Abstract
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.
Highlights
This article is an open access articleThe members of Picornaviridae including, foot-and-mouth disease virus (FMDV), human enterovirus 71, and hand, foot-and-mouth disease virus, are positive-sense single-strandedRNA viruses
The FMDV minigenome provides an groups to study the replication of RNA viruses
Replicons or minigenomes are defined as opportunity for molecular biology research related to FMDV replication, transcription, self-replicating and but non-infectious
Summary
This article is an open access articleThe members of Picornaviridae including, foot-and-mouth disease virus (FMDV), human enterovirus 71, and hand, foot-and-mouth disease virus, are positive-sense single-strandedRNA viruses. The genomic RNA of approximately 8500 nucleotides is enclosed within a non-enveloped capsid with an icosahedral structure [1,2]. By viral proteases to yield three intermediate protein precursors (P1-3). The P1 protein precursor encodes four structural proteins, VP1-4, to form the viral capsid, while L protein and those encoded by the P2 (2A, 2B, 2C) and P3. The coding region is flanked by untranslated regions (UTRs) at 50 and 30 ends, so-called 50 UTR and 30 UTR, respectively, and downstream of the 30 UTR is a poly A tail. These non-coding regions are the crucial cis-acting elements involved in viral replication, transcription, and translation
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