Publications by authors named "Ralf Bartenschlager"

324 Publications

The compound SBI-0090799 inhibits Zika virus (ZIKV) infection by blocking formation of the membranous replication compartment.

J Virol 2021 Sep 1:JVI0099621. Epub 2021 Sep 1.

Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, USA.

Zika virus (ZIKV) is a mosquito-borne pathogen classified by the World Health Organization (WHO) as a public health emergency of international concern in 2016, and it is still identified as a priority disease. Although most infected individuals are asymptomatic or show mild symptoms, a risk of neurologic complications is associated with infection in adults. Additionally, infection during pregnancy is directly linked to microcephaly and other congenital malformations. Since there are no currently available vaccines or approved therapeutics for this virus, there is a critical unmet need in developing treatments to prevent future ZIKV outbreaks. Towards this end, we performed a large-scale cell-based high-content screen of 51,520 chemical compounds to identify potential antiviral drug candidates. The compound (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) was found to inhibit replication of multiple ZIKV strains and in different cell systems. SBI-0090799 did not affect viral entry or RNA translation but suppressed RNA replication by preventing the formation of the membranous replication compartment. Selection of drug-resistant viruses identified single amino acid substitions in the N-terminal region of non-structural protein NS4A arguing this to be the likely drug target. These resistance mutations rescued viral RNA replication and restored the formation of the membranous replication compartment. This mechanism of action is similar to clinically-approved NS5A inhibitors for hepatitis C virus (HCV). Taken together, SBI-0090799 represents a promising lead candidate for the development of an antiviral treatment against ZIKV infection for the mitigation of severe complications and potential resurgent outbreaks of the virus. This study describes the elucidation of (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) as selective and potent inhibitor of Zika virus (ZIKV) replication using a high throughput screening approach. Mapping and resistance studies, supported by electron microscopy observations, indicate that the small molecule is functioning through inhibition of NS4A-mediated formation of ZIKV replication compartments in the endoplasmic reticulum (ER). Intriguingly, this defines a novel non-enzymatic target and chemical matter for the development of a new class of ZIKV antivirals. Moreover, chemical modulation affecting this non-structural protein mirrors the identification and development of hepatitis C virus (HCV) NS5A inhibitor daclatasvir and its derivatives, similarly interfering with the formation of the viral replication compartment and also targeting a protein with no enzymatic activity, which have been part of a curative strategy for HCV.
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http://dx.doi.org/10.1128/JVI.00996-21DOI Listing
September 2021

Evaluation of accuracy, exclusivity, limit-of-detection and ease-of-use of LumiraDx™: An antigen-detecting point-of-care device for SARS-CoV-2.

Infection 2021 Aug 12. Epub 2021 Aug 12.

Division of Clinical Tropical Medicine, Heidelberg University Hospital, Heidelberg, Germany.

Purpose: Rapid antigen-detecting tests (Ag-RDTs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transform pandemic control. Thus far, sensitivity (≤ 85%) of lateral-flow assays has limited scale-up. Conceivably, microfluidic immunofluorescence Ag-RDTs could increase sensitivity for SARS-CoV-2 detection.

Methods: This multi-centre diagnostic accuracy study investigated performance of the microfluidic immunofluorescence LumiraDx™ assay, enrolling symptomatic and asymptomatic participants with suspected SARS-CoV-2 infection. Participants collected a supervised nasal mid-turbinate (NMT) self-swab for Ag-RDT testing, in addition to a professionally collected nasopharyngeal (NP) swab for routine testing with reverse transcriptase polymerase chain reaction (RT-PCR). Results were compared to calculate sensitivity and specificity. Sub-analyses investigated the results by viral load, symptom presence and duration. An analytical study assessed exclusivity and limit-of-detection (LOD). In addition, we evaluated ease-of-use.

Results: The study was conducted between November 2nd 2020 and 4th of December 2020. 761 participants were enrolled, with 486 participants reporting symptoms on testing day. 120 out of 146 RT-PCR positive cases were detected positive by LumiraDx™, resulting in a sensitivity of 82.2% (95% CI 75.2-87.5%). Specificity was 99.3% (CI 98.3-99.7%). Sensitivity was increased in individuals with viral load ≥ 7 log10 SARS-CoV2 RNA copies/ml (93.8%; CI 86.2-97.3%). Testing against common respiratory commensals and pathogens showed no cross-reactivity and LOD was estimated to be 2-56 PFU/mL. The ease-of-use-assessment was favourable for lower throughput settings.

Conclusion: The LumiraDx™ assay showed excellent analytical sensitivity, exclusivity and clinical specificity with good clinical sensitivity using supervised NMT self-sampling.

Trial Registration Number And Registration Date: DRKS00021220 and 01.04.2020.
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http://dx.doi.org/10.1007/s15010-021-01681-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8358901PMC
August 2021

Determinants in non-structural protein 4A of dengue virus required for RNA replication and replication organelle biogenesis.

J Virol 2021 Aug 11:JVI0131021. Epub 2021 Aug 11.

Department of Infectious Diseases, Molecular Virology, Heidelberg University, 69120 Heidelberg, Germany.

Dengue virus (DENV) constitutes one of the most important arboviral pathogens affecting humans. The high prevalence of DENV infections, which cause more than twenty thousand deaths annually, and the lack of effective vaccines or direct-acting antiviral drugs make it a global health concern. DENV genome replication occurs in close association with the host endomembrane system, which is remodeled to form the viral replication organelle that originates from ER membranes. To date, the viral and cellular determinants responsible for the biogenesis of DENV replication organelles are still poorly defined. The viral nonstructural protein (NS) 4A can remodel membranes and has been shown to associate with numerous host factors in DENV replicating cells. In the present study we used reverse and forward genetic screens and identified sites within NS4A required for DENV replication. We also mapped the determinants in NS4A required for interactions with other viral proteins. Moreover, taking advantage of our recently developed polyprotein expression system, we evaluated the role of NS4A in the formation of DENV replication organelles. Together, we report a detailed map of determinants within NS4A required for RNA replication, interaction with other viral proteins and replication organelle formation. Our results suggest that NS4A might be an attractive target for antiviral therapy. DENV is the most prevalent mosquito-borne virus, causing around 390 million infections each year. There are no approved therapies to treat DENV infection and the only available vaccine shows limited efficacy. The viral non-structural proteins have emerged as attractive drug targets, due to their pivotal role in RNA replication and establishment of virus-induced membranous compartments, designated replication organelles (ROs). The transmembrane protein NS4A, generated by cleavage of the NS4A-2K-4B precursor, contributes to DENV replication by unknown mechanisms. Here, we report a detailed genetic interaction map of NS4A and identify residues required for RNA replication and interaction between NS4A-2K-4B and NS2B-3 as well as NS1. Importantly, by means of an expression-based system we demonstrate the essential role of NS4A in ROs biogenesis and identify determinants in NS4A required for this process. Our data suggest that NS4A is an attractive target for antiviral therapy.
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http://dx.doi.org/10.1128/JVI.01310-21DOI Listing
August 2021

