A Novel Plasmid DNA-Based Foot and Mouth Disease Virus Minigenome for Intracytoplasmic mRNA Production.
Ploypailin SemkumChallika KaewborisuthNattarat ThangthamniyomSirin TheerawatanasirikulChalermpol LekcharoensukPayuda HansoongnernPongrama RamasootaPorntippa LekcharoensukPublished in: Viruses (2021)
Picornaviruses are non-enveloped, single-stranded RNA viruses that cause highly contagious diseases, such as polio and hand, foot-and-mouth disease (HFMD) in human, and foot-and-mouth disease (FMD) in animals. Reverse genetics and minigenome of picornaviruses mainly depend on in vitro transcription and RNA transfection; however, this approach is inefficient due to the rapid degradation of RNA template. Although DNA-based reverse genetics systems driven by mammalian RNA polymerase I and/or II promoters display the advantage of rescuing the engineered FMDV, the enzymatic functions are restricted in the nuclear compartment. To overcome these limitations, we successfully established a novel DNA-based vector, namely pKLS3, an FMDV minigenome containing the minimum cis-acting elements of FMDV essential for intracytoplasmic transcription and translation of a foreign gene. A combination of pKLS3 minigenome and the helper plasmids yielded the efficient production of uncapped-green florescent protein (GFP) mRNA visualized in the transfected cells. We have demonstrated the application of the pKLS3 for cell-based antiviral drug screening. Not only is the DNA-based FMDV minigenome system useful for the FMDV research and development but it could be implemented for generating other picornavirus minigenomes. Additionally, the prospective applications of this viral minigenome system as a vector for DNA and mRNA vaccines are also discussed.
Keyphrases
- circulating tumor
- nucleic acid
- cell free
- single molecule
- binding protein
- escherichia coli
- endothelial cells
- induced apoptosis
- gene expression
- stem cells
- cell death
- sars cov
- single cell
- genome wide
- immune response
- hydrogen peroxide
- dna methylation
- cell therapy
- circulating tumor cells
- small molecule
- signaling pathway
- regulatory t cells
- genetic diversity
- genome wide identification
- protein protein
- tandem mass spectrometry
- klebsiella pneumoniae