Publications by authors named "Konstantin M J Sparrer"

28 Publications

  • Page 1 of 1

HIV protease: late action to prevent immune detection.

Signal Transduct Target Ther 2021 Apr 16;6(1):157. Epub 2021 Apr 16.

Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany.

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http://dx.doi.org/10.1038/s41392-021-00588-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8052432PMC
April 2021

Alpha-1 antitrypsin inhibits TMPRSS2 protease activity and SARS-CoV-2 infection.

Nat Commun 2021 03 19;12(1):1726. Epub 2021 Mar 19.

Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany.

SARS-CoV-2 is a respiratory pathogen and primarily infects the airway epithelium. As our knowledge about innate immune factors of the respiratory tract against SARS-CoV-2 is limited, we generated and screened a peptide/protein library derived from bronchoalveolar lavage for inhibitors of SARS-CoV-2 spike-driven entry. Analysis of antiviral fractions revealed the presence of α-antitrypsin (αAT), a highly abundant circulating serine protease inhibitor. Here, we report that αAT inhibits SARS-CoV-2 entry at physiological concentrations and suppresses viral replication in cell lines and primary cells including human airway epithelial cultures. We further demonstrate that αAT binds and inactivates the serine protease TMPRSS2, which enzymatically primes the SARS-CoV-2 spike protein for membrane fusion. Thus, the acute phase protein αAT is an inhibitor of TMPRSS2 and SARS-CoV-2 entry, and may play an important role in the innate immune defense against the novel coronavirus. Our findings suggest that repurposing of αAT-containing drugs has prospects for the therapy of COVID-19.
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http://dx.doi.org/10.1038/s41467-021-21972-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979852PMC
March 2021

SARS-CoV-2 infects and replicates in cells of the human endocrine and exocrine pancreas.

Nat Metab 2021 02 3;3(2):149-165. Epub 2021 Feb 3.

Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Neuherberg, Germany.

Infection-related diabetes can arise as a result of virus-associated β-cell destruction. Clinical data suggest that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the coronavirus disease 2019 (COVID-19), impairs glucose homoeostasis, but experimental evidence that SARS-CoV-2 can infect pancreatic tissue has been lacking. In the present study, we show that SARS-CoV-2 infects cells of the human exocrine and endocrine pancreas ex vivo and in vivo. We demonstrate that human β-cells express viral entry proteins, and SARS-CoV-2 infects and replicates in cultured human islets. Infection is associated with morphological, transcriptional and functional changes, including reduced numbers of insulin-secretory granules in β-cells and impaired glucose-stimulated insulin secretion. In COVID-19 full-body postmortem examinations, we detected SARS-CoV-2 nucleocapsid protein in pancreatic exocrine cells, and in cells that stain positive for the β-cell marker NKX6.1 and are in close proximity to the islets of Langerhans in all four patients investigated. Our data identify the human pancreas as a target of SARS-CoV-2 infection and suggest that β-cell infection could contribute to the metabolic dysregulation observed in patients with COVID-19.
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http://dx.doi.org/10.1038/s42255-021-00347-1DOI Listing
February 2021

RINT1 Regulates SUMOylation and the DNA Damage Response to Preserve Cellular Homeostasis in Pancreatic Cancer.

Cancer Res 2021 Apr 2;81(7):1758-1774. Epub 2021 Feb 2.

Department of Internal Medicine I, University Medical Centre Ulm, Ulm, Germany.

Pancreatic ductal adenocarcinoma (PDAC) still presents with a dismal prognosis despite intense research. Better understanding of cellular homeostasis could identify druggable targets to improve therapy. Here we propose RAD50-interacting protein 1 (RINT1) as an essential mediator of cellular homeostasis in PDAC. In a cohort of resected PDAC, low RINT1 protein expression correlated significantly with better survival. Accordingly, RINT1 depletion caused severe growth defects associated with accumulation of DNA double-strand breaks (DSB), G cell cycle arrest, disruption of Golgi-endoplasmic reticulum homeostasis, and cell death. Time-resolved transcriptomics corroborated by quantitative proteome and interactome analyses pointed toward defective SUMOylation after RINT1 loss, impairing nucleocytoplasmic transport and DSB response. Subcutaneous xenografts confirmed tumor response by RINT1 depletion, also resulting in a survival benefit when transferred to an orthotopic model. Primary human PDAC organoids licensed RINT1 relevance for cell viability. Taken together, our data indicate that RINT1 loss affects PDAC cell fate by disturbing SUMOylation pathways. Therefore, a RINT1 interference strategy may represent a new putative therapeutic approach. SIGNIFICANCE: These findings provide new insights into the aggressive behavior of PDAC, showing that RINT1 directly correlates with survival in patients with PDAC by disturbing the SUMOylation process, a crucial modification in carcinogenesis.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2633DOI Listing
April 2021

Natural cystatin C fragments inhibit GPR15-mediated HIV and SIV infection without interfering with GPR15L signaling.