Neutralizing antibody response against variants of concern after vaccination of dialysis patients with BNT162b2.

Kidney Int 2021 09 12;100(3):700-702. Epub 2021 Jul 12.

Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany; German Center for Infection Research, Heidelberg partner site, Heidelberg, Germany; Division Virus-Associated Carcinogenesis, German Cancer Research Center, Heidelberg, Germany.

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http://dx.doi.org/10.1016/j.kint.2021.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8274271PMC
September 2021

Deep probabilistic tracking of particles in fluorescence microscopy images.

Med Image Anal 2021 08 8;72:102128. Epub 2021 Jun 8.

Biomedical Computer Vision Group, Heidelberg University, BioQuant, IPMB, and DKFZ Heidelberg, Heidelberg 69120, Germany. Electronic address:

Tracking of particles in temporal fluorescence microscopy image sequences is of fundamental importance to quantify dynamic processes of intracellular structures as well as virus structures. We introduce a probabilistic deep learning approach for fluorescent particle tracking, which is based on a recurrent neural network that mimics classical Bayesian filtering. Compared to previous deep learning methods for particle tracking, our approach takes into account uncertainty, both aleatoric and epistemic uncertainty. Thus, information about the reliability of the computed trajectories is determined. Manual tuning of tracking parameters is not necessary and prior knowledge about the noise statistics is not required. Short and long-term temporal dependencies of individual object dynamics are exploited for state prediction, and assigned detections are used to update the predicted states. For correspondence finding, we introduce a neural network which computes assignment probabilities jointly across multiple detections as well as determines the probabilities of missing detections. Training requires only simulated data and therefore tedious manual annotation of ground truth is not needed. We performed a quantitative performance evaluation based on synthetic and real 2D as well as 3D fluorescence microscopy images. We used image data of the Particle Tracking Challenge as well as real time-lapse fluorescence microscopy images displaying virus structures and chromatin structures. It turned out that our approach yields state-of-the-art results or improves the tracking results compared to previous methods.
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http://dx.doi.org/10.1016/j.media.2021.102128DOI Listing
August 2021

Global analysis of protein-RNA interactions in SARS-CoV-2-infected cells reveals key regulators of infection.

Mol Cell 2021 07 24;81(13):2851-2867.e7. Epub 2021 May 24.

MRC-University of Glasgow Centre for Virus Research, G61 1QH Glasgow, Scotland, UK; Department of Biochemistry, University of Oxford, South Parks Road, OX1 3QU Oxford, UK. Electronic address:

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 relies on cellular RNA-binding proteins (RBPs) to replicate and spread, although which RBPs control its life cycle remains largely unknown. Here, we employ a multi-omic approach to identify systematically and comprehensively the cellular and viral RBPs that are involved in SARS-CoV-2 infection. We reveal that SARS-CoV-2 infection profoundly remodels the cellular RNA-bound proteome, which includes wide-ranging effects on RNA metabolic pathways, non-canonical RBPs, and antiviral factors. Moreover, we apply a new method to identify the proteins that directly interact with viral RNA, uncovering dozens of cellular RBPs and six viral proteins. Among them are several components of the tRNA ligase complex, which we show regulate SARS-CoV-2 infection. Furthermore, we discover that available drugs targeting host RBPs that interact with SARS-CoV-2 RNA inhibit infection. Collectively, our results uncover a new universe of host-virus interactions with potential for new antiviral therapies against COVID-19.
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http://dx.doi.org/10.1016/j.molcel.2021.05.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8142890PMC
July 2021

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

EMBO Rep 2021 06 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
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8183418PMC
June 2021

From Multiplex Serology to Serolomics-A Novel Approach to the Antibody Response against the SARS-CoV-2 Proteome.

Viruses 2021 04 24;13(5). Epub 2021 Apr 24.

Infections and Cancer Epidemiology, German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ), 69120 Heidelberg, Germany.

The emerging SARS-CoV-2 pandemic entails an urgent need for specific and sensitive high-throughput serological assays to assess SARS-CoV-2 epidemiology. We, therefore, aimed at developing a fluorescent-bead based SARS-CoV-2 multiplex serology assay for detection of antibody responses to the SARS-CoV-2 proteome. Proteins of the SARS-CoV-2 proteome and protein N of SARS-CoV-1 and common cold Coronaviruses (ccCoVs) were recombinantly expressed in or HEK293 cells. Assay performance was assessed in a COVID-19 case cohort ( = 48 hospitalized patients from Heidelberg) as well as = 85 age- and sex-matched pre-pandemic controls from the ESTHER study. Assay validation included comparison with home-made immunofluorescence and commercial enzyme-linked immunosorbent (ELISA) assays. A sensitivity of 100% (95% CI: 86-100%) was achieved in COVID-19 patients 14 days post symptom onset with dual sero-positivity to SARS-CoV-2 N and the receptor-binding domain of the spike protein. The specificity obtained with this algorithm was 100% (95% CI: 96-100%). Antibody responses to ccCoVs N were abundantly high and did not correlate with those to SARS-CoV-2 N. Inclusion of additional SARS-CoV-2 proteins as well as separate assessment of immunoglobulin (Ig) classes M, A, and G allowed for explorative analyses regarding disease progression and course of antibody response. This newly developed SARS-CoV-2 multiplex serology assay achieved high sensitivity and specificity to determine SARS-CoV-2 sero-positivity. Its high throughput ability allows epidemiologic SARS-CoV-2 research in large population-based studies. Inclusion of additional pathogens into the panel as well as separate assessment of Ig isotypes will furthermore allow addressing research questions beyond SARS-CoV-2 sero-prevalence.
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http://dx.doi.org/10.3390/v13050749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8147094PMC
April 2021

Antiviral drug screen identifies DNA-damage response inhibitor as potent blocker of SARS-CoV-2 replication.

