Biology subjects

Thiel, V.

Publications and source records attributed to Thiel, V..

At least 37 records · Page 2Linked to original sources

bioRxiv2021

Post-entry, spike-dependent replication advantage of B.1.1.7 and B.1.617.2 over B.1 SARS-CoV-2 in an ACE2-deficient human lung cell line

Epidemiological data demonstrate that SARS-CoV-2 variants of concern (VOC) B.1.1.7 and B.1.617.2 are more transmissible and infections are associated with a higher mortality than non-VOC virus infections. Phenotypic properties underlying their enhanced spread in the human population remain unknown. B.1.1.7 virus isolates displayed inferior or equivalent spread in most cell lines and primary cells compared to an ancestral B.1 SARS-CoV-2, and were outcompeted by the latter. Lower infectivity and delayed entry kinetics of B.1.1.7 viruses were accompanied by inefficient proteolytic processing of spike. B.1.1.7 viruses failed to escape from neutralizing antibodies, but slightly dampened induction of innate immunity. The bronchial cell line NCI-H1299 supported 24- and 595-fold increased growth of B.1.1.7 and B.1.617.2 viruses, respectively, in the absence of detectable ACE2 expression and in a spike-determined fashion. Superior spread in NCI-H1299 cells suggests that VOCs employ a distinct set of cellular cofactors that may be unavailable in standard cell lines.

microbiology↗

bioRxiv2021

Recombinant Lloviu virus as a model to study inaccessible zoonotic viruses

Next generation sequencing has revealed the presence of many RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, many of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Recombinant LLOV (rLLOV) displays typical filovirus features, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal against pandemic preparedness.

microbiology↗

bioRxiv2021

Enhanced fitness of SARS-CoV-2 variant of concern B.1.1.7, but not B.1.351, in animal models

Emerging variants of concern (VOCs) drive the SARS-CoV-2 pandemic. We assessed VOC B.1.1.7, now prevalent in several countries, and VOC B.1.351, representing the greatest threat to populations with immunity to the early SARS-CoV-2 progenitors. B.1.1.7 showed a clear fitness advantage over the progenitor variant (wt-S614G) in ferrets and two mouse models, where the substitutions in the spike glycoprotein were major drivers for fitness advantage. In the "superspreader" hamster model, B.1.1.7 and wt-S614G had comparable fitness, whereas B.1.351 was outcompeted. The VOCs had similar replication kinetics as compared to wt-S614G in human airway epithelial cultures. Our study highlights the importance of using multiple models for complete fitness characterization of VOCs and demonstrates adaptation of B.1.1.7 towards increased upper respiratory tract replication and enhanced transmission in vivo. Summary sentenceB.1.1.7 VOC outcompetes progenitor SARS-CoV-2 in upper respiratory tract replication competition in vivo.

microbiology↗

bioRxiv2021

Effective interferon (IFN)-lambda treatment regimen to control lethal MERS-CoV infection in mice

Effective broad-spectrum antivirals are critical to prevent and control emerging human coronavirus (hCoV) infections. Despite considerable progress made towards identifying and evaluating several synthetic broad-spectrum antivirals against hCoV infections, a narrow therapeutic window has limited their success. Enhancing the endogenous interferon (IFN) and interferon-stimulated gene (ISG) response is another antiviral strategy known for decades. However, the side effects of pegylated type-I IFNs (IFN-Is) and the pro-inflammatory response detected after delayed IFN-I therapy have discouraged their clinical use. In contrast to IFN-Is, IFN-{lambda}, a dominant IFN at the epithelial surface, is shown to be less pro-inflammatory. Consequently, we evaluated the prophylactic and therapeutic efficacy of IFN-{lambda} in hCoV infected airway epithelial cells and mice. Human primary airway epithelial cells treated with a single dose of IFN-I (IFN-) and IFN-{lambda} showed similar ISG expression, whereas cells treated with two doses of IFN-{lambda} expressed elevated levels of ISG compared to IFN-a treated cells. Similarly, mice treated with two dose IFN-{lambda} were better protected compared to mice receiving a single dose, and a combination of prophylactic and delayed therapeutic regimens completely protected mice from lethal MERS-CoV-infection. A two dose IFN-{lambda} regimen significantly reduced lung viral RNA and inflammatory cytokine levels with marked improvement in lung inflammation. Collectively, we identify an ideal regimen for IFN-{lambda} use and demonstrate the protective efficacy of IFN-{lambda} in MERS-CoV infected mice.