Proc Natl Acad Sci U S A 2021 01;118(3)

Institute of Molecular Virology, Ulm University Medical Center, 89081 Ulm, Germany;

GPR15 is a G protein-coupled receptor (GPCR) proposed to play a role in mucosal immunity that also serves as a major entry cofactor for HIV-2 and simian immunodeficiency virus (SIV). To discover novel endogenous GPR15 ligands, we screened a hemofiltrate (HF)-derived peptide library for inhibitors of GPR15-mediated SIV infection. Our approach identified a C-terminal fragment of cystatin C (CysC95-146) that specifically inhibits GPR15-dependent HIV-1, HIV-2, and SIV infection. In contrast, GPR15L, the chemokine ligand of GPR15, failed to inhibit virus infection. We found that cystatin C fragments preventing GPR15-mediated viral entry do not interfere with GPR15L signaling and are generated by proteases activated at sites of inflammation. The antiretroviral activity of CysC95-146 was confirmed in primary CD4 T cells and is conserved in simian hosts of SIV infection. Thus, we identified a potent endogenous inhibitor of GPR15-mediated HIV and SIV infection that does not interfere with the physiological function of this GPCR.
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http://dx.doi.org/10.1073/pnas.2023776118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826402PMC
January 2021

Drug Inhibition of SARS-CoV-2 Replication in Human Pluripotent Stem Cell-Derived Intestinal Organoids.

Cell Mol Gastroenterol Hepatol 2021 10;11(4):935-948. Epub 2020 Nov 10.

Department of Internal Medicine I, Ulm University Hospital, Ulm, Germany. Electronic address:

Background And Aims: The COVID-19 pandemic has spread worldwide and poses a severe health risk. While most patients present mild symptoms, descending pneumonia can lead to severe respiratory insufficiency. Up to 50% of patients show gastrointestinal symptoms like diarrhea or nausea, intriguingly associating with prolonged symptoms and increased severity. Thus, models to understand and validate drug efficiency in the gut of COVID-19 patients are of urgent need.

Methods: Human intestinal organoids derived from pluripotent stem cells (PSC-HIOs) have led, due to their complexity in mimicking human intestinal architecture, to an unprecedented number of successful disease models including gastrointestinal infections. Here, we employed PSC-HIOs to dissect SARS-CoV-2 pathogenesis and its inhibition by remdesivir, one of the leading drugs investigated for treatment of COVID-19.

Results: Immunostaining for viral entry receptor ACE2 and SARS-CoV-2 spike protein priming protease TMPRSS2 showed broad expression in the gastrointestinal tract with highest levels in the intestine, the latter faithfully recapitulated by PSC-HIOs. Organoids could be readily infected with SARS-CoV-2 followed by viral spread across entire PSC-HIOs, subsequently leading to organoid deterioration. However, SARS-CoV-2 spared goblet cells lacking ACE2 expression. Importantly, we challenged PSC-HIOs for drug testing capacity. Specifically, remdesivir effectively inhibited SARS-CoV-2 infection dose-dependently at low micromolar concentration and rescued PSC-HIO morphology.

Conclusions: Thus, PSC-HIOs are a valuable tool to study SARS-CoV-2 infection and to identify and validate drugs especially with potential action in the gut.
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http://dx.doi.org/10.1016/j.jcmgh.2020.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7655023PMC
April 2021

SARS-CoV-2 Is Restricted by Zinc Finger Antiviral Protein despite Preadaptation to the Low-CpG Environment in Humans.

mBio 2020 10 16;11(5). Epub 2020 Oct 16.

Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany

Recent evidence shows that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is sensitive to interferons (IFNs). However, the most effective types of IFNs and the underlying antiviral effectors remain to be defined. Here, we show that zinc finger antiviral protein (ZAP), which preferentially targets CpG dinucleotides in viral RNA sequences, restricts SARS-CoV-2. We further demonstrate that ZAP and its cofactors KHNYN and TRIM25 are expressed in human lung cells. Type I, II, and III IFNs all strongly inhibited SARS-CoV-2 and further induced ZAP expression. Comprehensive sequence analyses revealed that SARS-CoV-2 and its closest relatives from horseshoe bats showed the strongest CpG suppression among all known human and bat coronaviruses, respectively. Nevertheless, endogenous ZAP expression restricted SARS-CoV-2 replication in human lung cells, particularly upon treatment with IFN-α or IFN-γ. Both the long and the short isoforms of human ZAP reduced SARS-CoV-2 RNA expression levels, but the former did so with greater efficiency. Finally, we show that the ability to restrict SARS-CoV-2 is conserved in ZAP orthologues of the reservoir bat and potential intermediate pangolin hosts of human coronaviruses. Altogether, our results show that ZAP is an important effector of the innate response against SARS-CoV-2, although this pandemic pathogen emerged from zoonosis of a coronavirus that was preadapted to the low-CpG environment in humans. Although interferons inhibit SARS-CoV-2 and have been evaluated for treatment of coronavirus disease 2019 (COVID-19), the most effective types and antiviral effectors remain to be defined. Here, we show that IFN-γ is particularly potent in restricting SARS-CoV-2 and in inducing expression of the antiviral factor ZAP in human lung cells. Knockdown experiments revealed that endogenous ZAP significantly restricts SARS-CoV-2. We further show that CpG dinucleotides which are specifically targeted by ZAP are strongly suppressed in the SARS-CoV-2 genome and that the two closest horseshoe bat relatives of SARS-CoV-2 show the lowest genomic CpG content of all coronavirus sequences available from this reservoir host. Nonetheless, both the short and long isoforms of human ZAP reduced SARS-CoV-2 RNA levels, and this activity was conserved in horseshoe bat and pangolin ZAP orthologues. Our findings indicating that type II interferon is particularly efficient against SARS-CoV-2 and that ZAP restricts this pandemic viral pathogen might promote the development of effective immune therapies against COVID-19.
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http://dx.doi.org/10.1128/mBio.01930-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7569149PMC
October 2020

HIV-1 infection activates endogenous retroviral promoters regulating antiviral gene expression.

