The rocaglate CR-31-B (-) inhibits SARS-CoV-2 replication at non-cytotoxic, low nanomolar concentrations in vitro and ex vivo

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a betacoronavirus in the subgenus Sarbecovirus causes a respiratory disease with varying symptoms referred to as coronavirus disease 2019 (COVID-19) and is responsible for a pandemic that started in early 2020. With no vaccines or effective antiviral treatments available, and infection and fatality numbers continuing to increase globally, the quest for novel therapeutic solutions remains an urgent priority. Rocaglates, a class of plant-derived cyclopenta[b]benzofurans, exhibit broad-spectrum antiviral activity against positive- and negative-sense RNA viruses. This compound class inhibits eukaryotic initiation factor 4A (eIF4A)-dependent mRNA translation initiation, resulting in strongly reduced viral RNA translation. The synthetic rocaglate CR-31-B (-) has previously been shown to inhibit the replication of human coronaviruses, such as HCoV-229E and MERS-CoV, as well as Zika-, Lassa-, Crimean Congo hemorrhagic fever virus in primary cells. Here, we assessed the antiviral activity of CR-31-B (-) against SARS-CoV-2 using both in vitro and ex vivo cell culture models. In African green monkey Vero E6 cells, CR-31-B (-) inhibited SARS-CoV-2 replication with an EC50 of ~1.8 nM. In line with this, viral protein accumulation and replication/transcription complex formation were found to be strongly reduced by this compound. In an ex vivo infection system using human airway epithelial cells, CR-31-B (-) was found to cause a massive reduction of SARS-CoV-2 titers by about 4 logs to nearly non-detectable levels. The data reveal a potent anti-SARS-CoV-2 activity by CR-31-B (-), corroborating previous results obtained for other coronaviruses and supporting the idea that rocaglates may be used in first-line antiviral intervention strategies against novel and emerging RNA virus outbreaks.

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Antagonistic Antiviral Activity between IFN-Lambda and IFN-Alpha against Lethal Crimean-Congo Hemorrhagic Fever Virus In Vitro

PLoS ONE ◽

10.1371/journal.pone.0116816 ◽

2015 ◽

Vol 10 (2) ◽

pp. e0116816 ◽

Cited By ~ 8

Author(s):

Licia Bordi ◽

Eleonora Lalle ◽

Claudia Caglioti ◽

Damiano Travaglini ◽

Daniele Lapa ◽

...

Keyword(s):

Antiviral Activity ◽

Hemorrhagic Fever ◽

Fever Virus ◽

Crimean Congo Hemorrhagic Fever ◽

Hemorrhagic Fever Virus ◽

Ifn Alpha ◽

Ifn Lambda

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Development and Characterization of a cDNA-Launch Recombinant Simian Hemorrhagic Fever Virus Expressing Enhanced Green Fluorescent Protein: ORF 2b’ Is Not Required for In Vitro Virus Replication

Viruses ◽

10.3390/v13040632 ◽

2021 ◽

Vol 13 (4) ◽

pp. 632

Author(s):

Yingyun Cai ◽

Shuiqing Yu ◽

Ying Fang ◽

Laura Bollinger ◽

Yanhua Li ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Cell Lines ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Infectious Clone ◽

Hemorrhagic Fever ◽

Simian Hemorrhagic Fever Virus ◽

Hemorrhagic Fever Virus ◽

Green Fluorescent

Simian hemorrhagic fever virus (SHFV) causes acute, lethal disease in macaques. We developed a single-plasmid cDNA-launch infectious clone of SHFV (rSHFV) and modified the clone to rescue an enhanced green fluorescent protein-expressing rSHFV-eGFP that can be used for rapid and quantitative detection of infection. SHFV has a narrow cell tropism in vitro, with only the grivet MA-104 cell line and a few other grivet cell lines being susceptible to virion entry and permissive to infection. Using rSHFV-eGFP, we demonstrate that one cricetid rodent cell line and three ape cell lines also fully support SHFV replication, whereas 55 human cell lines, 11 bat cell lines, and three rodent cells do not. Interestingly, some human and other mammalian cell lines apparently resistant to SHFV infection are permissive after transfection with the rSHFV-eGFP cDNA-launch plasmid. To further demonstrate the investigative potential of the infectious clone system, we introduced stop codons into eight viral open reading frames (ORFs). This approach suggested that at least one ORF, ORF 2b’, is dispensable for SHFV in vitro replication. Our proof-of-principle experiments indicated that rSHFV-eGFP is a useful tool for illuminating the understudied molecular biology of SHFV.

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Hepatocyte pathway alterations in response to in vitro Crimean Congo hemorrhagic fever virus infection

Virus Research ◽

10.1016/j.virusres.2013.10.013 ◽

2014 ◽

Vol 179 ◽

pp. 187-203 ◽

Cited By ~ 15

Author(s):

Christophe Fraisier ◽

Raquel Rodrigues ◽

Vinh Vu Hai ◽

Maya Belghazi ◽

Stéphanie Bourdon ◽

...