immunology↗

bioRxiv2021

Structure-function analysis of the nsp14 N7-guanine methyltransferase reveals an essential role in betacoronavirus replication

As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their mRNAs, protect them from degradation by cellular 5 exoribonucleases, and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bi-functional replicase subunit harboring an N-terminal 3'-to-5' exoribonuclease (ExoN) domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14s enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome-CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum. Significance StatementThe ongoing SARS-CoV-2 pandemic emphasizes the urgent need to develop efficient broad-spectrum anti-CoV drugs. The structure-function characterization of conserved CoV replicative enzymes is key to identifying the most suitable drug targets. Using a multidisciplinary comparative approach and different betacoronaviruses, we characterized the key conserved residues of the nsp14 (N7-guanine)-methyltransferase, a poorly defined subunit of the CoV mRNA-synthesizing machinery. Our study highlights the unique structural features of this enzyme and establishes its essential role in betacoronavirus replication, while identifying two residues that are critical for the replication of the four betacoronaviruses tested, including SARS-CoV-2.

molecular biology↗

bioRxiv2021

Convergent use of phosphatidic acid for Hepatitis C virus and SARS-CoV-2 replication organelle formation

Double membrane vesicles (DMVs) are used as replication organelles by phylogenetically and biologically distant pathogenic RNA viruses such as hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Viral DMVs are morphologically analogous to DMVs formed during autophagy, and although the proteins required for DMV formation are extensively studied, the lipids driving their biogenesis are largely unknown. Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral replication and autophagy. Using DMVs in HCV-replicating cells as model, we found that AGPATs are recruited to and critically contribute to HCV replication and DMV formation. AGPAT1/2 double knockout also impaired SARS-CoV-2 replication and the formation of autophagosome-like structures. By using correlative light and electron microscopy, we observed the relocalization of AGPAT proteins to HCV and SARS-CoV-2 induced DMVs. In addition, an intracellular PA sensor accumulated at viral DMV formation sites, consistent with elevated levels of PA in fractions of purified DMVs analyzed by lipidomics. Apart from AGPATs, PA is generated by alternative pathways via phosphotidylcholine (PC) and diacylglycerol (DAG). Pharmacological inhibition of these synthesis pathways also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as an important lipid used for replication organelle formation by HCV and SARS-CoV-2, two phylogenetically disparate viruses causing very different diseases, i.e. chronic liver disease and COVID-19, respectively. In addition, our data argue that host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses. One Sentence SummaryPhosphatidic acid is important for the formation of double membrane vesicles, serving as replication organelles of hepatitis C virus and SARS-CoV-2, and offering a possible host-targeting strategy to treat SARS-CoV-2 infection.

microbiology↗

bioRxiv2021

Exploiting synergistic interactions of Medicago sativa L. and Paraburkholderia tropica for enhanced biodegradation of diesel fuel hydrocarbons

The biotechnological application of microorganisms for rhizoremediation of contaminated sites requires the development of plant-microbe symbionts capable of plant growth promotion and hydrocarbon degradation. Studies focusing on microbial consortia are often difficult to reproduce, thereby necessitating the need for culturable single bacterial species for biotechnological applications. Through genomic analyses and plant growth experiments, we examined the synergistic interactions of Medicago sativa L. and Paraburkholderia tropica for enhanced remediation of diesel fuel-contaminated soils. Comparative genomics revealed strong potential of P. tropica for plant growth-promotion, chemotaxis and motility, root nodulation and colonization, and diesel fuel degradation. Plant growth experiments confirmed that P. tropica thrived in the contaminated soils and effectively enhanced M. sativa growth. Geochemical analysis showed that the M. sativa + P. tropica treatment resulted in an efficient degradation of diesel fuel hydrocarbons within two months, offering great prospects for enhanced biodegradation of organic pollutants.