Nucleic Acids Res 2020 11;48(19):10890-10908

Institute of Molecular Virology, Ulm University Medical Center, Ulm 89081, Germany.

Although endogenous retroviruses (ERVs) are known to harbor cis-regulatory elements, their role in modulating cellular immune responses remains poorly understood. Using an RNA-seq approach, we show that several members of the ERV9 lineage, particularly LTR12C elements, are activated upon HIV-1 infection of primary CD4+ T cells. Intriguingly, HIV-1-induced ERVs harboring transcription start sites are primarily found in the vicinity of immunity genes. For example, HIV-1 infection activates LTR12C elements upstream of the interferon-inducible genes GBP2 and GBP5 that encode for broad-spectrum antiviral factors. Reporter assays demonstrated that these LTR12C elements drive gene expression in primary CD4+ T cells. In line with this, HIV-1 infection triggered the expression of a unique GBP2 transcript variant by activating a cryptic transcription start site within LTR12C. Furthermore, stimulation with HIV-1-induced cytokines increased GBP2 and GBP5 expression in human cells, but not in macaque cells that naturally lack the GBP5 gene and the LTR12C element upstream of GBP2. Finally, our findings suggest that GBP2 and GBP5 have already been active against ancient viral pathogens as they suppress the maturation of the extinct retrovirus HERV-K (HML-2). In summary, our findings uncover how human cells can exploit remnants of once-infectious retroviruses to regulate antiviral gene expression.
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http://dx.doi.org/10.1093/nar/gkaa832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641743PMC
November 2020

Nuclear PYHIN proteins target the host transcription factor Sp1 thereby restricting HIV-1 in human macrophages and CD4+ T cells.

PLoS Pathog 2020 08 6;16(8):e1008752. Epub 2020 Aug 6.

Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany.

Members of the family of pyrin and HIN domain containing (PYHIN) proteins play an emerging role in innate immunity. While absent in melanoma 2 (AIM2) acts a cytosolic sensor of non-self DNA and plays a key role in inflammasome assembly, the γ-interferon-inducible protein 16 (IFI16) restricts retroviral gene expression by sequestering the transcription factor Sp1. Here, we show that the remaining two human PYHIN proteins, i.e. myeloid cell nuclear differentiation antigen (MNDA) and pyrin and HIN domain family member 1 (PYHIN1 or IFIX) share this antiretroviral function of IFI16. On average, knock-down of each of these three nuclear PYHIN proteins increased infectious HIV-1 yield from human macrophages by more than an order of magnitude. Similarly, knock-down of IFI16 strongly increased virus transcription and production in primary CD4+ T cells. The N-terminal pyrin domain (PYD) plus linker region containing a nuclear localization signal (NLS) were generally required and sufficient for Sp1 sequestration and anti-HIV-1 activity of IFI16, MNDA and PYHIN1. Replacement of the linker region of AIM2 by the NLS-containing linker of IFI16 resulted in a predominantly nuclear localization and conferred direct antiviral activity to AIM2 while attenuating its ability to form inflammasomes. The reverse change caused nuclear-to-cytoplasmic relocalization of IFI16 and impaired its antiretroviral activity but did not result in inflammasome assembly. We further show that the Zn-finger domain of Sp1 is critical for the interaction with IFI16 supporting that pyrin domains compete with DNA for Sp1 binding. Finally, we found that human PYHIN proteins also inhibit Hepatitis B virus and simian vacuolating virus 40 as well as the LINE-1 retrotransposon. Altogether, our data show that IFI16, PYHIN1 and MNDA restrict HIV-1 and other viral pathogens by interfering with Sp1-dependent gene expression and support an important role of nuclear PYHIN proteins in innate antiviral immunity.
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http://dx.doi.org/10.1371/journal.ppat.1008752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7433898PMC
August 2020

An enzyme-based immunodetection assay to quantify SARS-CoV-2 infection.

Antiviral Res 2020 09 29;181:104882. Epub 2020 Jul 29.

