Construction and characterization of a recombinant yellow fever virus stably expressing Gaussia luciferase

Kassar, Telissa C.; Magalhães, Tereza; Júnior, José Valter Joaquim Silva; Carvalho, Amanda G. O.; Silva, Andréa N.M.R. Da; Queiroz, Sabrina R.A.; Bertani, Giovani R.; Gil, Laura Helena Vega Gonzales

doi:10.1590/0001-3765201720160196

Cited by 7 publications

(4 citation statements)

References 33 publications

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“…CHIKV remains a potential threat to public health with no specific antiviral available [ 9 ]. In this study, we successfully constructed a replicative CHIKV reporter replicon using homologous recombination in yeast, a strategy previously used to obtain reverse genetics systems for RNA viruses, such as dengue, yellow fever, bovine viral diarrhea virus and infectious bursal disease virus (IBDV) [ 29 , 32 , 33 , 34 ]. A BHK-21 cell line expressing the GLuc-Neo-CHIKV system, the BHK-21-GLuc-nsP-CHIKV-99659, was developed and demonstrated to persistently express the replicon RNA with no change in GLuc activity over 10 passages.…”

Section: Discussionmentioning

confidence: 99%

The Antifungal Itraconazole Is a Potent Inhibitor of Chikungunya Virus Replication

Policastro

Dolci

Godoy

et al. 2022

Viruses

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Chikungunya virus (CHIKV) is the causative agent of chikungunya fever, a disabling disease that can cause long-term severe arthritis. Since the last large CHIKV outbreak in 2015, the reemergence of the virus represents a serious public health concern. The morbidity associated with viral infection emphasizes the need for the development of specific anti-CHIKV drugs. Herein, we describe the development and characterization of a CHIKV reporter replicon cell line and its use in replicon-based screenings. We tested 960 compounds from MMV/DNDi Open Box libraries and identified four candidates with interesting antiviral activities, which were confirmed in viral infection assays employing CHIKV-nanoluc and BHK-21 cells. The most noteworthy compound identified was itraconazole (ITZ), an orally available, safe, and cheap antifungal, that showed high selectivity indexes of >312 and >294 in both replicon-based and viral infection assays, respectively. The antiviral activity of this molecule has been described against positive-sense single stranded RNA viruses (+ssRNA) and was related to cholesterol metabolism that could affect the formation of the replication organelles. Although its precise mechanism of action against CHIKV still needs to be elucidated, our results demonstrate that ITZ is a potent inhibitor of the viral replication that could be repurposed as a broad-spectrum antiviral.

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Section: Discussionmentioning

confidence: 99%

The Antifungal Itraconazole Is a Potent Inhibitor of Chikungunya Virus Replication

Policastro

Dolci

Godoy

et al. 2022

Viruses

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“…Given these considerations, we chose to pursue a function-based approach to study the productive entry of a recombinant YFV that expresses a reporter enzyme only after viral entry and translation. Several flavivirus reporter systems have been developed, mainly for high-throughput screening of viral replication (17,26,(48)(49)(50)(51)(52)(53)(54)(55). In one remarkable study, Byk et al adapted a Renilla luciferase-expressing DENV reporter virus to show that the incoming DENV capsid protein must be ubiquitylated prior to viral gene expression (17).…”

Section: Discussionmentioning

confidence: 99%

A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating

Ramanathan

Zhang

Douam

et al. 2020

mBio

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While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replicationdefective yellow fever virus (YFV) entry reporter, YFVΔSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVΔSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin-and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry. IMPORTANCE Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.

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“…To generate the YFV-17D-mScarlet plasmid, the first 27 nucleotides of the sequence encoding YFV-17D NS1, the Gaussia luciferase (GLuc) gene, and a dengue virus E linker coding sequence (E stem and TMD) were amplified by PCR from pBSC-YFV-GLuc ( 75 ) (provided by L. Gil, Oswaldo Cruz Foundation). The amplified gene cassette was then inserted between the E and NS1 coding sequences of pACNR-FLYF-17D-RL (provided by C. Rice, the Rockefeller University) through Infusion-based molecular cloning, yielding pACNR-FLYF-17D-GLuc.…”

Section: Methodsmentioning

confidence: 99%

Membrane-proximal motifs encode differences in signaling strength between type I and III interferon receptors

Mesev,

Lin,

Guare

et al. 2023

Sci. Signal.

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Interferons (IFNs) play crucial roles in antiviral defenses. Despite using the same Janus-activated kinase (JAK)–signal transducer and activator of transcription (STAT) signaling cascade, type I and III IFN receptors differ in the magnitude and dynamics of their signaling in terms of STAT phosphorylation, gene transcription, and antiviral responses. These differences are not due to ligand-binding affinity and receptor abundance. Here, we investigated the ability of the intracellular domains (ICDs) of IFN receptors to differentiate between type I and III IFN signaling. We engineered synthetic, heterodimeric type I and III IFN receptors that were stably expressed at similar amounts in human cells and responded to a common ligand. We found that our synthetic type I IFN receptors stimulated STAT phosphorylation and gene expression to greater extents than did the corresponding type III IFN receptors. Furthermore, we identified short “box motifs” within ICDs that bind to JAK1 that were sufficient to encode differences between the type I and III IFN receptors. Together, our results indicate that specific regions within the ICDs of IFN receptor subunits encode different downstream signaling strengths that enable type I and III IFN receptors to produce distinct signaling outcomes.

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Construction and characterization of a recombinant yellow fever virus stably expressing Gaussia luciferase

Cited by 7 publications

References 33 publications

The Antifungal Itraconazole Is a Potent Inhibitor of Chikungunya Virus Replication

The Antifungal Itraconazole Is a Potent Inhibitor of Chikungunya Virus Replication

A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating

Membrane-proximal motifs encode differences in signaling strength between type I and III interferon receptors

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