microbiology↗

bioRxiv2021

Metagenomic insight into the plant growth-promoting potential of a diesel-degrading bacterial consortium for enhanced rhizoremediation application

The slow rate of natural attenuation of organic pollutants, together with unwanted environmental impacts of traditional remediation strategies, has necessitated the exploration of plant-microbe systems for enhanced bioremediation applications. The identification of microorganisms capable of promoting both plant growth and hydrocarbon degradation is crucial to the success of plant-based remediation techniques. Through successive enrichments of a soil sample from a historic oil-contaminated site in Wietze, Germany, we isolated a plant growth-promoting and hydrocarbon-degrading bacterial consortium. Metagenome analysis of the consortium led to the identification of genes and taxa putatively associated with these processes. The majority of the coding DNA sequences involved in these reactions were affiliated to Acidocella aminolytica and Acidobacterium capsulatum. In microcosm experiments performed in association with Medicago sativa L., the consortium achieved 91% rhizodegradation of diesel fuel hydrocarbons within 60 days, indicating its potential for biotechnological applications in the remediation of sites contaminated by organic pollutants.

microbiology↗

bioRxiv2021

A genome-wide CRISPR screen identifies interactors of the autophagy pathway as conserved coronavirus targets

Over the past 20 years, the emergence of three highly pathogenic coronaviruses (CoV) - SARS-CoV, MERS-CoV, and most recently SARS-CoV-2 - has shown that CoVs pose a serious risk to human health and highlighted the importance of developing effective therapies against them. Similar to other viruses, CoVs are dependent on host factors for their survival and replication. We hypothesized that evolutionarily distinct CoVs may exploit similar host factors and pathways to support their replication cycle. Here, we conducted two independent genome-wide CRISPR/Cas9 knockout screens to identify pan-CoV host factors required for the replication of both endemic and emerging CoVs, including the novel CoV SARS-CoV-2. Strikingly, we found that several autophagy-related genes, including the immunophilin FKBP8, TMEM41B, and MINAR1, were common host factors required for CoV replication. Importantly, inhibition of the immunophilin family with the compounds Tacrolimus, Cyclosporin A, and the non-immunosuppressive derivative Alisporivir, resulted in dose-dependent inhibition of CoV replication in primary human nasal epithelial cell cultures that resemble the natural site of virus replication. Overall, we identified host factors that are crucial for CoV replication and demonstrate that these factors constitute potential targets for therapeutic intervention by clinically approved drugs.

microbiology↗

bioRxiv2020

No evidence for human monocyte-derived macrophage infection and antibody-mediated enhancement of SARS-CoV-2 infection

Vaccines are essential to control the spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and to protect the vulnerable population. However, one safety concern of vaccination is the possible development of antibody-dependent enhancement (ADE) of SARS-CoV-2 infection. The potential infection of Fc receptor bearing cells such as macrophages, would support continued virus replication and inflammatory responses, and thereby potentially worsen the clinical outcome of COVID-19. Here we demonstrate that SARS-CoV-2 and SARS-CoV-1 neither infect human monocyte-derived macrophages nor induce inflammatory cytokines in these cells, in sharp contrast to Middle East respiratory syndrome (MERS) coronavirus and the common cold human coronavirus 229E. Furthermore, serum from convalescent COVID-19 patients neither induced enhancement of SARS-CoV-2 infection nor innate immune response in human macrophages. These results support the view that ADE may not be involved in the immunopathological processes associated with COVID-19, however, more studies are necessary to understand the potential contribution of antibodies-virus complexes with other cells expressing FcR receptors.