Cell Rep 2021 04 18;35(1):108940. Epub 2021 Mar 18.

Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, UCLA, Los Angeles, CA 90095, USA; California NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address:

SARS-CoV-2 has currently precipitated the COVID-19 global health crisis. We developed a medium-throughput drug-screening system and identified a small-molecule library of 34 of 430 protein kinase inhibitors that were capable of inhibiting the SARS-CoV-2 cytopathic effect in human epithelial cells. These drug inhibitors are in various stages of clinical trials. We detected key proteins involved in cellular signaling pathways mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-damage response that are critical for SARS-CoV-2 infection. A drug-protein interaction-based secondary screen confirmed compounds, such as the ATR kinase inhibitor berzosertib and torin2 with anti-SARS-CoV-2 activity. Berzosertib exhibited potent antiviral activity against SARS-CoV-2 in multiple cell types and blocked replication at the post-entry step. Berzosertib inhibited replication of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus (MERS-CoV) as well. Our study highlights key promising kinase inhibitors to constrain coronavirus replication as a host-directed therapy in the treatment of COVID-19 and beyond as well as provides an important mechanism of host-pathogen interactions.
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http://dx.doi.org/10.1016/j.celrep.2021.108940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7969873PMC
April 2021

The Basicity Makes the Difference: Improved Canavanine-Derived Inhibitors of the Proprotein Convertase Furin.

ACS Med Chem Lett 2021 Mar 9;12(3):426-432. Epub 2021 Feb 9.

Institute of Pharmaceutical Chemistry, Philipps University, Marbacher Weg 6, 35032 Marburg, Germany.

Furin activates numerous viral glycoproteins, and its inhibition prevents virus replication and spread. Through the replacement of arginine by the less basic canavanine, new inhibitors targeting furin in the trans-Golgi network were developed. These inhibitors exert potent antiviral activity in cell culture with much lower toxicity than arginine-derived analogues, most likely due to their reduced protonation in the blood circulation. Thus, despite its important physiological functions, furin might be a suitable antiviral drug target.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7957917PMC
March 2021

TLR3 activation by Zika virus stimulates inflammatory cytokine production which dampens the antiviral response induced by RIG-I-like receptors.

J Virol 2021 Mar 3. Epub 2021 Mar 3.

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

Infection with the Zika virus (ZIKV), a member of the family, can cause serious neurological disorders, most notably microcephaly in newborns. Here we investigated the innate immune response to ZIKV infection in cells of the nervous system. In human neural progenitor cells (hNPCs), a target for ZIKV infection and likely involved in ZIKV-associated neuropathology, viral infection failed to elicit an antiviral interferon (IFN) response. However, pharmacological inhibition of TLR3 partially restored this deficit. Analogous results were obtained in human iPSC-derived astrocytes, which are capable of mounting a strong antiviral cytokine response. There, ZIKV is sensed by both RIG-I and MDA5 and induces an IFN response as well as expression of pro-inflammatory cytokines such as interleukin-6 (IL-6). Upon inhibition of TLR3, also in astrocytes the antiviral cytokine response was enhanced, whereas amounts of pro-inflammatory cytokines were reduced. To study the underlying mechanism, we used human epithelial cells as an easy to manipulate model system. We found that ZIKV is sensed in these cells by RIG-I to induce a robust IFN response and by TLR3 to trigger the expression of pro-inflammatory cytokines, including IL-6. ZIKV induced upregulation of IL-6 activated the STAT3 pathway, which decreased STAT1 phosphorylation in a SOCS-3 dependent manner, thus reducing the IFN response. In conclusion, we show that TLR3 activation by ZIKV suppresses IFN responses triggered by RIG-I-like receptors.Zika virus (ZIKV) has a pronounced neurotropism and infections with this virus can cause serious neurological disorders, most notably microcephaly and the Guillain-Barré syndrome. Our studies reveal that during ZIKV infection, recognition of viral RNA by TLR3 enhances the production of inflammatory cytokines and suppresses the interferon response triggered by RIG-I-like receptors (RLR) in a SOCS3-dependent manner, thus facilitating virus replication. The discovery of this crosstalk between antiviral (RLR) and inflammatory (TLR) responses may have important implications for our understanding of ZIKV-induced pathogenesis.
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http://dx.doi.org/10.1128/JVI.01050-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8139665PMC
March 2021

SARS-CoV-2 infection remodels the host protein thermal stability landscape.

Mol Syst Biol 2021 02;17(2):e10188

Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global threat to human health and has compromised economic stability. In addition to the development of an effective vaccine, it is imperative to understand how SARS-CoV-2 hijacks host cellular machineries on a system-wide scale so that potential host-directed therapies can be developed. In situ proteome-wide abundance and thermal stability measurements using thermal proteome profiling (TPP) can inform on global changes in protein activity. Here we adapted TPP to high biosafety conditions amenable to SARS-CoV-2 handling. We discovered pronounced temporal alterations in host protein thermostability during infection, which converged on cellular processes including cell cycle, microtubule and RNA splicing regulation. Pharmacological inhibition of host proteins displaying altered thermal stability or abundance during infection suppressed SARS-CoV-2 replication. Overall, this work serves as a framework for expanding TPP workflows to globally important human pathogens that require high biosafety containment and provides deeper resolution into the molecular changes induced by SARS-CoV-2 infection.
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http://dx.doi.org/10.15252/msb.202010188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7885171PMC
February 2021

Challenges for Targeting SARS-CoV-2 Proteases as a Therapeutic Strategy for COVID-19.

ACS Infect Dis 2021 06 11;7(6):1457-1468. Epub 2021 Feb 11.

