Comparative Analysis of Ebola Virus Glycoprotein Interactions With Human and Bat Cells

Kühl, Annika; Hoffmann, Markus; Müller, Marcel A.; Munster, Vincent J.; Gnirß, Kerstin; Kiene, Miriam; Tsegaye, Theodros Solomon; Behrens, Georg; Herrler, Georg; Feldmann, Heinz; Drosten, Christian; Pöhlmann, Stefan

doi:10.1093/infdis/jir306

Cited by 64 publications

(70 citation statements)

References 42 publications

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“…For example, our attempts to establish a reliable T7-based filoviral minigenome systems in a fruit bat cell line, the Rousettus aegyptiacus cell line RoNi/7.1 (Kuhl et al, 2011), failed because we were not able to express plasmid-encoded T7 in these cells (data not shown). Because fruit bats are a suspected reservoir species for EBOV (Leroy et al, 2005), bat cell-based minigenome assays would be highly beneficial for filovirus research.…”

Section: Resultsmentioning

confidence: 99%

“…Cell lines used in this study include: human embryonic kidney cells (HEK 293T; ATCC CRL-3216), African green monkey kidney cells (Vero; ATCC CRL-1586), human epithelial osteosarcoma cells (U20S; ATCC HTB-96), human hepatocellular carcinoma (Huh7) cells, human cervical carcinoma (HeLa; ATCC CCL-2), the hamster baby kidney cell line BSR-T7/5 constitutively expressing T7 (Buchholz et al, 1999), and the kidney fibroblast cell line RoNi/7.1 from the fruit bat Rousettus aegyptiacus (Kuhl et al, 2011)). All cell lines with the exception of the BSR-T7/5 and the RoNi/7.1 cells were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (50 units/ml), streptomycin (50 mg/ml) and L-glutamine (200 mM).…”

Section: Methodsmentioning

confidence: 99%

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An RNA polymerase II-driven Ebola virus minigenome system as an advanced tool for antiviral drug screening

et al. 2017

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Ebola virus (EBOV) causes a severe disease in humans with the potential for significant international public health consequences. Currently, treatments are limited to experimental vaccines and therapeutics. Therefore, research into prophylaxis and antiviral strategies to combat EBOV infections is of utmost importance. The requirement for high containment laboratories to study EBOV infection is a limiting factor for conducting EBOV research. To overcome this issue, minigenome systems have been used as valuable tools to study EBOV replication and transcription mechanisms and to screen for antiviral compounds at biosafety level 2. The most commonly used EBOV minigenome system relies on the ectopic expression of the T7 RNA polymerase (T7), which can be limiting for certain cell types. We have established an improved EBOV minigenome system that utilizes endogenous RNA polymerase II (pol II) as a driver for the synthesis of minigenome RNA. We show here that this system is as efficient as the T7-based minigenome system, but works in a wider range of cell types, including biologically relevant cell types such as bat cells. Importantly, we were also able to adapt this system to a reliable and cost-effective 96-well format antiviral screening assay with a Z-factor of 0.74, indicative of a robust assay. Using this format, we identified JG40, an inhibitor of Hsp70, as an inhibitor of EBOV replication, highlighting the potential for this system as a tool for antiviral drug screening. In summary, this updated EBOV minigenome system provides a convenient and effective means of advancing the field of EBOV research.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

An RNA polymerase II-driven Ebola virus minigenome system as an advanced tool for antiviral drug screening