immunology↗

bioRxiv2020

Betulonic acid derivatives inhibiting coronavirus replication in cell culture via the nsp15 endoribonuclease

The lack of medication to suppress coronavirus infections is a main reason for the dramatic course of the COVID-19 pandemic. There is an urgent need to identify suitable coronavirus drug targets and corresponding lead molecules. Here we describe the discovery of a class of coronavirus inhibitors acting on nsp15, a hexameric protein component of the viral replication-transcription complexes, endowed with immune evasion-associated endoribonuclease activity. SAR exploration of these 1,2,3-triazolo fused betulonic acid derivatives yielded lead molecule 5h as a strong inhibitor (antiviral EC50: 0.6 M) of human coronavirus 229E replication. An nsp15 endoribonuclease active site mutant virus was markedly less sensitive to 5h, and selected resistance to the compound mapped to mutations in the N-terminal part of nsp15, at an interface between two nsp15 monomers. The biological findings were substantiated by the nsp15 binding mode for 5h, predicted by docking. Hence, besides delivering a distinct class of inhibitors, our study revealed a druggable pocket in the nsp15 hexamer with relevance for anti-coronavirus drug development.

microbiology↗

bioRxiv2020

Susceptibility of well-differentiated airway epithelial cell cultures from domestic and wildlife animals to SARS-CoV-2

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally, and the number of cases continues to rise all over the world. Besides humans, the zoonotic origin, as well as intermediate and potential spillback host reservoirs of SARS-CoV-2 are unknown. To circumvent ethical and experimental constraints, and more importantly, to reduce and refine animal experimentation, we employed our airway epithelial cell (AEC) culture repository composed of various domesticated and wildlife animal species to assess their susceptibility to SARS-CoV-2. In this study, we inoculated well-differentiated animal AEC cultures of monkey, cat, ferret, dog, rabbit, pig, cattle, goat, llama, camel, and two neotropical bat species with SARS-CoV-2. We observed that SARS-CoV-2 only replicated efficiently in monkey and cat AEC culture models. Whole-genome sequencing of progeny virus revealed no obvious signs of nucleotide transitions required for SARS-CoV-2 to productively infect monkey and cat epithelial airway cells. Our findings, together with the previously reported human-to-animal spillover events warrants close surveillance to understand the potential role of cats, monkeys, and closely related species as spillback reservoirs for SARS-CoV-2.

microbiology↗

bioRxiv2020

SARS-CoV-2 spike D614G variant confers enhanced replication and transmissibility

During the evolution of SARS-CoV-2 in humans a D614G substitution in the spike (S) protein emerged and became the predominant circulating variant (S-614G) of the COVID-19 pandemic1. However, whether the increasing prevalence of the S-614G variant represents a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains elusive. Here, we generated isogenic SARS-CoV-2 variants and demonstrate that the S-614G variant has (i) enhanced binding to human ACE2, (ii) increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a novel human ACE2 knock-in mouse model, and (iii) markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Collectively, our data show that while the S-614G substitution results in subtle increases in binding and replication in vitro, it provides a real competitive advantage in vivo, particularly during the transmission bottle neck, providing an explanation for the global predominance of S-614G variant among the SARS-CoV-2 viruses currently circulating.

microbiology↗

bioRxiv2020

Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome

Programmed ribosomal frameshifting is the key event during translation of the SARS-CoV-2 RNA genome allowing synthesis of the viral RNA-dependent RNA polymerase and downstream viral proteins. Here we present the cryo-EM structure of the mammalian ribosome in the process of translating viral RNA paused in a conformation primed for frameshifting. We observe that the viral RNA adopts a pseudoknot structure lodged at the mRNA entry channel of the ribosome to generate tension in the mRNA that leads to frameshifting. The nascent viral polyprotein that is being synthesized by the ribosome paused at the frameshifting site forms distinct interactions with the ribosomal polypeptide exit tunnel. We use biochemical experiments to validate our structural observations and to reveal mechanistic and regulatory features that influence the frameshifting efficiency. Finally, a compound previously shown to reduce frameshifting is able to inhibit SARS-CoV-2 replication in infected cells, establishing coronavirus frameshifting as target for antiviral intervention.