Department of Pathology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, California 94305, United States.

Two proteases produced by the SARS-CoV-2 virus, the main protease and papain-like protease, are essential for viral replication and have become the focus of drug development programs for treatment of COVID-19. We screened a highly focused library of compounds containing covalent warheads designed to target cysteine proteases to identify new lead scaffolds for both M and PL proteases. These efforts identified a small number of hits for the M protease and no viable hits for the PL protease. Of the M hits identified as inhibitors of the purified recombinant protease, only two compounds inhibited viral infectivity in cellular infection assays. However, we observed a substantial drop in antiviral potency upon expression of TMPRSS2, a transmembrane serine protease that acts in an alternative viral entry pathway to the lysosomal cathepsins. This loss of potency is explained by the fact that our lead M inhibitors are also potent inhibitors of host cell cysteine cathepsins. To determine if this is a general property of M inhibitors, we evaluated several recently reported compounds and found that they are also effective inhibitors of purified human cathepsins L and B and showed similar loss in activity in cells expressing TMPRSS2. Our results highlight the challenges of targeting M and PL proteases and demonstrate the need to carefully assess selectivity of SARS-CoV-2 protease inhibitors to prevent clinical advancement of compounds that function through inhibition of a redundant viral entry pathway.
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http://dx.doi.org/10.1021/acsinfecdis.0c00815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901237PMC
June 2021

Prevalence of SARS-CoV-2 Infection in Children and Their Parents in Southwest Germany.

JAMA Pediatr 2021 06;175(6):586-593

Institute of Virology, University Medical Centre and Faculty of Medicine, University of Freiburg, Freiburg im Breisgau, Germany.

Importance: School and daycare closures were enforced as measures to confine the novel coronavirus disease 2019 (COVID-19) pandemic, based on the assumption that young children may play a key role in severe acute respiratory coronavirus 2 (SARS-CoV-2) spread. Given the grave consequences of contact restrictions for children, a better understanding of their contribution to the COVID-19 pandemic is of great importance.

Objective: To describe the rate of SARS-CoV-2 infections and the seroprevalence of SARS-CoV-2 antibodies in children aged 1 to 10 years, compared with a corresponding parent of each child, in a population-based sample.

Design, Setting, And Participants: This large-scale, multicenter, cross-sectional investigation (the COVID-19 BaWü study) enrolled children aged 1 to 10 years and a corresponding parent between April 22 and May 15, 2020, in southwest Germany.

Exposures: Potential exposure to SARS-CoV-2.

Main Outcomes And Measures: The main outcomes were infection and seroprevalence of SARS-CoV-2. Participants were tested for SARS-CoV-2 RNA from nasopharyngeal swabs by reverse transcription-polymerase chain reaction and SARS-CoV-2 specific IgG antibodies in serum by enzyme-linked immunosorbent assays and immunofluorescence tests. Discordant results were clarified by electrochemiluminescence immunoassays, a second enzyme-linked immunosorbent assay, or an in-house Luminex-based assay.

Results: This study included 4964 participants: 2482 children (median age, 6 [range, 1-10] years; 1265 boys [51.0%]) and 2482 parents (median age, 40 [range, 23-66] years; 615 men [24.8%]). Two participants (0.04%) tested positive for SARS-CoV-2 RNA. The estimated SARS-CoV-2 seroprevalence was low in parents (1.8% [95% CI, 1.2-2.4%]) and 3-fold lower in children (0.6% [95% CI, 0.3-1.0%]). Among 56 families with at least 1 child or parent with seropositivity, the combination of a parent with seropositivity and a corresponding child with seronegativity was 4.3 (95% CI, 1.19-15.52) times higher than the combination of a parent who was seronegative and a corresponding child with seropositivity. We observed virus-neutralizing activity for 66 of 70 IgG-positive serum samples (94.3%).

Conclusions And Relevance: In this cross-sectional study, the spread of SARS-CoV-2 infection during a period of lockdown in southwest Germany was particularly low in children aged 1 to 10 years. Accordingly, it is unlikely that children have boosted the pandemic. This SARS-CoV-2 prevalence study, which appears to be the largest focusing on children, is instructive for how ad hoc mass testing provides the basis for rational political decision-making in a pandemic.
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http://dx.doi.org/10.1001/jamapediatrics.2021.0001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823424PMC
June 2021

Memory-like HCV-specific CD8 T cells retain a molecular scar after cure of chronic HCV infection.

Nat Immunol 2021 02 4;22(2):229-239. Epub 2021 Jan 4.

Department of Medicine II, University Hospital Freiburg, Freiburg, Germany.

In chronic hepatitis C virus (HCV) infection, exhausted HCV-specific CD8 T cells comprise memory-like and terminally exhausted subsets. However, little is known about the molecular profile and fate of these two subsets after the elimination of chronic antigen stimulation by direct-acting antiviral (DAA) therapy. Here, we report a progenitor-progeny relationship between memory-like and terminally exhausted HCV-specific CD8 T cells via an intermediate subset. Single-cell transcriptomics implicated that memory-like cells are maintained and terminally exhausted cells are lost after DAA-mediated cure, resulting in a memory polarization of the overall HCV-specific CD8 T cell response. However, an exhausted core signature of memory-like CD8 T cells was still detectable, including, to a smaller extent, in HCV-specific CD8 T cells targeting variant epitopes. These results identify a molecular signature of T cell exhaustion that is maintained as a chronic scar in HCV-specific CD8 T cells even after the cessation of chronic antigen stimulation.
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http://dx.doi.org/10.1038/s41590-020-00817-wDOI Listing
February 2021

Microscopy-based assay for semi-quantitative detection of SARS-CoV-2 specific antibodies in human sera: A semi-quantitative, high throughput, microscopy-based assay expands existing approaches to measure SARS-CoV-2 specific antibody levels in human sera.

Bioessays 2021 03 30;43(3):e2000257. Epub 2020 Dec 30.