et al. 2017

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“…Andrew Rambauthttp://www.virological.org and https://github.com/ebov/space-time Experimental Models: Cell Lines Bat kidney cells from Hypsignathus monstrosusKuhl et al., 2011HypNi/1.1Bat lung cells from Hypsignathus monstrosusKuhl et al., 2011HypLu/45.1Bat lung cells from Hippsideros abaeKuhl et al., 2011HipaLu/24.3Bat kidney cells from Rousettus aegyptiacusKuhl et al., 2011RoNi/7.1Bat kidney cells from Epomops buettikoferiKuhl et al., 2011EpoNi/22.1Bat lung cells from Myotis daubentoniiKuhl et al., 2011MyDauLu/47.1Human embryonic kidney cellsECACCHEK293T; RRID: CVCL_0063Human bronchial epithelial cellsReddel et al., 1989BEAS-2B; RRID: CVCL_0168Human lung alveolus cellsSmith, 1977A549; RRID: CVCL_0023Human hepatoma cell lineNakabayashi et al., 1982HuH7; RRID: CVCL_0336 Recombinant DNA GIN_1316_opt_pcDNA3.1(+): Codon optimized version of GenBank: AKG65286.1 in pCDNA3.1 plasmid vectorGenScriptGIN_1316_optpTG126 Luciferase encoding plasmidFrançois-Loïc CossetpTG126phCMV-5349 MLV Gag/Pol-encoding plasmidFrançois-Loïc CossetphCMV-5349 Sequence-Based Reagents Primers for SDM, see Table S1N/AN/A Software and Algorithms NEBaseChanger site directed mutagenesis softwareNew England Biolabshttp://nebasechanger.neb.com/GTR+I+Γ model in PhyMLGuindon et al., 2010N/APyMOL Molecular Graphics System, Version 1.8Schrödinger…”

Section: Methodsmentioning

confidence: 99%

Human Adaptation of Ebola Virus during the West African Outbreak

Urbanowicz

McClure

Sakuntabhai

et al. 2016

Cell

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192

188

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SummaryThe 2013–2016 outbreak of Ebola virus (EBOV) in West Africa was the largest recorded. It began following the cross-species transmission of EBOV from an animal reservoir, most likely bats, into humans, with phylogenetic analysis revealing the co-circulation of several viral lineages. We hypothesized that this prolonged human circulation led to genomic changes that increased viral transmissibility in humans. We generated a synthetic glycoprotein (GP) construct based on the earliest reported isolate and introduced amino acid substitutions that defined viral lineages. Mutant GPs were used to generate a panel of pseudoviruses, which were used to infect different human and bat cell lines. These data revealed that specific amino acid substitutions in the EBOV GP have increased tropism for human cells, while reducing tropism for bat cells. Such increased infectivity may have enhanced the ability of EBOV to transmit among humans and contributed to the wide geographic distribution of some viral lineages.

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“…EpoNi/22.1 African fruit bat (Epomops buettikoferi) cells (adult kidney-derived) were maintained in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U penicillin per ml, 100 mg streptomycin per ml, 1 mM sodium pyruvate, and 0.1 mM non-essential amino acids (Kuhl et al, 2011). Sendai virus (SeV) strain Cantell was grown in 10-day-old embryonating chicken eggs at 37 1C for 48 h (hours).…”

Section: Cell Lines and Virusesmentioning

confidence: 99%

Lloviu virus VP24 and VP35 proteins function as innate immune antagonists in human and bat cells

Feagins

Basler

2015

Virology

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Lloviu virus (LLOV) is a new member of the filovirus family that also includes Ebola virus (EBOV) and Marburg virus (MARV). LLOV has not been cultured; however, its genomic RNA sequence indicates the coding capacity to produce homologs of the EBOV and MARV VP24, VP35, and VP40 proteins. EBOV and MARV VP35 proteins inhibit interferon (IFN)-alpha/beta production and EBOV VP35 blocks activation of the antiviral kinase PKR. The EBOV VP24 and MARV VP40 proteins inhibit IFN signaling, albeit by different mechanisms. Here we demonstrate that LLOV VP35 suppresses Sendai virus induced IFN regulatory factor 3 (IRF3) phosphorylation, IFN-α/β production, and PKR phosphorylation. Additionally, LLOV VP24 blocks tyrosine phosphorylated STAT1 binding to karyopherin alpha 5 (KPNA5), STAT1 nuclear accumulation, and IFN-induced gene expression. LLOV VP40 lacks detectable IFN antagonist function. These activities parallel EBOV IFN inhibitory functions. EBOV and LLOV VP35 and VP24 proteins also inhibit IFN responses in bat cells. These data suggest that LLOV infection will block innate immune responses in a manner similar to EBOV.

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Comparative Analysis of Ebola Virus Glycoprotein Interactions With Human and Bat Cells

Cited by 64 publications

References 42 publications

An RNA polymerase II-driven Ebola virus minigenome system as an advanced tool for antiviral drug screening

An RNA polymerase II-driven Ebola virus minigenome system as an advanced tool for antiviral drug screening

Human Adaptation of Ebola Virus during the West African Outbreak

Lloviu virus VP24 and VP35 proteins function as innate immune antagonists in human and bat cells

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