War II. Currently, SFSV is recognized as one of the most widespread phleboviruses, exhibiting high seroprevalence rates in humans and domestic animals and causing a self-limiting but incapacitating disease predominantly in immunologically naive troops and travelers. We show how the nonstructural NSs protein of SFSV counteracts the upregulation of the antiviral interferon (IFN) system. SFSV NSs specifically inhibits promoter binding by IFN transcription factor 3 (IRF3), a molecular strategy which is unique among phleboviruses and, to our knowledge, among human pathogenic RNA viruses in general. This IRF3-specific and stoichiometric mechanism, greatly distinct from the ones exhibited by the highly virulent phleboviruses, correlates with the intermediate level of pathogenicity of SFSV.
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http://dx.doi.org/10.1128/JVI.01202-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6232482PMC
December 2018

An orthogonal proteomic survey uncovers novel Zika virus host factors.

Nature 2018 09 3;561(7722):253-257. Epub 2018 Sep 3.

Max-Planck Institute of Biochemistry, Innate Immunity Laboratory, Martinsried, Germany.

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 newborns. 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.
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http://dx.doi.org/10.1038/s41586-018-0484-5DOI Listing
September 2018

Oxeiptosis-a cell death pathway to mitigate damage caused by radicals.

Cell Death Differ 2018 07;25(7):1191-1193

Immunopathology of Virus Infections, Institute of Virology, Technical University of Munich, Munich, Germany.

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http://dx.doi.org/10.1038/s41418-018-0134-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030169PMC
July 2018

Viral targeting of TFIIB impairs de novo polymerase II recruitment and affects antiviral immunity.

PLoS Pathog 2018 04 30;14(4):e1006980. Epub 2018 Apr 30.

Innate Immunity Laboratory, Max-Planck Institute of Biochemistry, Martinsried/Munich, Germany.

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.
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http://dx.doi.org/10.1371/journal.ppat.1006980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5927403PMC
April 2018

Oxeiptosis, a ROS-induced caspase-independent apoptosis-like cell-death pathway.

Nat Immunol 2018 02 18;19(2):130-140. Epub 2017 Dec 18.

Innate Immunity Laboratory, Max-Planck Institute of Biochemistry, Martinsried, Munich, Germany.

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.
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http://dx.doi.org/10.1038/s41590-017-0013-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5786482PMC
February 2018

Viperin Targets Flavivirus Virulence by Inducing Assembly of Noninfectious Capsid Particles.

J Virol 2018 01 14;92(1). Epub 2017 Dec 14.

Department of Clinical Microbiology, Virology, Umeå University, Umeå, Sweden

Efficient antiviral immunity requires interference with virus replication at multiple layers targeting diverse steps in the viral life cycle. We describe here a novel flavivirus inhibition mechanism that results in interferon-mediated obstruction of tick-borne encephalitis virus particle assembly and involves release of malfunctioning 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 knockdown and reduced by elevated levels of GBF1. Our results suggest that viperin targets flavivirus virulence by inducing the secretion of unproductive noninfectious virus particles via a GBF1-dependent mechanism. This as-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 the interference of flavivirus assembly. We show here that interferon, through the action of viperin, can disturb the 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 appears to vary between the different viruses, indicating that cellular targets utilized by several viruses might be involved. In this study, we show that viperin induces 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 is essential in the life cycle of many viruses, also targeted by viperin, implicating GBF1 as a novel putative drug target.
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http://dx.doi.org/10.1128/JVI.01751-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5730767PMC
January 2018

Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions.

J Virol 2017 09 10;91(17). Epub 2017 Aug 10.

School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom

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.
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http://dx.doi.org/10.1128/JVI.00805-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5553161PMC
September 2017

Molluscum Contagiosum Virus Protein MC005 Inhibits NF-κB Activation by Targeting NEMO-Regulated IκB Kinase Activation.

J Virol 2017 08 12;91(15). Epub 2017 Jul 12.

School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland

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.
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http://dx.doi.org/10.1128/JVI.00545-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5512260PMC
August 2017

Discrimination of Self and Non-Self Ribonucleic Acids.

J Interferon Cytokine Res 2017 05;37(5):184-197

Innate Immunity Laboratory, Max-Planck Institute of Biochemistry , Munich, Germany .

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.
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http://dx.doi.org/10.1089/jir.2016.0092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5439445PMC
May 2017

Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2'-O methylations.

Proc Natl Acad Sci U S A 2017 03 1;114(11):E2106-E2115. Epub 2017 Mar 1.

Department of Biochemistry and Groupe de Recherche Axe sur la Structure des Proteines, McGill University, Montreal, QC, Canada H3G 0B1;

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.
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http://dx.doi.org/10.1073/pnas.1612444114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5358387PMC
March 2017