molecular biology↗

bioRxiv2020

SARS-CoV-2 Nsp1 binds ribosomal mRNA channel to inhibit translation

The non-structural protein 1 (Nsp1), also referred to as the host shutoff factor, is the first viral protein that is synthesized in SARS-CoV-2 infected human cells to suppress host innate immune functions1,2. By combining cryo-electron microscopy and biochemical experiments, we show that SARS-CoV-2 Nsp1 binds to the human 40S subunit in ribosomal complexes including the 43S pre-initiation complex. The protein inserts its C-terminal domain at the entrance to the mRNA channel where it interferes with mRNA binding. We observe potent translation inhibition in the presence of Nsp1 in lysates from human cells. Based on the high-resolution structure of the 40S-Nsp1 complex, we identify residues of Nsp1 crucial for mediating translation inhibition. We further show that the full-length 5’ untranslated region of the genomic viral mRNA stimulates translation in vitro, suggesting that SARS-CoV-2 combines inhibition of translation by Nsp1 with efficient translation of the viral mRNA to achieve expression of viral genes3.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2020

Multi-level proteomics reveals host-perturbation strategies of SARS-CoV-2 and SARS-CoV

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

systems biology↗

bioRxiv2020

Physiologic RNA Targets and Refined Sequence Specificity of Coronavirus EndoU

Coronavirus EndoU inhibits dsRNA-activated antiviral responses; however, the physiologic RNA substrates of EndoU are unknown. In this study, we used mouse hepatitis virus (MHV)-infected bone-marrow-derived macrophage (BMM) and cyclic phosphate cDNA sequencing to identify the RNA targets of EndoU. EndoU targeted viral RNA, cleaving the 3' side of pyrimidines with a strong preference for UA and CA sequences (endoYA). EndoU-dependent cleavage was detected in every region of MHV RNA, from the 5' NTR to the 3' NTR, including transcriptional regulatory sequences (TRS). Cleavage at two CA dinucleotides immediately adjacent to the MHV poly(A) tail suggest a mechanism to suppress negative-strand RNA synthesis and the accumulation of viral dsRNA. MHV with EndoU (EndoUmut) or 2'-5' phosphodiesterase (PDEmut) mutations provoked the activation of RNase L in BMM, with corresponding cleavage of RNAs by RNase L. The physiologic targets of EndoU are viral RNA templates required for negative-strand RNA synthesis and dsRNA accumulation. ImpactCoronavirus EndoU cleaves UA and CA sequences (endoYA) within viral (+) strand RNA to evade dsRNA-activated host responses.

microbiology↗

bioRxiv2020

Identification of five antiviral compounds from the Pandemic Response Box targeting SARS-CoV-2

With currently over 4 million confirmed cases worldwide, including more than 300000 deaths, the current Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic has a major impact on the economy and health care system. Currently, a limited amount of prophylactic or therapeutic intervention options are available against SARS-CoV-2. In this study, we screened 400 compounds from the antimicrobial Pandemic Response Box library for inhibiting properties against SARS-CoV-2. We identified sixteen compounds that potently inhibited SARS-CoV-2 replication, of which five compounds displayed equal or even higher antiviral activity compared to Remdesivir. These results show that five compounds should be further investigated for their mode of action, safety and efficacy against SARS-CoV-2. HighlightsO_LI400 compounds from the pandemic response box were tested for antiviral activity against SARS-CoV-2. C_LIO_LI5 compounds had an equal or higher antiviral efficacy towards SARS-CoV-2, compared to the nucleoside analogue Remdesivir. C_LI

microbiology↗