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

Emergence of the novel pathogenic coronavirus SARS-CoV-2 and its rapid pandemic spread presents challenges that demand immediate attention. Here, we describe the development of a semi-quantitative high-content microscopy-based assay for detection of three major classes (IgG, IgA, and IgM) of SARS-CoV-2 specific antibodies in human samples. The possibility to detect antibodies against the entire viral proteome together with a robust semi-automated image analysis workflow resulted in specific, sensitive and unbiased assay that complements the portfolio of SARS-CoV-2 serological assays. Sensitive, specific and quantitative serological assays are urgently needed for a better understanding of humoral immune response against the virus as a basis for developing public health strategies to control viral spread. The procedure described here has been used for clinical studies and provides a general framework for the application of quantitative high-throughput microscopy to rapidly develop serological assays for emerging virus infections.
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http://dx.doi.org/10.1002/bies.202000257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7883048PMC
March 2021

Replication-Independent Generation and Morphological Analysis of Flavivirus Replication Organelles.

STAR Protoc 2020 Dec 25;1(3):100173. Epub 2020 Nov 25.

Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, 69120, Germany.

Positive-strand RNA viruses replicate in distinct membranous structures called replication organelles (ROs). Mechanistic studies of RO formation have been difficult because perturbations affecting viral replication have an impact on viral protein amounts, thus affecting RO biogenesis. Here, we present a detailed guide on how to use a replication-independent expression system, designated pIRO (plasmid-induced replication organelle formation), inducing bona fide flavivirus ROs in transfected cells. This will be useful for mechanistic studies of viral and cellular factors driving flavivirus RO biogenesis. For complete details on the use and execution of this protocol, please refer to Cerikan et al. (2020).
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http://dx.doi.org/10.1016/j.xpro.2020.100173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757399PMC
December 2020

A Versatile Reporter System To Monitor Virus-Infected Cells and Its Application to Dengue Virus and SARS-CoV-2.

J Virol 2021 01 28;95(4). Epub 2021 Jan 28.

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

Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of this include flaviviruses, such as dengue virus and Zika virus, which cause millions of yearly infections around the globe, and coronaviruses, such as SARS-CoV-2, the source of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of research aimed at determining methods to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective treatments. Here, we describe the generation and characterization of a reporter system that can be used to visualize and identify cells infected with dengue virus or SARS-CoV-2. This system is based on viral protease activity that mediates cleavage and nuclear translocation of an engineered fluorescent protein stably expressed in cells. We show the suitability of this system for live cell imaging, for visualization of single infected cells, and for screening and testing of antiviral compounds. With the integrated modular building blocks, this system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility. Reporter systems are useful tools for fast and quantitative visualization of virus-infected cells within a host cell population. Here, we describe a reporter system that takes advantage of virus-encoded proteases expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the GFP moiety translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the and the families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.
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http://dx.doi.org/10.1128/JVI.01715-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851548PMC
January 2021

Integrative Imaging Reveals SARS-CoV-2-Induced Reshaping of Subcellular Morphologies.

Cell Host Microbe 2020 12 17;28(6):853-866.e5. Epub 2020 Nov 17.

Department of Infectious Diseases, Molecular Virology, Heidelberg University, 69120 Heidelberg, Germany; German Center for Infection Research, Heidelberg partner site, Heidelberg, Germany; Division Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany. Electronic address:

Pathogenesis induced by SARS-CoV-2 is thought to result from both an inflammation-dominated cytokine response and virus-induced cell perturbation causing cell death. Here, we employ an integrative imaging analysis to determine morphological organelle alterations induced in SARS-CoV-2-infected human lung epithelial cells. We report 3D electron microscopy reconstructions of whole cells and subcellular compartments, revealing extensive fragmentation of the Golgi apparatus, alteration of the mitochondrial network and recruitment of peroxisomes to viral replication organelles formed by clusters of double-membrane vesicles (DMVs). These are tethered to the endoplasmic reticulum, providing insights into DMV biogenesis and spatial coordination of SARS-CoV-2 replication. Live cell imaging combined with an infection sensor reveals profound remodeling of cytoskeleton elements. Pharmacological inhibition of their dynamics suppresses SARS-CoV-2 replication. We thus report insights into virus-induced cytopathic effects and provide alongside a comprehensive publicly available repository of 3D datasets of SARS-CoV-2-infected cells for download and smooth online visualization.
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http://dx.doi.org/10.1016/j.chom.2020.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7670925PMC
December 2020

SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography.

Nat Commun 2020 11 18;11(1):5885. Epub 2020 Nov 18.

Schaller Research Groups, Department of Infectious Diseases-Virology, Heidelberg University, Heidelberg, Germany.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID19 pandemic, is a highly pathogenic β-coronavirus. As other coronaviruses, SARS-CoV-2 is enveloped, replicates in the cytoplasm and assembles at intracellular membranes. Here, we structurally characterize the viral replication compartment and report critical insights into the budding mechanism of the virus, and the structure of extracellular virions close to their native state by in situ cryo-electron tomography and subtomogram averaging. We directly visualize RNA filaments inside the double membrane vesicles, compartments associated with viral replication. The RNA filaments show a diameter consistent with double-stranded RNA and frequent branching likely representing RNA secondary structures. We report that assembled S trimers in lumenal cisternae do not alone induce membrane bending but laterally reorganize on the envelope during virion assembly. The viral ribonucleoprotein complexes (vRNPs) are accumulated at the curved membrane characteristic for budding sites suggesting that vRNP recruitment is enhanced by membrane curvature. Subtomogram averaging shows that vRNPs are distinct cylindrical assemblies. We propose that the genome is packaged around multiple separate vRNP complexes, thereby allowing incorporation of the unusually large coronavirus genome into the virion while maintaining high steric flexibility between the vRNPs.
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http://dx.doi.org/10.1038/s41467-020-19619-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7676268PMC
November 2020

Dimer Organization of Membrane-Associated NS5A of Hepatitis C Virus as Determined by Highly Sensitive H-Detected Solid-State NMR.

Angew Chem Int Ed Engl 2021 03 18;60(10):5339-5347. Epub 2021 Jan 18.

Molecular Microbiology and Structural Biochemistry, Labex Ecofect, UMR 5086 CNRS, Université de Lyon 1, 7 passage du Vercors, 69367, Lyon, France.