Keyword(s):

Virus Infection ◽

Hemorrhagic Fever ◽

Fever Virus ◽

Crimean Congo Hemorrhagic Fever ◽

Hemorrhagic Fever Virus

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Multiple Mechanisms Control Phosphorylation of PHAS-I in Five (S/T)P Sites That Govern Translational Repression

Molecular and Cellular Biology ◽

10.1128/mcb.20.10.3558-3567.2000 ◽

2000 ◽

Vol 20 (10) ◽

pp. 3558-3567 ◽

Cited By ~ 137

Author(s):

Isabelle Mothe-Satney ◽

Daqing Yang ◽

Patrick Fadden ◽

Timothy A. J. Haystead ◽

John C. Lawrence

Keyword(s):

Mrna Translation ◽

Hek293 Cells ◽

Initiation Factor ◽

Translational Repression ◽

Phosphorylation Sites ◽

Translational Repressor ◽

Eukaryotic Initiation Factor 4E ◽

Dependent Processes ◽

Constitutive Phosphorylation

ABSTRACT Control of the translational repressor, PHAS-I, was investigated by expressing proteins with Ser/Thr → Ala mutations in the five (S/T)P phosphorylation sites. Results of experiments with HEK293 cells reveal at least three levels of control. At one extreme is nonregulated phosphorylation, exemplified by constitutive phosphorylation of Ser82. At an intermediate level, amino acids and insulin stimulate the phosphorylation of Thr36, Thr45, and Thr69 via mTOR-dependent processes that function independently of other sites in PHAS-I. At the third level, the extent of phosphorylation of one site modulates the phosphorylation of another. This control is represented by Ser64 phosphorylation, which depends on the phosphorylation of all three TP sites. The five sites have different influences on the electrophoretic properties of PHAS-I and on the affinity of PHAS-I for eukaryotic initiation factor 4E (eIF4E). Phosphorylation of Thr45 or Ser64 results in the most dramatic decreases in eIF4E binding in vitro. However, each of the sites influences mRNA translation, either directly by modulating the binding affinity of PHAS-I and eIF4E or indirectly by affecting the phosphorylation of other sites.

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Small interfering RNAs are highly effective inhibitors regarding Crimean-Congo hemorrhagic fever virus replication in vitro

10.1101/2020.05.13.093047 ◽

2020 ◽

Author(s):

Fanni Földes ◽

Mónika Madai ◽

Henrietta Papp ◽

Gábor Kemenesi ◽

Brigitta Zana ◽

...

Keyword(s):

Rna Interference ◽

Hemorrhagic Fever ◽

Fever Virus ◽

World Health ◽

Dependent Manner ◽

Small Interfering Rnas ◽

Crimean Congo Hemorrhagic Fever ◽

Hemorrhagic Fever Virus ◽

Antiviral Therapies

AbstractCrimean-Congo hemorrhagic fever virus (CCHFV) is one of the prioritized diseases of World Health Organization, considering its potential to create a public health emergency and more importantly, the absence of efficacious drugs and/or vaccines regarding treatment. The highly lethal nature characteristic to CCHFV restricts research to BSL-4 laboratories, which complicates effective research and developmental strategies. In consideration of antiviral therapies, RNA interference can be used to suppress viral replication by targeting viral genes. RNA interference uses small interfering RNAs (siRNAs) to silence genes. The aim of our study was to design siRNAs that inhibit CCHFV replication and can serve as a basis for further antiviral therapies. A549 cells were infected with CCHFV after transfection with the siRNAs. Following 72 hours, nucleic acid from the supernatant was extracted for Droplet Digital PCR analysis. Among the investigated siRNAs we identified four effective candidates against all three segments of CCHF genome: one for the S and M segments, whilst two for the L segment. Consequently, blocking any segment of CCHFV leads to changes in the virus copy number that indicates an antiviral effect of the siRNAs in vitro. The most active siRNAs were demonstrated a specific inhibitory effect against CCHFV in a dose-dependent manner. In summary, we demonstrated the ability of specific siRNAs to inhibit CCHFV replication in vitro. This promising result can be used in future anti-CCHFV therapy developments.

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Nanodroplet-Benzalkonium Chloride Formulation Demonstrates In Vitro and Ex- Vivo Broad-Spectrum Antiviral Activity Against SARS-CoV-2 and other Enveloped Viruses

10.21203/rs.3.rs-362864/v1 ◽

2021 ◽

Author(s):

Jessie Pannu ◽

Susan Ciotti ◽

Shyamala Ganesan ◽

George Arida ◽

Chad Costley ◽

...

