Category Ranking
98%
Total Visits
921
Avg Visit Duration
2 minutes
Citations
20
Article Abstract
Porcine reproductive and respiratory syndrome virus (PRRSV), an Arteriviridae family enveloped RNA virus, is a major swine pathogen. Using yeast transformation-associated recombination (TAR) cloning, we efficiently generated infectious PRRSV and GFP-expressing clones, identifying transcription-regulating sequences as essential for stable foreign gene expression. Screening SARS-CoV-2 antivirals showed potent inhibition by the multitarget drug ribavirin, the polymerase inhibitors remdesivir and its metabolite GS-441524. Molnupiravir, targeting the polymerase by a different mechanism, showed reduced efficacy against PRRSV, while the protease inhibitor GC376 was ineffective. The AlphaFold-predicted structure of the PRRSV polymerase revealed conserved catalytic architecture with the SARS-CoV-2 polymerases, explaining cross-family inhibitor activity. In contrast, structural divergence in proteases correlated with GC376's inefficacy. These findings underscore the utility of the TAR cloning for arterivirus engineering, with potential applications in vector vaccine development.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC12413465 | PMC |
| http://dx.doi.org/10.1038/s44298-025-00148-3 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Development of GFP-expressing infectious clones for PRRSV using TAR cloning for antiviral drug screening.
Npj Viruses
September 2025
Faculty of Veterinary Medicine, Institute of Virology, Freie Universität Berlin, Berlin, Germany.
Porcine reproductive and respiratory syndrome virus (PRRSV), an Arteriviridae family enveloped RNA virus, is a major swine pathogen. Using yeast transformation-associated recombination (TAR) cloning, we efficiently generated infectious PRRSV and GFP-expressing clones, identifying transcription-regulating sequences as essential for stable foreign gene expression. Screening SARS-CoV-2 antivirals showed potent inhibition by the multitarget drug ribavirin, the polymerase inhibitors remdesivir and its metabolite GS-441524.
View Article and Find Full Text PDFDevelopment of a yeast-based CRISPR genome editing system for feline coronavirus.
Front Microbiol
August 2025
State Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, China.
Introduction: Feline infectious peritonitis (FIP), caused by feline coronavirus (FCoV), is a highly lethal disease characterized by systemic organ infection in cats. Current challenges of FIP include the absence of definitive diagnostic criteria, effective vaccines, and targeted therapies. Developing a robust genome editing toolkit is therefore critical to unraveling FCoV replication and pathogenesis mechanisms, elucidating viral protein functions, and identifying promising diagnostic and therapeutic targets.
View Article and Find Full Text PDFAssembly and Mutagenesis of Human Coronavirus OC43 Genomes in Yeast via Transformation-Associated Recombination.
Bio Protoc
August 2025
Department of Microbiology & Immunology, Dalhousie University, Halifax, NS, Canada.
Human coronavirus OC43 (HCoV-OC43) is an endemic "common cold" coronavirus widely used to study fundamental aspects of coronavirus biology and to test therapeutic interventions. Recently, we used a yeast-based reverse genetics strategy to create recombinant HCoV-OC43 and fluorescent reporter viruses. We assembled a DNA copy of the HCoV-OC43 genome from six linear dsDNA fragments and a linearized yeast centromeric plasmid/bacterial artificial chromosome (YCpBAC) vector in using transformation-associated recombination (TAR).
View Article and Find Full Text PDFRapid generation of HCoV-229E and HCoV-OC43 reporter viruses and replicons for antiviral research.
Front Cell Infect Microbiol
August 2025
Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Shanghai Institute of Infectious Disease and Biosecurity, Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China.
Introduction: The large size of coronavirus genome, along with the instability of certain genomic sequences, makes the construction of reverse genetics for coronaviruses particularly challenging. The rapid development and application of reverse genetics systems for coronaviruses require further exploration.
Methods: Using transformation-associated recombination (TAR) cloning in yeast and the CRISPR-Cas9 system, reverse genetics systems of two mild coronaviruses HCoV-OC43 and HCoV-229E were rapidly established.
A Review of DNA Restriction-Free Overlapping Sequence Cloning Techniques for Synthetic Biology.
Biotechnol J
July 2025
Laboratório de Biologia Molecular e Computacional, Centro de Biotecnologia da UFRGS, Departamento de Biologia Molecular e Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
DNA cloning methods are fundamental tools in molecular biology, synthetic biology, and genetic engineering that enable precise DNA manipulation for various scientific and biotechnological applications. This review systematically summarizes the major restriction-free overlapping sequence cloning (RFOSC) techniques currently used in synthetic biology and examines their development, efficiency, practicality, and specific applications. In vitro methods, including Gibson Assembly, Circular Polymerase Extension Cloning (CPEC), Polymerase Incomplete Primer Extension (PIPE), Overlap Extension Cloning (OEC), Uracil DNA Glycosylase-based Cloning (UDG-Cloning), and commercially available techniques such as In-Fusion, have been discussed alongside hybrid approaches such as Ligation-Independent Cloning (LIC), Sequence-Independent Cloning (SLIC), and T5 Exonuclease-Dependent Assembly (TEDA).
View Article and Find Full Text PDF