The Hepatitis C virus nonstructural protein 5A (NS5A) is a membrane-associated protein involved in multiple steps of the viral life cycle. Direct-acting antivirals (DAAs) targeting NS5A are a cornerstone of antiviral therapy, but the mode-of-action of these drugs is poorly understood. This is due to the lack of information on the membrane-bound NS5A structure. Herein, we present the structural model of an NS5A AH-linker-D1 protein reconstituted as proteoliposomes. We use highly sensitive proton-detected solid-state NMR methods suitable to study samples generated through synthetic biology approaches. Spectra analyses disclose that both the AH membrane anchor and the linker are highly flexible. Paramagnetic relaxation enhancements (PRE) reveal that the dimer organization in lipids requires a new type of NS5A self-interaction not reflected in previous crystal structures. In conclusion, we provide the first characterization of NS5A AH-linker-D1 in a lipidic environment shedding light onto the mode-of-action of clinically used NS5A inhibitors.
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http://dx.doi.org/10.1002/anie.202013296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986703PMC
March 2021

A Randomized Open label Phase-II Clinical Trial with or without Infusion of Plasma from Subjects after Convalescence of SARS-CoV-2 Infection in High-Risk Patients with Confirmed Severe SARS-CoV-2 Disease (RECOVER): A structured summary of a study protocol for a randomised controlled trial.

Trials 2020 Oct 6;21(1):828. Epub 2020 Oct 6.

Department of Internal Medicine V, Heidelberg University Hospital, Heidelberg, Germany.

Objectives: Primary objectives • To assess the time from randomisation until an improvement within 84 days defined as two points on a seven point ordinal scale or live discharge from the hospital in high-risk patients (group 1 to group 4) with SARS-CoV-2 infection requiring hospital admission by infusion of plasma from subjects after convalescence of SARS-CoV-2 infection or standard of care. Secondary objectives • To assess overall survival, and the overall survival rate at 28 56 and 84 days. • To assess SARS-CoV-2 viral clearance and load as well as antibody titres. • To assess the percentage of patients that required mechanical ventilation. • To assess time from randomisation until discharge.

Trial Design: Randomised, open-label, multicenter phase II trial, designed to assess the clinical outcome of SARS-CoV-2 disease in high-risk patients (group 1 to group 4) following treatment with anti-SARS-CoV-2 convalescent plasma or standard of care.

Participants: High-risk patients >18 years of age hospitalized with SARS-CoV-2 infection in 10-15 university medical centres will be included. High-risk is defined as SARS-CoV-2 positive infection with Oxygen saturation at ≤ 94% at ambient air with additional risk features as categorised in 4 groups: • Group 1, pre-existing or concurrent hematological malignancy and/or active cancer therapy (incl. chemotherapy, radiotherapy, surgery) within the last 24 months or less. • Group 2, chronic immunosuppression not meeting the criteria of group 1. • Group 3, age ≥ 50 - 75 years meeting neither the criteria of group 1 nor group 2 and at least one of these criteria: Lymphopenia < 0.8 x G/l and/or D-dimer > 1μg/mL. • Group 4, age ≥ 75 years meeting neither the criteria of group 1 nor group 2. Observation time for all patients is expected to be at least 3 months after entry into the study. Patients receive convalescent plasma for two days (day 1 and day 2) or standard of care. For patients in the standard arm, cross over is allowed from day 10 in case of not improving or worsening clinical condition. Nose/throat swabs for determination of viral load are collected at day 0 and day 1 (before first CP administration) and subsequently at day 2, 3, 5, 7, 10, 14, 28 or until discharge. Serum for SARS-Cov-2 diagnostic is collected at baseline and subsequently at day 3, 7, 14 and once during the follow-up period (between day 35 and day 84). There is a regular follow-up of 3 months. All discharged patients are followed by regular phone calls. All visits, time points and study assessments are summarized in the Trial Schedule (see full protocol Table 1). All participating trial sites will be supplied with study specific visit worksheets that list all assessments and procedures to be completed at each visit. All findings including clinical and laboratory data are documented by the investigator or an authorized member of the study team in the patient's medical record and in the electronic case report forms (eCRFs).

Intervention And Comparator: This trial will analyze the effects of convalescent plasma from recovered subjects with SARS-CoV-2 antibodies in high-risk patients with SARS-CoV-2 infection. Patients at high risk for a poor outcome due to underlying disease, age or condition as listed above are eligible for enrollment. In addition, eligible patients have a confirmed SARS-CoV-2 infection and O saturation ≤ 94% while breathing ambient air. Patients are randomised to receive (experimental arm) or not receive (standard arm) convalescent plasma in two bags (238 - 337 ml plasma each) from different donors (day 1, day 2). A cross over from the standard arm into the experimental arm is possible after day 10 in case of not improving or worsening clinical condition.

Main Outcomes: Primary endpoints: The main purpose of the study is to assess the time from randomisation until an improvement within 84 days defined as two points on a seven-point ordinal scale or live discharge from the hospital in high-risk patients (group 1 to group 4) with SARS-CoV-2 infection requiring hospital admission by infusion of plasma from subjects after convalescence of a SARS-CoV-2 infection or standard of care. Secondary endpoints: • Overall survival, defined as the time from randomisation until death from any cause 28-day, 56-day and 84-day overall survival rates. • SARS-CoV-2 viral clearance and load as well as antibody titres. • Requirement mechanical ventilation at any time during hospital stay (yes/no). • Time until discharge from randomisation. • Viral load, changes in antibody titers and cytokine profiles are analysed in an exploratory manner using paired non-parametric tests (before - after treatment).

Randomisation: Upon confirmation of eligibility (patients must meet all inclusion criteria and must not meet exclusion criteria described in section 5.3 and 5.4 of the full protocol), the clinical site must contact a centralized internet randomization system ( https://randomizer.at/ ). Patients are randomized using block randomisation to one of the two arms, experimental arm or standard arm, in a 1:1 ratio considering a stratification according to the 4 risk groups (see Participants).

Blinding (masking): The study is open-label, no blinding will be performed.

Numbers To Be Randomised (sample Size): A total number of 174 patients is required for the entire trial, n=87 per group.

