Sindbis virus (SINV), a positive-sense single stranded RNA virus that causes mild symptoms in humans, is transmitted by mosquito bites. SINV reverse genetics have many implications, not only in understanding alphavirus transmission, replication cycle, and virus-host interactions, but also in biotechnology and biomedical applications. The rescue of SINV infectious particles is usually achieved by transfecting susceptible cells (BHK-21) with SINV-infectious mRNA genomes generated from cDNA constructed via in vitro translation (IVT). That procedure is time consuming, costly, and relies heavily on reagent quality. Here, we constructed a novel infectious SINV cDNA construct that expresses its genomic RNA in yeast cells controlled by galactose induction. Using spheroplasts made from this yeast, we established a robust polyethylene glycol-mediated yeast: BHK-21 fusion protocol to rescue infectious SINV particles. Our approach is timesaving and utilizes common lab reagents for SINV rescue. It could be a useful tool for the rescue of large single strand RNA viruses, such as SARS-CoV-2.
【저자키워드】 reverse genetics, Saccharomyces cerevisiae, alphavirus, positive-sense single strand RNA virus, Sindbis virus, galactose induction, transformation-associated recombination (TAR) cloning, frozen yeast spheroplasts, in vitro transfection, PEG-mediated fusion, 【초록키워드】 SARS-CoV-2, protocol, translation, Genome, Transmission, in vitro, virus, genetics, Particle, Replication, mRNA, humans, RNA viruses, Virus-host interactions, RNA virus, yeast, genomic RNA, cDNA, Particles, Implications, Mild symptom, reagent, lab, approach, positive-sense, robust, transmitted, cause, costly, susceptible cell, expresse, BHK-21, spheroplast, transfecting, yeast cell, 【제목키워드】 fusion, rescue,