Abstract
Reverse genetics is a key methodology for producing genetically modified RNA viruses and deciphering cellular and viral biological properties, but methods based on the preparation of plasmid-based complete viral genomes are laborious and unpredictable. Here, both wild-type and genetically modified infectious RNA viruses were generated in days using the newly described ISA (infectious-subgenomic-amplicons) method. This new versatile and simple procedure may enhance our capacity to obtain infectious RNA viruses from PCR-amplified genetic material.
Highlights
Development of molecular methods that enable production of infectious virus from DNA copies of their genomes has significantly improved our knowledge of RNA virus life cycles and pathogenesis, by permitting the development of ‘reverse genetics’, i.e. studies of the impact of specific mutations on the biological properties of viruses (Gorchakov et al, 2012; Ruggli & Rice, 1999)
We describe a simple and versatile reverse genetics method designated ISA that facilitates the rescue of infectious RNA viruses from genomic DNA material without requiring cloning, propagation of cDNA into bacteria or in vitro RNA transcription
We first tested this hypothesis with six flaviviruses that represent major flaviviral evolutionary lineages
Summary
Development of molecular methods that enable production of infectious virus from DNA copies of their genomes has significantly improved our knowledge of RNA virus life cycles and pathogenesis, by permitting the development of ‘reverse genetics’, i.e. studies of the impact of specific mutations on the biological properties of viruses (Gorchakov et al, 2012; Ruggli & Rice, 1999). Current methodologies for construction of infectious cDNA clones are unpredictable and laborious processes frequently associated with undesirable mutations or unstable/toxic clones in bacteria (Ruggli & Rice, 1999) Various methodological improvements, such as the use of alternative hosts (Csorgo et al, 2012; Polo et al, 1997; Schoggins et al, 2012), lowcopy-number plasmids (Bredenbeek et al, 2003; Gritsun & Gould, 1998), cosmid vectors (Zhang et al, 2001), bacterial artificial chromosomes (Suzuki et al, 2007), modified promoters (Mishin et al, 2001) or modified viral genome sequences with reduced cryptic bacterial promoter activity (Pu et al, 2011) have been proposed. By including at the 59 terminus of the first (59) DNA fragment a promoter of DNA-dependent RNA polymerases, and at the 39 terminus of the last (39) DNA fragment a ribozyme sequence and a signal sequence for RNA poly-adenylation, we anticipated that this genomic DNA copy would be transcribed as a full-length RNA genome with authentic 59 and 39 termini that would be efficiently exported out of the nucleus (in the case of a virus replicating in the cytoplasmic compartment)
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