Keyword(s):

Antiviral Activity ◽

Broad Spectrum ◽

Benzalkonium Chloride ◽

Ex Vivo ◽

Viral Disease ◽

Influenza B ◽

Human Coronavirus ◽

Enveloped Viruses ◽

Nasal Irritation

Abstract Objective: The Covid-19 pandemic has highlighted the importance of aerosolized droplets inhaled into the nose in the transmission of respiratory viral disease. Inactivating pathogenic viruses at the nasal port of entry may reduce viral loads, thereby reducing infection, transmission and spread. In this communication, we demonstrate safe and broad anti-viral activity of oil-in-water nanoemulsion (nanodroplet) formulation containing the potent antiseptic 0.13% Benzalkonium Chloride (NE-BZK). Results: We have demonstrated that NE-BZK exhibits broad-spectrum, long-lasting antiviral activity with >99.9% in vitro killing of enveloped viruses including SARS-CoV-2, human coronavirus, RSV, and influenza B. In vitro and ex-vivo studies demonstrated continued killing of >99.99% of human coronavirus with diluted NE-BZK and persistent for 8 hours post application, respectively. The repeated application of NE-BZK, twice daily for 2 weeks into rabbit nostrils demonstrated its safety with no nasal irritation. These findings demonstrate that formulating BZK into the proprietary nanodroplets offers a safe and effective antiviral and a significant addition to strategies to combat the spread of respiratory viral infectious diseases.

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Binding of eukaryotic translation initiation factor 4E (eIF4E) to eIF4G represses translation of uncapped mRNA.

Molecular and Cellular Biology ◽

10.1128/mcb.17.12.6876 ◽

1997 ◽

Vol 17 (12) ◽

pp. 6876-6886 ◽

Cited By ~ 52

Author(s):

S Z Tarun ◽

A B Sachs

Keyword(s):

Translation Initiation ◽

Mrna Translation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Eukaryotic Translation Initiation Factor ◽

Eukaryotic Translation ◽

Eukaryotic Translation Initiation ◽

Stimulation Of

mRNA translation in crude extracts from the yeast Saccharomyces cerevisiae is stimulated by the cap structure and the poly(A) tail through the binding of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) and the poly(A) tail-binding protein Pab1p. These proteins also bind to the translation initiation factor eIF4G and thereby link the mRNA to the general translational apparatus. In contrast, uncapped, poly(A)-deficient mRNA is translated poorly in yeast extracts, in part because of the absence of eIF4E and Pab1p binding sites on the mRNA. Here, we report that uncapped-mRNA translation is also repressed in yeast extracts due to the binding of eIF4E to eIF4G. Specifically, we find that mutations which weaken the eIF4E binding site on the yeast eIF4G proteins Tif4631p and Tif4632p lead to temperature-sensitive growth in vivo and the stimulation of uncapped-mRNA translation in vitro. A mutation in eIF4E which disturbs its ability to interact with eIF4G also leads to a stimulation of uncapped-mRNA translation in vitro. Finally, overexpression of eIF4E in vivo or the addition of excess eIF4E in vitro reverses these effects of the mutations. These data support the hypothesis that the eIF4G protein can efficiently stimulate translation of exogenous uncapped mRNA in extracts but is prevented from doing so as a result of its association with eIF4E. They also suggest that some mRNAs may be translationally regulated in vivo in response to the amount of free eIF4G in the cell.

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Small Interfering RNAs Are Highly Effective Inhibitors of Crimean-Congo Hemorrhagic Fever Virus Replication In Vitro

Molecules ◽

10.3390/molecules25235771 ◽

2020 ◽

Vol 25 (23) ◽

pp. 5771

Author(s):

Fanni Földes ◽

Mónika Madai ◽

Henrietta Papp ◽

Gábor Kemenesi ◽

Brigitta Zana ◽

...

Keyword(s):

Rna Interference ◽

A549 Cells ◽

Hemorrhagic Fever ◽

Fever Virus ◽

World Health ◽

Small Interfering Rnas ◽

Crimean Congo Hemorrhagic Fever ◽

Hemorrhagic Fever Virus ◽

Antiviral Therapies

Crimean-Congo hemorrhagic fever virus (CCHFV) is one of the prioritized diseases of the World Health Organization, considering its potential to create a public health emergency and, more importantly, the absence of efficacious drugs and/or vaccines for treatment. The highly pathogenic characteristic of CCHFV restricts research to BSL-4 laboratories, which complicates effective research and developmental strategies. In consideration of antiviral therapies, RNA interference can be used to suppress viral replication by targeting viral genes. RNA interference uses small interfering RNAs (siRNAs) to silence genes. The aim of our study was to design and test siRNAs in vitro that inhibit CCHFV replication and can serve as a basis for further antiviral therapies. A549 cells were infected with CCHFV after transfection with the siRNAs. Following 72 h, nucleic acid from the supernatant was extracted for RT Droplet Digital PCR analysis. Among the investigated siRNAs we identified effective candidates against all three segments of the CCHF genome. Consequently, blocking any segment of CCHFV leads to changes in the virus copy number that indicates an antiviral effect of the siRNAs. In summary, we demonstrated the ability of specific siRNAs to inhibit CCHFV replication in vitro. This promising result can be integrated into future anti-CCHFV therapy developments.

