Construction of yellow fever virus subgenomic replicons by yeast-based homologous recombination cloning technique

Queiroz, Sabrina R.A.; Silva, Andréa N.M.R.; Santos, Jefferson; Marques, Ernesto T. A.; Bertani, Giovani R.; Gil, Laura Helena Vega Gonzales

doi:10.1590/s0001-37652013005000008

Cited by 7 publications

(9 citation statements)

References 41 publications

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“…This interaction occurs in a region (the interaction domain of RNApol domain) that is conserved in several flaviviruses and that is, therefore, likely to be relevant to the genus. Although eIF3L overexpression and plaque reduction assays (using an YFV replicon [13]) demonstrated a slight (if any) inhibition of YFV replication, we were able to demonstrate that eIF3L overexpression facilitates translation, with potential implications in viral replication.…”

Section: Introductionmentioning

confidence: 65%

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The eukaryotic translation initiation factor 3 subunit L protein interacts with FlavivirusNS5 and may modulate yellow fever virus replication

Morais

Terzian

Duarte

et al. 2013

Virol J

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BackgroundYellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and the expansion of YFV-endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, which are essential for viral replication, and the interactions between NS5 and cellular proteins have been studied to better understand viral replication. The aim of this study was to characterize the interaction of the NS5 protein with eukaryotic translation initiation factor 3 subunit L (eIF3L) and to evaluate the role of eIF3L in yellow fever replication.MethodsTo identify interactions of YFV NS5 with cellular proteins, we performed a two-hybrid screen using the YFV NS5 RdRp domain as bait with a human cDNA library, and RNApol deletion mutants were generated and analyzed using the two-hybrid system for mapping the interactions. The RNApol region involved was segmented into three fragments and analyzed using an eIF3L-expressing yeast strain. To map the NS5 residues that are critical for the interactions, we performed site-direct mutagenesis in segment 3 of the interaction domain (ID) and confirmed the interaction using in vitro assays and in vivo coimmunoprecipitation. The significance of eIF3L for YFV replication was investigated using eIF3L overexpression and RNA interference.ResultsIn this work, we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed using in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs at a region (the interaction domain of the RNApol domain) that is conserved in several flaviviruses and that is, therefore, likely to be relevant to the genus. eIF3L overexpression and plaque reduction assays showed a slight effect on YFV replication, indicating that the interaction of eIF3L with YFV NS5 may play a role in YFV replication.ConclusionsAlthough the precise function of eIF3L on interactions with viral proteins is not entirely understood, these results indicate an interaction of eIF3L with YF NS5 and that eIF3L overexpression facilitates translation, which has potential implications for virus replication.

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Section: Introductionmentioning

confidence: 65%

“…For overexpression, BHK-21-Rep-YFV17D LucNeoIres cells, a stable bicistronic dual-reporter YFV replicon cell line expressing the neomycin phosphotransferase resistance gene and the reporter gene firefly luciferase [13], were used. The cells were transfected with the pCDNAFlag-eIF3L plasmid or an empty vector.…”

Section: Resultsmentioning

confidence: 99%

The eukaryotic translation initiation factor 3 subunit L protein interacts with FlavivirusNS5 and may modulate yellow fever virus replication

Morais

Terzian

Duarte

et al. 2013

Virol J

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“…Thus, we report the first reverse genetics system for IBDV constructed from homologous recombination in yeast. This method has been described in the literature for the genetic manipulation of other viruses, such as Yellow Fever ( Queiroz et al , 2013 ) and HIV-1 ( Marozsan and Arts, 2003 ) and it has shown to be efficient and easier to perform compared to the use of restriction enzymes and in vitro ligation.…”

Section: Discussionmentioning

confidence: 99%

Efficient assembly of full-length infectious clone of Brazilian IBDV isolate by homologous recombination in yeast

Silva¹,

Arenhart²,

Santos³

et al. 2014

Braz. J. Microbiol.

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The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world’s first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br) was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br) showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development.

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“…1c and Table 2). Self-replicating subgenomic replicons are genetically engineered viral RNA molecules that are shorter than full-length viral genomes and are capable of replication but incapable of producing virions [22,29,62,70]. However, they can be packaged into viral particles if viral coat proteins are provided [62].…”

Section: General Principles Of Live Cell Reporter Systemsmentioning

confidence: 99%

“…Self-replicating subgenomic replicons are genetically engineered viral RNA molecules that are shorter than full-length viral genomes and are capable of replication but incapable of producing virions [22,29,62,70]. However, they can be packaged into viral particles if viral coat proteins are provided [62]. For Flaviviridae viruses, such as CSFV, two self-replicating RNAs (replicons) were produced by replacing the coding region for C to E2 of CSFV with the Rluc sequence or the coding region for C to E1 with the Rluc-2A sequence [63].…”

Section: General Principles Of Live Cell Reporter Systemsmentioning

confidence: 99%

Live Cell Reporter Systems for Positive-Sense Single Strand RNA Viruses

Ren

Peng

Chen

et al. 2016

Appl Biochem Biotechnol

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Cell-based reporter systems have facilitated studies of viral replication and pathogenesis, virus detection, and drug susceptibility testing. There are three types of cell-based reporter systems that express certain reporter protein for positive-sense single strand RNA virus infections. The first type is classical reporter system, which relies on recombinant virus, reporter virus particle, or subgenomic replicon. During infection with the recombinant virus or reporter virus particle, the reporter protein is expressed and can be detected in real time in a dose-dependent manner. Using subgenomic replicon, which are genetically engineered viral RNA molecules that are capable of replication but incapable of producing virions, the translation and replication of the replicon could be tracked by the accumulation of reporter protein. The second type of reporter system involves genetically engineered cells bearing virus-specific protease cleavage sequences, which can sense the incoming viral protease. The third type is based on viral replicase, which can report the specific virus infection via detection of the incoming viral replicase. This review specifically focuses on the major technical breakthroughs in the design of cell-based reporter systems and the application of these systems to the further understanding and control of viruses over the past few decades.

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Construction of yellow fever virus subgenomic replicons by yeast-based homologous recombination cloning technique

Cited by 7 publications

References 41 publications

The eukaryotic translation initiation factor 3 subunit L protein interacts with FlavivirusNS5 and may modulate yellow fever virus replication

The eukaryotic translation initiation factor 3 subunit L protein interacts with FlavivirusNS5 and may modulate yellow fever virus replication

Efficient assembly of full-length infectious clone of Brazilian IBDV isolate by homologous recombination in yeast

Live Cell Reporter Systems for Positive-Sense Single Strand RNA Viruses

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