Subgenomic Reporter RNA System for Detection of Alphavirus Infection in Mosquitoes

Steel, J. Jordan; Franz, Alexander W. E.; Sánchez-Vargas, Irma; Olson, Ken E.; Geiss, Brian J.

doi:10.1371/journal.pone.0084930

Cited by 7 publications

(13 citation statements)

References 48 publications

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“…However, the use of arbovirus trans-replicase systems has mostly been limited to mammalian cells. Thus far, this approach has been used to study only SFV in Drosophila cells (42) and SINV in mosquito cells (43).…”

mentioning

confidence: 99%

A Chikungunya Virus trans -Replicase System Reveals the Importance of Delayed Nonstructural Polyprotein Processing for Efficient Replication Complex Formation in Mosquito Cells

Bartholomeeusen

Utt

Coppens

et al. 2018

J Virol

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Chikungunya virus (CHIKV) is a medically important alphavirus that is transmitted by and mosquitoes. The viral replicase complex consists of four nonstructural proteins (nsPs) expressed as a polyprotein precursor and encompasses all enzymatic activities required for viral RNA replication. nsPs interact with host components of which most are still poorly understood, especially in mosquitos. A CHIKV -replicase system that allows the uncoupling of RNA replication and nsP expression was adapted to mosquito cells and subsequently used for analysis of universal and host-specific effects of 17 different nonstructural polyprotein (ns-polyprotein) mutations. It was found that mutations blocking nsP enzymatic activities as well as insertions of enhanced green fluorescent protein (EGFP) into different nsPs had similar effects on-replicase activity regardless of the host (i.e., mammalian or mosquito). Mutations that slow down or accelerate ns-polyprotein processing generally had no effect or reduced -replicase activity in mammalian cells, while in mosquito cells most of them increased-replicase activity prominently. Increased RNA replication in mosquito cells was counteracted by an antiviral RNA interference (RNAi) response. Substitution of the W258 residue in the membrane binding peptide of nsP1 resulted in a temperature-sensitive defect, in the context of both the -replicase and infectious CHIKV. The defect was compensated for by secondary mutations selected during passaging of mutant CHIKV. These findings demonstrate the value of alphavirus-replicase systems for studies of viral RNA replication and virus-host interactions. Chikungunya virus is an important mosquito-transmitted human pathogen. This virus actively replicates in mosquitoes, but the underlying molecular mechanisms and interactions of viral and host components are poorly understood. This is partly due to the lack of reliable systems for functional analysis of viral nonstructural polyproteins (ns-polyproteins) and nonstructural proteins (nsPs) in mosquito cells. Adaption of a CHIKV -replicase system allowed study of the effects of mutations in the ns-polyprotein on RNA replication in cells derived from mammalian and mosquito hosts. We found that a slowdown of ns-polyprotein processing facilitates replication complex formation and/or functioning in mosquito cells and that this process is antagonized by the natural RNAi defense system present in mosquito cells. The mosquito-adapted CHIKV-replicase system represents a valuable tool to study alphavirus-mosquito interactions at the molecular level and to develop advanced antiviral strategies.

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mentioning

confidence: 99%

A Chikungunya Virus trans -Replicase System Reveals the Importance of Delayed Nonstructural Polyprotein Processing for Efficient Replication Complex Formation in Mosquito Cells

Bartholomeeusen

Utt

Coppens

et al. 2018

J Virol

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“…Although recombinant viruses can be infectious, the viruses expressing fluorescent proteins tend to be attenuated due to the modification of their genomes [72]. Furthermore, many viruses Reporter gene is fused with viral structural protein or placing the reporter gene cassette behind the 3' terminus of the viral structural protein gene.…”