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5′-UTR recruitment of the translation initiation factor eIF4GI or DAP5 drives cap-independent translation of a subset of human mRNAs

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013678 ◽

2020 ◽

Vol 295 (33) ◽

pp. 11693-11706 ◽

Cited By ~ 1

Author(s):

Solomon A. Haizel ◽

Usha Bhardwaj ◽

Ruben L. Gonzalez ◽

Somdeb Mitra ◽

Dixie J. Goss

Keyword(s):

Translation Initiation ◽

Tumor Hypoxia ◽

Mrna Translation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Luciferase Reporter ◽

Binding Studies ◽

Specific Subset ◽

Independent Translation

During unfavorable conditions (e.g. tumor hypoxia or viral infection), canonical, cap-dependent mRNA translation is suppressed in human cells. Nonetheless, a subset of physiologically important mRNAs (e.g. hypoxia-inducible factor 1α [HIF-1α], fibroblast growth factor 9 [FGF-9], and p53) is still translated by an unknown, cap-independent mechanism. Additionally, expression levels of eukaryotic translation initiation factor 4GI (eIF4GI) and of its homolog, death-associated protein 5 (DAP5), are elevated. By examining the 5′ UTRs of HIF-1α, FGF-9, and p53 mRNAs and using fluorescence anisotropy binding studies, luciferase reporter-based in vitro translation assays, and mutational analyses, we demonstrate here that eIF4GI and DAP5 specifically bind to the 5′ UTRs of these cap-independently translated mRNAs. Surprisingly, we found that the eIF4E-binding domain of eIF4GI increases not only the binding affinity but also the selectivity among these mRNAs. We further demonstrate that the affinities of eIF4GI and DAP5 binding to these 5′ UTRs correlate with the efficiency with which these factors drive cap-independent translation of these mRNAs. Integrating the results of our binding and translation assays, we conclude that eIF4GI or DAP5 is critical for recruitment of a specific subset of mRNAs to the ribosome, providing mechanistic insight into their cap-independent translation.

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Antiviral Activity of Favipiravir (T-705) against a Broad Range of ParamyxovirusesIn Vitroand against Human Metapneumovirus in Hamsters

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00709-16 ◽

2016 ◽

Vol 60 (8) ◽

pp. 4620-4629 ◽

Cited By ~ 20

Author(s):

D. Jochmans ◽

S. van Nieuwkoop ◽

S. L. Smits ◽

J. Neyts ◽

R. A. M. Fouchier ◽

...

Keyword(s):

Antiviral Activity ◽

Measles Virus ◽

Rna Virus ◽

Antiviral Drug ◽

Specific Effect ◽

Human Metapneumovirus ◽

Drug Candidate ◽

Emerging Viruses ◽

Syncytial Virus

ABSTRACTThe clinical impact of infections with respiratory viruses belonging to the familyParamyxoviridaeargues for the development of antiviral therapies with broad-spectrum activity. Favipiravir (T-705) has demonstrated potent antiviral activity against multiple RNA virus families and is presently in clinical evaluation for the treatment of influenza. Here we demonstratein vitroactivity of T-705 against the paramyxoviruses human metapneumovirus (HMPV), respiratory syncytial virus, human parainfluenza virus, measles virus, Newcastle disease virus, and avian metapneumovirus. In addition, we demonstrate activity against HMPV in hamsters. T-705 treatment inhibited replication of all paramyxoviruses testedin vitro, with 90% effective concentration (EC90) values of 8 to 40 μM. Treatment of HMPV-challenged hamsters with T-705 at 200 mg/kg of body weight/day resulted in 100% protection from infection of the lungs. In all treated and challenged animals, viral RNA remained detectable in the respiratory tract. The observation that T-705 treatment had a significant effect on infectious viral titers, with a limited effect on viral genome titers, is in agreement with its proposed mode of action of viral mutagenesis. However, next-generation sequencing of viral genomes isolated from treated and challenged hamsters did not reveal (hyper)mutation. Polymerase activity assays revealed a specific effect of T-705 on the activity of the HMPV polymerase. With the reported antiviral activity of T-705 against a broad range of RNA virus families, this small molecule is a promising broad-range antiviral drug candidate for limiting the viral burden of paramyxoviruses and for evaluation for treatment of infections with (re)emerging viruses, such as the henipaviruses.

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