Institute of Molecular Virology, Ulm University Medical Center, 89081, Ulm, Germany. Electronic address:

SARS-CoV-2 is a novel pandemic coronavirus that caused a global health and economic crisis. The development of efficient drugs and vaccines against COVID-19 requires detailed knowledge about SARS-CoV-2 biology. Several techniques to detect SARS-CoV-2 infection have been established, mainly based on counting infected cells by staining plaques or foci, or by quantifying the viral genome by PCR. These methods are laborious, time-consuming and expensive and therefore not suitable for a high sample throughput or rapid diagnostics. We here report a novel enzyme-based immunodetection assay that directly quantifies the amount of de novo synthesized viral spike protein within fixed and permeabilized cells. This in-cell ELISA enables a rapid and quantitative detection of SARS-CoV-2 infection in microtiter format, regardless of the virus isolate or target cell culture. It follows the established method of performing ELISA assays and does not require expensive instrumentation. Utilization of the in-cell ELISA allows to e.g. determine TCID of virus stocks, antiviral efficiencies (IC values) of drugs or neutralizing activity of sera. Thus, the in-cell spike ELISA represents a promising alternative to study SARS-CoV-2 infection and inhibition and may facilitate future research.
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http://dx.doi.org/10.1016/j.antiviral.2020.104882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7388004PMC
September 2020

An improved method for high-throughput quantification of autophagy in mammalian cells.

Sci Rep 2020 07 22;10(1):12241. Epub 2020 Jul 22.

Institute of Molecular Virology, Ulm University Medical Center, 89081, Ulm, Germany.

Autophagy is a cellular homeostatic pathway with functions ranging from cytoplasmic protein turnover to immune defense. Therapeutic modulation of autophagy has been demonstrated to positively impact the outcome of autophagy-dysregulated diseases such as cancer or microbial infections. However, currently available agents lack specificity, and new candidates for drug development or potential cellular targets need to be identified. Here, we present an improved method to robustly detect changes in autophagy in a high-throughput manner on a single cell level, allowing effective screening. This method quantifies eGFP-LC3B positive vesicles to accurately monitor autophagy. We have significantly streamlined the protocol and optimized it for rapid quantification of large numbers of cells in little time, while retaining accuracy and sensitivity. Z scores up to 0.91 without a loss of sensitivity demonstrate the robustness and aptness of this approach. Three exemplary applications outline the value of our protocols and cell lines: (I) Examining autophagy modulating compounds on four different cell types. (II) Monitoring of autophagy upon infection with e.g. measles or influenza A virus. (III) CRISPR/Cas9 screening for autophagy modulating factors in T cells. In summary, we offer ready-to-use protocols to generate sensitive autophagy reporter cells and quantify autophagy in high-throughput assays.
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http://dx.doi.org/10.1038/s41598-020-68607-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376206PMC
July 2020

Vpu modulates DNA repair to suppress innate sensing and hyper-integration of HIV-1.

Nat Microbiol 2020 10 20;5(10):1247-1261. Epub 2020 Jul 20.

Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany.

To avoid innate sensing and immune control, human immunodeficiency virus type 1 (HIV-1) has to prevent the accumulation of viral complementary DNA species. Here, we show that the late HIV-1 accessory protein Vpu hijacks DNA repair mechanisms to promote degradation of nuclear viral cDNA in cells that are already productively infected. Vpu achieves this by interacting with RanBP2-RanGAP1*SUMO1-Ubc9 SUMO E3-ligase complexes at the nuclear pore to reprogramme promyelocytic leukaemia protein nuclear bodies and reduce SUMOylation of Bloom syndrome protein, unleashing end degradation of viral cDNA. Concomitantly, Vpu inhibits RAD52-mediated homologous repair of viral cDNA, preventing the generation of dead-end circular forms of single copies of the long terminal repeat and permitting sustained nucleolytic attack. Our results identify Vpu as a key modulator of the DNA repair machinery. We show that Bloom syndrome protein eliminates nuclear HIV-1 cDNA and thereby suppresses immune sensing and proviral hyper-integration. Therapeutic targeting of DNA repair may facilitate the induction of antiviral immunity and suppress proviral integration replenishing latent HIV reservoirs.
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http://dx.doi.org/10.1038/s41564-020-0753-6DOI Listing
October 2020

Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2.

Science 2020 09 17;369(6508):1249-1255. Epub 2020 Jul 17.

Gene Center Munich, Department of Biochemistry, University of Munich, Munich, Germany.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. A major virulence factor of SARS-CoVs is the nonstructural protein 1 (Nsp1), which suppresses host gene expression by ribosome association. Here, we show that Nsp1 from SARS-CoV-2 binds to the 40 ribosomal subunit, resulting in shutdown of messenger RNA (mRNA) translation both in vitro and in cells. Structural analysis by cryo-electron microscopy of in vitro-reconstituted Nsp1-40 and various native Nsp1-40 and -80 complexes revealed that the Nsp1 C terminus binds to and obstructs the mRNA entry tunnel. Thereby, Nsp1 effectively blocks retinoic acid-inducible gene I-dependent innate immune responses that would otherwise facilitate clearance of the infection. Thus, the structural characterization of the inhibitory mechanism of Nsp1 may aid structure-based drug design against SARS-CoV-2.
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http://dx.doi.org/10.1126/science.abc8665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402621PMC
September 2020

CpG Frequency in the 5' Third of the Gene Determines Sensitivity of Primary HIV-1 Strains to the Zinc-Finger Antiviral Protein.

mBio 2020 01 14;11(1). Epub 2020 Jan 14.

Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany

CpG dinucleotide suppression has been reported to allow HIV-1 to evade inhibition by the zinc-finger antiviral protein (ZAP). Here, we show that primate lentiviruses display marked differences in CpG frequencies across their genome, ranging from 0.44% in simian immunodeficiency virus SIVwrc from Western red colobus to 2.3% in SIVmon infecting mona monkeys. Moreover, functional analyses of a large panel of human and simian immunodeficiency viruses revealed that the magnitude of CpG suppression does not correlate with their susceptibility to ZAP. However, we found that the number of CpG dinucleotides within a region of ∼700 bases at the 5' end of the gene determines ZAP sensitivity of primary HIV-1 strains but not of HIV-2. Increased numbers of CpGs in this region were associated with reduced mRNA expression and viral protein production. ZAP sensitivity profiles of chimeric simian-human immunodeficiency viruses (SHIVs) expressing different HIV-1 genes were highly similar to those of the corresponding HIV-1 strains. The frequency of CpGs in the identified region correlated with differences in clinical progression rates. Thus, the CpG frequency in a specific part of , rather than the overall genomic CpG content, governs the susceptibility of HIV-1 to ZAP and might affect viral pathogenicity Evasion of the zinc-finger antiviral protein (ZAP) may drive CpG dinucleotide suppression in HIV-1 and many other viral pathogens but the viral determinants of ZAP sensitivity are poorly defined. Here, we examined CpG suppression and ZAP sensitivity in a large number of primate lentiviruses and demonstrate that their genomic frequency of CpGs varies substantially and does not correlate with ZAP sensitivity. We further show that the number of CpG residues in a defined region at the 5' end of the gene together with structural features plays a key role in HIV-1 susceptibility to ZAP and correlates with differences in clinical progression rates in HIV-1-infected individuals. Our identification of a specific part of as a major determinant of HIV-1 susceptibility to ZAP restriction provides a basis for future studies of the underlying inhibitory mechanisms and their potential relevance in the pathogenesis of AIDS.
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http://dx.doi.org/10.1128/mBio.02903-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6960287PMC
January 2020

Guanylate-Binding Proteins 2 and 5 Exert Broad Antiviral Activity by Inhibiting Furin-Mediated Processing of Viral Envelope Proteins.

Cell Rep 2019 05;27(7):2092-2104.e10

Institute of Molecular Virology, Ulm University Medical Center, Ulm 89081, Germany. Electronic address:

Guanylate-binding protein (GBP) 5 is an interferon (IFN)-inducible cellular factor reducing HIV-1 infectivity by an incompletely understood mechanism. Here, we show that this activity is shared by GBP2, but not by other members of the human GBP family. GBP2/5 decrease the activity of the cellular proprotein convertase furin, which mediates conversion of the HIV-1 envelope protein (Env) precursor gp160 into mature gp120 and gp41. Because this process primes HIV-1 Env for membrane fusion, viral particles produced in the presence of GBP2/5 are poorly infectious due to increased incorporation of non-functional gp160. Furin activity is critical for the processing of envelope glycoproteins of many viral pathogens. Consistently, GBP2/5 also inhibit Zika, measles, and influenza A virus replication and decrease infectivity of viral particles carrying glycoproteins of Marburg and murine leukemia viruses. Collectively, our results show that GPB2/5 exert broad antiviral activity by suppressing the activity of the virus-dependency factor furin.
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http://dx.doi.org/10.1016/j.celrep.2019.04.063DOI Listing
May 2019

Centrosomal protein TRIM43 restricts herpesvirus infection by regulating nuclear lamina integrity.

Nat Microbiol 2019 01 12;4(1):164-176. Epub 2018 Nov 12.

Department of Microbiology, The University of Chicago, Chicago, IL, USA.

Tripartite motif (TRIM) proteins mediate antiviral host defences by either directly targeting viral components or modulating innate immune responses. Here we identify a mechanism of antiviral restriction in which a TRIM E3 ligase controls viral replication by regulating the structure of host cell centrosomes and thereby nuclear lamina integrity. Through RNAi screening we identified several TRIM proteins, including TRIM43, that control the reactivation of Kaposi's sarcoma-associated herpesvirus. TRIM43 was distinguished by its ability to restrict a broad range of herpesviruses and its profound upregulation during herpesvirus infection as part of a germline-specific transcriptional program mediated by the transcription factor DUX4. TRIM43 ubiquitinates the centrosomal protein pericentrin, thereby targeting it for proteasomal degradation, which subsequently leads to alterations of the nuclear lamina that repress active viral chromatin states. Our study identifies a role of the TRIM43-pericentrin-lamin axis in intrinsic immunity, which may be targeted for therapeutic intervention against herpesviral infections.
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http://dx.doi.org/10.1038/s41564-018-0285-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294671PMC
January 2019

TRIM25 Binds RNA to Modulate Cellular Anti-viral Defense.

J Mol Biol 2018 12 17;430(24):5280-5293. Epub 2018 Oct 17.

Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, USA. Electronic address:

TRIM25 is a multi-domain, RING-type E3 ubiquitin ligase of the tripartite motif family that has important roles in multiple RNA-dependent processes. In particular, TRIM25 functions as an effector of RIG-I and ZAP, which are innate immune sensors that recognize viral RNA and induce ubiquitin-dependent anti-viral response mechanisms. TRIM25 is reported to also bind RNA, but the molecular details of this interaction or its relevance to anti-viral defense have not been elucidated. Here, we characterize the RNA-binding activity of TRIM25 and find that the protein binds both single-stranded and double-stranded RNA. Multiple regions of TRIM25 contribute to this functionality, including the C-terminal SPRY domain and a lysine-rich motif in the linker segment connecting the SPRY and coiled-coil domains. RNA binding modulates TRIM25's ubiquitination activity in vitro, its localization in cells, and its anti-viral activity. Taken together with other studies, our results indicate that RNA binding by TRIM25 has at least three important functional consequences: by enhancing ubiquitination activity, either through allosteric effects or through clustering of multiple TRIM25 molecules; by modulating the multi-domain structure of the TRIM25 dimer, and thereby structural coupling of the SPRY and RBCC elements during the ubiquitination reaction; and by facilitating subcellular localization of the E3 ligase during virus infection.
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http://dx.doi.org/10.1016/j.jmb.2018.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6289755PMC
December 2018

SIVcol Nef counteracts SERINC5 by promoting its proteasomal degradation but does not efficiently enhance HIV-1 replication in human CD4+ T cells and lymphoid tissue.

PLoS Pathog 2018 08 20;14(8):e1007269. Epub 2018 Aug 20.

Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany.

SERINC5 is a host restriction factor that impairs infectivity of HIV-1 and other primate lentiviruses and is counteracted by the viral accessory protein Nef. However, the importance of SERINC5 antagonism for viral replication and cytopathicity remained unclear. Here, we show that the Nef protein of the highly divergent SIVcol lineage infecting mantled guerezas (Colobus guereza) is a potent antagonist of SERINC5, although it lacks the CD4, CD3 and CD28 down-modulation activities exerted by other primate lentiviral Nefs. In addition, SIVcol Nefs decrease CXCR4 cell surface expression, suppress TCR-induced actin remodeling, and counteract Colobus but not human tetherin. Unlike HIV-1 Nef proteins, SIVcol Nef induces efficient proteasomal degradation of SERINC5 and counteracts orthologs from highly divergent vertebrate species, such as Xenopus frogs and zebrafish. A single Y86F mutation disrupts SERINC5 and tetherin antagonism but not CXCR4 down-modulation by SIVcol Nef, while mutation of a C-proximal di-leucine motif has the opposite effect. Unexpectedly, the Y86F change in SIVcol Nef had little if any effect on viral replication and CD4+ T cell depletion in preactivated human CD4+ T cells and in ex vivo infected lymphoid tissue. However, SIVcol Nef increased virion infectivity up to 10-fold and moderately increased viral replication in resting peripheral blood mononuclear cells (PBMCs) that were first infected with HIV-1 and activated three or six days later. In conclusion, SIVcol Nef lacks several activities that are conserved in other primate lentiviruses and utilizes a distinct proteasome-dependent mechanism to counteract SERINC5. Our finding that evolutionarily distinct SIVcol Nefs show potent anti-SERINC5 activity supports a relevant role of SERINC5 antagonism for viral fitness in vivo. Our results further suggest this Nef function is particularly important for virion infectivity under conditions of limited CD4+ T cell activation.
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http://dx.doi.org/10.1371/journal.ppat.1007269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6117100PMC
August 2018

TRIM Proteins and Their Roles in Antiviral Host Defenses.

Annu Rev Virol 2018 09 27;5(1):385-405. Epub 2018 Jun 27.

Department of Microbiology, The University of Chicago, Chicago, Illinois 60637, USA; email: , ,

Tripartite motif (TRIM) proteins are a versatile family of ubiquitin E3 ligases involved in a multitude of cellular processes. Studies in recent years have demonstrated that many TRIM proteins play central roles in the host defense against viral infection. While some TRIM proteins directly antagonize distinct steps in the viral life cycle, others regulate signal transduction pathways induced by innate immune sensors, thereby modulating antiviral cytokine responses. Furthermore, TRIM proteins have been implicated in virus-induced autophagy and autophagy-mediated viral clearance. Given the important role of TRIM proteins in antiviral restriction, it is not surprising that several viruses have evolved effective maneuvers to neutralize the antiviral action of specific TRIM proteins. Here, we describe the major antiviral mechanisms of TRIM proteins as well as viral strategies to escape TRIM-mediated host immunity.
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http://dx.doi.org/10.1146/annurev-virology-092917-043323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6186430PMC
September 2018

TRIM proteins: New players in virus-induced autophagy.

PLoS Pathog 2018 02 1;14(2):e1006787. Epub 2018 Feb 1.

Department of Microbiology, University of Chicago, Chicago, Illinois, United States of America.

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http://dx.doi.org/10.1371/journal.ppat.1006787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5794181PMC
February 2018

Viral unmasking of cellular 5S rRNA pseudogene transcripts induces RIG-I-mediated immunity.

Nat Immunol 2018 Jan 27;19(1):53-62. Epub 2017 Nov 27.

Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA.