Section: General Principles Of Live Cell Reporter Systemsmentioning

confidence: 99%

“…This cell line expressed high levels of luciferase activity during infection with SINV and provided a sensitive assay for titering the virus [58]. Moreover, a species-specific insect cellbased system for detecting wild-type SINV infection was developed, which produced SINV minigenome containing a virus inducible promoter and a fluorescent gene [72]. During the virus infection, the fluorescent gene was specially induced and expressed in the cells [72].…”

Section: Replicase-sensor Reporter Systems For (+) Ssrna Virusmentioning

confidence: 99%

“…Moreover, a species-specific insect cellbased system for detecting wild-type SINV infection was developed, which produced SINV minigenome containing a virus inducible promoter and a fluorescent gene [72]. During the virus infection, the fluorescent gene was specially induced and expressed in the cells [72]. In another system, replicon-defective plasmids derived from the SINV were constructed by replacing the structural genes of SINV with reporter genes and deleting 1139 bp in the NS 4 gene of the virus [44].…”

Section: Replicase-sensor Reporter Systems For (+) Ssrna Virusmentioning

confidence: 99%

“…Cell-based reporter systems are also well suited for use in clinical examinations and epidemiological surveillance [44]. The subgenomic reporter RNA systems developed by Steel and his colleagues were relatively species specific and allowed for rapid and simple visual detection of the wild-type viruses in mosquito cells [72]. Moreover, the replicon-defective reporter gene assay could detect a variety of Alphaviruses from tissue cultures with a limit of detection between one and ten (plaque-forming unit) PFU for SINV while other RNA viruses, such as the Japanese encephalitis virus and Tahyna virus, displayed negative results with this system [44].…”

Section: Reporter Systems Focused On Antiviral Researchmentioning

confidence: 99%

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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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Transgene-induced cell death following dengue-2 virus infection in Aedes aegypti

Carvalho

Costa-da-Silva

Petersen

et al. 2023

Sci Rep

Self Cite

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Dengue viruses (DENVs) are mosquito-borne flaviviruses causing millions of human infections each year and pose a challenge for public health systems worldwide. Aedes aegypti is the principal vector species transmitting DENVs to humans. Controlling Ae. aegypti is difficult due to the abundance of breeding sites and increasing insecticide resistance in the vector populations. Developing new vector control strategies is critical for decreasing the disease burden. One potential approach is genetically replacing Ae. aegypti populations with vector populations highly resistant to DENV transmission. Here, we focus on an alternative strategy for generating dengue 2 virus (DENV-2) resistance in genetically-modified Ae. aegypti in which the mosquitoes express an inactive form of Michelob_x (Mx), an antagonist of the Inhibitor of Apoptosis (IAP), to induce apoptosis in those cells in which actively replicating DENV-2 is present. The inactive form of Mx was flanked by the RRRRSAG cleavage motif, which was recognized by the NS2B/NS3 protease of the infecting DENV-2 thereby releasing and activating Mx which then induced apoptosis. Our transgenic strain exhibited a significantly higher mortality rate than the non-transgenic control when infected with DENV-2. We also transfected a DNA construct containing inactive Mx fused to eGFP into C6/36 mosquito cells and indirectly observed Mx activation on days 3 and 6 post-DENV-2 infections. There were clear signs that the viral NS2B/NS3 protease cleaved the transgene, thereby releasing Mx protein into the cytoplasm, as was confirmed by the detection of eGFP expression in infected cells. The present study represents proof of the concept that virus infection can be used to induce apoptosis in infected mosquito cells.

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Subgenomic Reporter RNA System for Detection of Alphavirus Infection in Mosquitoes

Cited by 7 publications

References 48 publications

A Chikungunya Virus trans -Replicase System Reveals the Importance of Delayed Nonstructural Polyprotein Processing for Efficient Replication Complex Formation in Mosquito Cells

A Chikungunya Virus trans -Replicase System Reveals the Importance of Delayed Nonstructural Polyprotein Processing for Efficient Replication Complex Formation in Mosquito Cells

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

Transgene-induced cell death following dengue-2 virus infection in Aedes aegypti

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