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Construction of a Chikungunya Virus, Replicon, and Helper Plasmids for Transfection of Mammalian Cells.

ABSTRACT: The genome of Alphaviruses can be modified to produce self-replicating RNAs and virus-like particles, which are useful virological tools. In this work, we generated three plasmids for the transfection of mammalian cells: an infectious clone of Chikungunya virus (CHIKV), one that codes for the structural proteins (helper plasmid), and another one that codes nonstructural proteins (replicon plasmid). All of these plasmids contain a reporter gene (mKate2). The reporter gene in the replicon RNA and the infectious clone are synthesized from subgenomic RNA. Co-transfection with the helper and replicon plasmids has biotechnological/biomedical applications because they allow for the delivery of self-replicating RNA for the transient expression of one or more genes to the target cells.

SUBMITTER: Colunga-Saucedo M

PROVIDER: S-EPMC9864538 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Construction of a Chikungunya Virus, Replicon, and Helper Plasmids for Transfection of Mammalian Cells.

Colunga-Saucedo Mayra M Rubio-Hernandez Edson I EI Coronado-Ipiña Miguel A MA Rosales-Mendoza Sergio S Castillo Claudia G CG Comas-Garcia Mauricio M

Viruses 20221231 1

The genome of Alphaviruses can be modified to produce self-replicating RNAs and virus-like particles, which are useful virological tools. In this work, we generated three plasmids for the transfection of mammalian cells: an infectious clone of Chikungunya virus (CHIKV), one that codes for the structural proteins (helper plasmid), and another one that codes nonstructural proteins (replicon plasmid). All of these plasmids contain a reporter gene (mKate2). The reporter gene in the replicon RNA and ...[more]

PMID: 36680173

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Project description:Chikungunya virus (CHIKV), a mosquito-borne human pathogen, causes a disabling disease characterized by severe joint pain that can persist for weeks, months, or even years in patients. The nonstructural protein 3 (nsP3) plays essential roles during acute infection, but little is known about the function of nsP3 during chronic disease. Here, we used subdiffraction multicolor microscopy for spatial and temporal analysis of CHIKV nsP3 within human cells that persistently replicate replicon RNA. Round cytoplasmic granules of various sizes (i) contained nsP3 and stress granule assembly factors 1 and 2 (G3BP1/2), (ii) were next to double-stranded RNA foci and nsP1-positive structures, and (iii) were close to the nuclear membrane and the nuclear pore complex protein Nup98. Analysis of protein turnover and mobility by live-cell microscopy revealed that the granules could persist for hours to days, accumulated newly synthesized protein, and moved through the cytoplasm at various speeds. The granules also had a static internal architecture and were stable in cell lysates. Refractory cells that had cleared the noncytotoxic replicon regained the ability to respond to arsenite-induced stress. In summary, nsP3 can form uniquely stable granular structures that persist long-term within the host cell. This continued presence of viral and cellular protein complexes has implications for the study of the pathogenic consequences of lingering CHIKV infection and the development of strategies to mitigate the burden of chronic musculoskeletal disease brought about by a medically important arthropod-borne virus (arbovirus).IMPORTANCE Chikungunya virus (CHIKV) is a reemerging alphavirus transmitted by mosquitos and causes transient sickness but also chronic disease affecting muscles and joints. No approved vaccines or antivirals are available. Thus, a better understanding of the viral life cycle and the role of viral proteins can aid in identifying new therapeutic targets. Advances in microscopy and development of noncytotoxic replicons (A. Utt, P. K. Das, M. Varjak, V. Lulla, A. Lulla, A. Merits, J Virol 89:3145-3162, 2015, https://doi.org/10.1128/JVI.03213-14) have allowed researchers to study viral proteins within controlled laboratory environments over extended durations. Here we established human cells that stably replicate replicon RNA and express tagged nonstructural protein 3 (nsP3). The ability to track nsP3 within the host cell and during persistent replication can benefit fundamental research efforts to better understand long-term consequences of the persistence of viral protein complexes and thereby provide the foundation for new therapeutic targets to control CHIKV infection and treat chronic disease symptoms.

Dataset Information

Construction of a Chikungunya Virus, Replicon, and Helper Plasmids for Transfection of Mammalian Cells.

Publications

Construction of a Chikungunya Virus, Replicon, and Helper Plasmids for Transfection of Mammalian Cells.

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