The sensor RIG-I detects double-stranded RNA derived from RNA viruses. Although RIG-I is also known to have a role in the antiviral response to DNA viruses, physiological RNA species recognized by RIG-I during infection with a DNA virus are largely unknown. Using next-generation RNA sequencing (RNAseq), we found that host-derived RNAs, most prominently 5S ribosomal RNA pseudogene 141 (RNA5SP141), bound to RIG-I during infection with herpes simplex virus 1 (HSV-1). Infection with HSV-1 induced relocalization of RNA5SP141 from the nucleus to the cytoplasm, and virus-induced shutoff of host protein synthesis downregulated the abundance of RNA5SP141-interacting proteins, which allowed RNA5SP141 to bind RIG-I and induce the expression of type I interferons. Silencing of RNA5SP141 strongly dampened the antiviral response to HSV-1 and the related virus Epstein-Barr virus (EBV), as well as influenza A virus (IAV). Our findings reveal that antiviral immunity can be triggered by host RNAs that are unshielded following depletion of their respective binding proteins by the virus.
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http://dx.doi.org/10.1038/s41590-017-0005-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5815369PMC
January 2018

TRIM23 mediates virus-induced autophagy via activation of TBK1.

Nat Microbiol 2017 Nov 4;2(11):1543-1557. Epub 2017 Sep 4.

Department of Microbiology, The University of Chicago, Chicago, IL, 60637, USA.

Autophagy and interferon (IFN)-mediated innate immunity are critical antiviral defence mechanisms, and recent evidence indicated that tripartite motif (TRIM) proteins are important regulators of both processes. Although the role of TRIM proteins in modulating antiviral cytokine responses has been well established, much less is known about their involvement in autophagy in response to different viral pathogens. Through a targeted RNAi screen examining the relevance of selected TRIM proteins in autophagy induced by herpes simplex virus 1 (HSV-1), encephalomyocarditis virus (EMCV) and influenza A virus (IAV), we identified several TRIM proteins that regulate autophagy in a virus-species-specific manner, as well as a few TRIM proteins that were essential for autophagy triggered by all three viruses and rapamycin, among them TRIM23. TRIM23 was critical for autophagy-mediated restriction of multiple viruses, and this activity was dependent on both its RING E3 ligase and ADP-ribosylation factor (ARF) GTPase activity. Mechanistic studies revealed that unconventional K27-linked auto-ubiquitination of the ARF domain is essential for the GTP hydrolysis activity of TRIM23 and activation of TANK-binding kinase 1 (TBK1) by facilitating its dimerization and ability to phosphorylate the selective autophagy receptor p62. Our work identifies the TRIM23-TBK1-p62 axis as a key component of selective autophagy and further reveals a role for K27-linked ubiquitination in GTPase-dependent TBK1 activation.
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http://dx.doi.org/10.1038/s41564-017-0017-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5658249PMC
November 2017

Mechanism of TRIM25 Catalytic Activation in the Antiviral RIG-I Pathway.

Cell Rep 2016 08 14;16(5):1315-1325. Epub 2016 Jul 14.

Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908, USA. Electronic address:

Antiviral response pathways induce interferon by higher-order assembly of signaling complexes called signalosomes. Assembly of the RIG-I signalosome is regulated by K63-linked polyubiquitin chains, which are synthesized by the E3 ubiquitin ligase, TRIM25. We have previously shown that the TRIM25 coiled-coil domain is a stable, antiparallel dimer that positions two catalytic RING domains on opposite ends of an elongated rod. We now show that the RING domain is a separate self-association motif that engages ubiquitin-conjugated E2 enzymes as a dimer. RING dimerization is required for catalysis, TRIM25-mediated RIG-I ubiquitination, interferon induction, and antiviral activity. We also provide evidence that RING dimerization and E3 ligase activity are promoted by binding of the TRIM25 SPRY domain to the RIG-I effector domain. These results indicate that TRIM25 actively participates in higher-order assembly of the RIG-I signalosome and helps to fine-tune the efficiency of the RIG-I-mediated antiviral response.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5076470PMC
http://dx.doi.org/10.1016/j.celrep.2016.06.070DOI Listing
August 2016

ATP hydrolysis by the viral RNA sensor RIG-I prevents unintentional recognition of self-RNA.

Elife 2015 Nov 26;4. Epub 2015 Nov 26.

Gene Center, Department of Biochemistry, Ludwig Maximilian University of Munich, Munich, Germany.

The cytosolic antiviral innate immune sensor RIG-I distinguishes 5' tri- or diphosphate containing viral double-stranded (ds) RNA from self-RNA by an incompletely understood mechanism that involves ATP hydrolysis by RIG-I's RNA translocase domain. Recently discovered mutations in ATPase motifs can lead to the multi-system disorder Singleton-Merten Syndrome (SMS) and increased interferon levels, suggesting misregulated signaling by RIG-I. Here we report that SMS mutations phenocopy a mutation that allows ATP binding but prevents hydrolysis. ATPase deficient RIG-I constitutively signals through endogenous RNA and co-purifies with self-RNA even from virus infected cells. Biochemical studies and cryo-electron microscopy identify a 60S ribosomal expansion segment as a dominant self-RNA that is stably bound by ATPase deficient RIG-I. ATP hydrolysis displaces wild-type RIG-I from this self-RNA but not from 5' triphosphate dsRNA. Our results indicate that ATP-hydrolysis prevents recognition of self-RNA and suggest that SMS mutations lead to unintentional signaling through prolonged RNA binding.
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http://dx.doi.org/10.7554/eLife.10859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4733034PMC
November 2015

Intracellular detection of viral nucleic acids.