Trial Status: Protocol version 1.2 dated 09/07/2020. A recruitment period of approximately 9 months and an overall study duration of approximately 12 months is anticipated. Recruitment of patients starts in the third quarter of 2020. The study duration of an individual patient is planned to be 3 months. After finishing all study-relevant procedures, therapy, and follow-up period, the patient is followed in terms of routine care and treated if necessary. Total trial duration: 18 months Duration of the clinical phase: 12 months First patient first visit (FPFV): 3 Quarter 2020 Last patient first visit (LPFV): 2 Quarter 2021 Last patient last visit (LPLV): 3 Quarter 2021 Trial Report completed: 4 Quarter 2021 TRIAL REGISTRATION: EudraCT Number: 2020-001632-10, https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-001632-10/DE , registered on 04/04/2020.

Full Protocol: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines (Additional file 2). The eCRF is attached (Additional file 3).
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http://dx.doi.org/10.1186/s13063-020-04735-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7538058PMC
October 2020

Identification of Interleukin1β as an Amplifier of Interferon alpha-induced Antiviral Responses.

PLoS Pathog 2020 10 1;16(10):e1008461. Epub 2020 Oct 1.

Division Systems Biology of Signal Transduction, German Cancer Research Center (DKFZ), Heidelberg, Germany.

The induction of an interferon-mediated response is the first line of defense against pathogens such as viruses. Yet, the dynamics and extent of interferon alpha (IFNα)-induced antiviral genes vary remarkably and comprise three expression clusters: early, intermediate and late. By mathematical modeling based on time-resolved quantitative data, we identified mRNA stability as well as a negative regulatory loop as key mechanisms endogenously controlling the expression dynamics of IFNα-induced antiviral genes in hepatocytes. Guided by the mathematical model, we uncovered that this regulatory loop is mediated by the transcription factor IRF2 and showed that knock-down of IRF2 results in enhanced expression of early, intermediate and late IFNα-induced antiviral genes. Co-stimulation experiments with different pro-inflammatory cytokines revealed that this amplified expression dynamics of the early, intermediate and late IFNα-induced antiviral genes can also be achieved by co-application of IFNα and interleukin1 beta (IL1β). Consistently, we found that IL1β enhances IFNα-mediated repression of viral replication. Conversely, we observed that in IL1β receptor knock-out mice replication of viruses sensitive to IFNα is increased. Thus, IL1β is capable to potentiate IFNα-induced antiviral responses and could be exploited to improve antiviral therapies.
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http://dx.doi.org/10.1371/journal.ppat.1008461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553310PMC
October 2020

A Novel Cis-Acting RNA Structural Element Embedded in the Core Coding Region of the Hepatitis C Virus Genome Directs Internal Translation Initiation of the Overlapping Core+1 ORF.

Int J Mol Sci 2020 Sep 22;21(18). Epub 2020 Sep 22.

Molecular Virology Laboratory, Hellenic Pasteur Institute (HPI), 11521 Athens, Greece.

Hepatitis C virus (HCV) genome translation is initiated via an internal ribosome entry site (IRES) embedded in the 5'-untranslated region (5'UTR). We have earlier shown that the conserved RNA stem-loops (SL) SL47 and SL87 of the HCV core-encoding region are important for viral genome translation in cell culture and in vivo. Moreover, we have reported that an open reading frame overlapping the core gene in the +1 frame (core+1 ORF) encodes alternative translation products, including a protein initiated at the internal AUG codons 85/87 of this frame (nt 597-599 and 603-605), downstream of SL87, which is designated core+1/Short (core+1/S). Here, we provide evidence for SL47 and SL87 possessing a novel cis-acting element that directs the internal translation initiation of core+1/S. Firstly, using a bicistronic dual luciferase reporter system and RNA-transfection experiments, we found that nucleotides 344-596 of the HCV genotype-1a and -2a genomes support translation initiation at the core+1 frame AUG codons 85/87, when present in the sense but not the opposite orientation. Secondly, site-directed mutagenesis combined with an analysis of ribosome-HCV RNA association elucidated that SL47 and SL87 are essential for this alternative translation mechanism. Finally, experiments using cells transfected with JFH1 replicons or infected with virus-like particles showed that core+1/S expression is independent from the 5'UTR IRES and does not utilize the polyprotein initiation codon, but it requires intact SL47 and SL87 structures. Thus, SL47 and SL87, apart from their role in viral polyprotein translation, are necessary elements for mediating the internal translation initiation of the alternative core+1/S ORF.
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http://dx.doi.org/10.3390/ijms21186974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7554737PMC
September 2020

Structures and distributions of SARS-CoV-2 spike proteins on intact virions.

Nature 2020 12 17;588(7838):498-502. Epub 2020 Aug 17.

Structural Studies Division, Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude. Heavily glycosylated S trimers bind to the angiotensin-converting enzyme 2 receptor and mediate entry of virions into target cells. S exhibits extensive conformational flexibility: it modulates exposure of its receptor-binding site and subsequently undergoes complete structural rearrangement to drive fusion of viral and cellular membranes. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy, but the structure and distribution of S on the virion surface remain unknown. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions and determine the high-resolution structure, conformational flexibility and distribution of S trimers in situ on the virion surface. These results reveal the conformations of S on the virion, and provide a basis from which to understand interactions between S and neutralizing antibodies during infection or vaccination.
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http://dx.doi.org/10.1038/s41586-020-2665-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116492PMC
December 2020

A Non-Replicative Role of the 3' Terminal Sequence of the Dengue Virus Genome in Membranous Replication Organelle Formation.

Cell Rep 2020 07;32(1):107859

Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany; German Center for Infection Research (DZIF), Heidelberg Partner Site, University, 69120 Heidelberg, Germany. Electronic address:

Dengue virus (DENV) and Zika virus (ZIKV), members of the Flavivirus genus, rearrange endoplasmic reticulum membranes to induce invaginations known as vesicle packets (VPs), which are the assumed sites for viral RNA replication. Mechanistic information on VP biogenesis has so far been difficult to attain due to the necessity of studying their formation under conditions of viral replication, where perturbations reducing replication will inevitably impact VP formation. Here, we report a replication-independent expression system, designated pIRO (plasmid-induced replication organelle formation) that induces bona fide DENV and ZIKV VPs that are morphologically indistinguishable from those in infected cells. Using this system, we demonstrate that sequences in the 3' terminal RNA region of the DENV, but not the ZIKV genome, contribute to VP formation in a non-replicative manner. These results validate the pIRO system that opens avenues for mechanistically dissecting virus replication from membrane reorganization.
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http://dx.doi.org/10.1016/j.celrep.2020.107859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7351112PMC
July 2020

Critical Role of Type III Interferon in Controlling SARS-CoV-2 Infection in Human Intestinal Epithelial Cells.