Curr Opin Microbiol 2015 Aug 18;26:1-9. Epub 2015 Mar 18.

Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Successful clearance of a microbial infection depends on the concerted action of both the innate and adaptive arms of the immune system. Accurate recognition of an invading pathogen is the first and most crucial step in eliciting effective antimicrobial defense mechanisms. In recent years, remarkable progress has been made towards understanding the molecular details of how the innate immune system recognizes microbial signatures, commonly called pathogen-associated molecular patterns (PAMPs). For viral pathogens, nucleic acids-both viral genomes and viral replication products-represent a major class of PAMPs that trigger antiviral host responses via activation of germline-encoded innate immune receptors. Here we summarize recent advances in intracellular innate sensing mechanisms of viral RNA and DNA.
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http://dx.doi.org/10.1016/j.mib.2015.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5084527PMC
August 2015

Complete Genome Sequence of a Wild-Type Measles Virus Isolated during the Spring 2013 Epidemic in Germany.

Genome Announc 2014 Apr 17;2(2). Epub 2014 Apr 17.

Max von Pettenkofer-Institute, Ludwig-Maximilians-University, Munich, Germany.

Measles virus induces an acute disease with rash and fever. Despite ongoing vaccination and elimination campaigns, the measles virus still sustains long-lasting transmission chains in Europe. Here we report the complete genome sequence of a wild-type measles virus isolated from a patient in Munich (MVi/Muenchen.DEU/19.13[D8]) during a German measles outbreak in 2013.
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http://dx.doi.org/10.1128/genomeA.00157-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3990739PMC
April 2014

In vivo ligands of MDA5 and RIG-I in measles virus-infected cells.

PLoS Pathog 2014 Apr 17;10(4):e1004081. Epub 2014 Apr 17.

Gene Center and Department of Biochemistry, Ludwig-Maximilians University Munich, Munich, Germany; Center for Integrated Protein Science Munich, Munich, Germany.

RIG-I-like receptors (RLRs: RIG-I, MDA5 and LGP2) play a major role in the innate immune response against viral infections and detect patterns on viral RNA molecules that are typically absent from host RNA. Upon RNA binding, RLRs trigger a complex downstream signaling cascade resulting in the expression of type I interferons and proinflammatory cytokines. In the past decade extensive efforts were made to elucidate the nature of putative RLR ligands. In vitro and transfection studies identified 5'-triphosphate containing blunt-ended double-strand RNAs as potent RIG-I inducers and these findings were confirmed by next-generation sequencing of RIG-I associated RNAs from virus-infected cells. The nature of RNA ligands of MDA5 is less clear. Several studies suggest that double-stranded RNAs are the preferred agonists for the protein. However, the exact nature of physiological MDA5 ligands from virus-infected cells needs to be elucidated. In this work, we combine a crosslinking technique with next-generation sequencing in order to shed light on MDA5-associated RNAs from human cells infected with measles virus. Our findings suggest that RIG-I and MDA5 associate with AU-rich RNA species originating from the mRNA of the measles virus L gene. Corresponding sequences are poorer activators of ATP-hydrolysis by MDA5 in vitro, suggesting that they result in more stable MDA5 filaments. These data provide a possible model of how AU-rich sequences could activate type I interferon signaling.
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http://dx.doi.org/10.1371/journal.ppat.1004081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3990713PMC
April 2014

Measles virus C protein interferes with Beta interferon transcription in the nucleus.

J Virol 2012 Jan 9;86(2):796-805. Epub 2011 Nov 9.

Max von Pettenkofer-Institute and Gene Center, Ludwig-Maximilians-University Munich, Munich, Germany.

Transcriptional induction of beta interferon (IFN-β) through pattern recognition receptors is a key event in the host defense against invading viruses. Infection of cells by paramyxoviruses, like measles virus (MV) (genus Morbillivirus), is sensed predominantly by the ubiquitous cytoplasmic helicase RIG-I, recognizing viral 5'-triphosphate RNAs, and to some degree by MDA5. While MDA5 activation is effectively prevented by the MV V protein, the viral mechanisms for inhibition of MDA5-independent induction of IFN-β remained obscure. Here, we identify the 186-amino-acid MV C protein, which shuttles between the nucleus and the cytoplasm, as a major viral inhibitor of IFN-β transcription in human cells. Activation of the transcription factor IRF3 by upstream kinases and nuclear import of activated IRF3 were not affected in the presence of C protein, suggesting a nuclear target. Notably, C proteins of wild-type MV isolates, which are poor IFN-β inducers, were found to comprise a canonical nuclear localization signal (NLS), whereas the NLSs of all vaccine strains, irrespective of their origins, were mutated. Site-directed mutagenesis of the C proteins from an MV wild-type isolate and from the vaccine virus strain Schwarz confirmed a correlation of nuclear localization and inhibition of IFN-β transcription. A functional NLS and efficient nuclear accumulation are therefore critical for MV C to retain its potential to downregulate IFN-β induction. We suggest that a defect in efficient nuclear import of C protein contributes to attenuation of MV vaccine strains.
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http://dx.doi.org/10.1128/JVI.05899-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3255862PMC
January 2012