Cell Rep 2020 07 19;32(1):107863. Epub 2020 Jun 19.

Research Group "Cellular polarity and viral infection," German Cancer Research Center (DKFZ), Heidelberg 69120, Germany; Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg 69120, Germany. Electronic address:

Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) is an unprecedented worldwide health problem that requires concerted and global approaches to stop the coronavirus 2019 (COVID-19) pandemic. Although SARS-CoV-2 primarily targets lung epithelium cells, there is growing evidence that the intestinal epithelium is also infected. Here, using both colon-derived cell lines and primary non-transformed colon organoids, we engage in the first comprehensive analysis of the SARS-CoV-2 life cycle in human intestinal epithelial cells (hIECs). Our results demonstrate that hIECs fully support SARS-CoV-2 infection, replication, and production of infectious de novo virus particles. We found that viral infection elicits an extremely robust intrinsic immune response where interferon-mediated responses are efficient at controlling SARS-CoV-2 replication and de novo virus production. Taken together, our data demonstrate that hIECs are a productive site of SARS-CoV-2 replication and suggest that the enteric phase of SARS-CoV-2 may participate in the pathologies observed in COVID-19 patients by contributing to increasing patient viremia and fueling an exacerbated cytokine response.
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http://dx.doi.org/10.1016/j.celrep.2020.107863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7303637PMC
July 2020

The Hepatitis B Virus Envelope Proteins: Molecular Gymnastics Throughout the Viral Life Cycle.

Annu Rev Virol 2020 09 29;7(1):263-288. Epub 2020 Jun 29.

Department of Infectious Diseases, University of Heidelberg, 69120 Heidelberg, Germany; email:

New hepatitis B virions released from infected hepatocytes are the result of an intricate maturation process that starts with the formation of the nucleocapsid providing a confined space where the viral DNA genome is synthesized via reverse transcription. Virion assembly is finalized by the enclosure of the icosahedral nucleocapsid within a heterogeneous envelope. The latter contains integral membrane proteins of three sizes, collectively known as hepatitis B surface antigen, and adopts multiple conformations in the course of the viral life cycle. The nucleocapsid conformation depends on the reverse transcription status of the genome, which in turn controls nucleocapsid interaction with the envelope proteins for virus exit. In addition, after secretion the virions undergo a distinct maturation step during which a topological switch of the large envelope protein confers infectivity. Here we review molecular determinants for envelopment and models that postulate molecular signals encoded in the capsid scaffold conducive or adverse to the recruitment of envelope proteins.
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http://dx.doi.org/10.1146/annurev-virology-092818-015508DOI Listing
September 2020

Hepatitis C virus exploits cyclophilin A to evade PKR.

Elife 2020 06 16;9. Epub 2020 Jun 16.

Division of Infection and Immunity, University College London, London, United Kingdom.

Counteracting innate immunity is essential for successful viral replication. Host cyclophilins (Cyps) have been implicated in viral evasion of host antiviral responses, although the mechanisms are still unclear. Here, we show that hepatitis C virus (HCV) co-opts the host protein CypA to aid evasion of antiviral responses dependent on the effector protein kinase R (PKR). Pharmacological inhibition of CypA rescues PKR from antagonism by HCV NS5A, leading to activation of an interferon regulatory factor-1 (IRF1)-driven cell intrinsic antiviral program that inhibits viral replication. These findings further the understanding of the complexity of Cyp-virus interactions, provide mechanistic insight into the remarkably broad antiviral spectrum of Cyp inhibitors, and uncover novel aspects of PKR activity and regulation. Collectively, our study identifies a novel antiviral mechanism that harnesses cellular antiviral immunity to suppress viral replication.
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http://dx.doi.org/10.7554/eLife.52237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7297535PMC
June 2020

A Coupled Mathematical Model of the Intracellular Replication of Dengue Virus and the Host Cell Immune Response to Infection.

Front Microbiol 2020 29;11:725. Epub 2020 Apr 29.

Center for Functional Genomics of Microbes, Institute of Bioinformatics, University Medicine Greifswald, Greifswald, Germany.

Dengue virus (DV) is a positive-strand RNA virus of the genus. It is one of the most prevalent mosquito-borne viruses, infecting globally 390 million individuals per year. The clinical spectrum of DV infection ranges from an asymptomatic course to severe complications such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), the latter because of severe plasma leakage. Given that the outcome of infection is likely determined by the kinetics of viral replication and the antiviral host cell immune response (HIR) it is of importance to understand the interaction between these two parameters. In this study, we use mathematical modeling to characterize and understand the complex interplay between intracellular DV replication and the host cells' defense mechanisms. We first measured viral RNA, viral protein, and virus particle production in Huh7 cells, which exhibit a notoriously weak intrinsic antiviral response. Based on these measurements, we developed a detailed intracellular DV replication model. We then measured replication in IFN competent A549 cells and used this data to couple the replication model with a model describing IFN activation and production of IFN stimulated genes (ISGs), as well as their interplay with DV replication. By comparing the cell line specific DV replication, we found that host factors involved in replication complex formation and virus particle production are crucial for replication efficiency. Regarding possible modes of action of the HIR, our model fits suggest that the HIR mainly affects DV RNA translation initiation, cytosolic DV RNA degradation, and naïve cell infection. We further analyzed the potential of direct acting antiviral drugs targeting different processes of the DV lifecycle and found that targeting RNA synthesis and virus assembly and release are the most promising anti-DV drug targets.
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http://dx.doi.org/10.3389/fmicb.2020.00725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7200986PMC
April 2020
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