Rescue of Infectious Sindbis Virus by Yeast Spheroplast-Mammalian Cell Fusion

Abstract

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.

Improved Combinatorial Assembly and Barcode Sequencing for Gene-Sized DNA Constructs

“…Standard recombinant DNA methods were used. The details are found in the Supplementary Methods document. − …”

Reinventing positive-strand RNA virus reverse genetics

“…Other promising genome engineering approaches include retron-associated recombineering, which uses the bacterial retron reverse transcriptase to continuously produce mutagenic oligos in vivo ( Lopez et al, 2022 ; Schubert et al, 2021 ), as well as synthetic base editing enzymes ( Gaudelli et al, 2017 ; Komor et al, 2016 ; Mok et al, 2020 ) and prime-editing enzymes ( Anzalone et al, 2019 ). I am also excited by the recent work of Ding, et al, who showed that yeast harboring a Sindbis virus cDNA driven by the strong, galactose-inducible Gal1 promoter can produce infectious transcripts and initiate replication upon fusion of the induced yeast with mammalian cells, thereby avoiding the need to isolate a cDNA and transcribe RNA in vitro ( Ding et al, 2021 ). I imagine a nearby future whereby nearly all standard molecular virology may be outsourced to yeast: we will be able to program yeast to create mutants of interest, such as with eMAGE, and to then produce infectious RNAs by feeding them galactose ( Fig.…”

Construction and verification of an infectious cDNA clone of coxsackievirus B5

“…Reverse genetics is a conventional method for studying the pathogenesis and virulence of viruses ( Hao et al., 2021 ; Ding et al., 2021 ). Recently, complementary DNA (cDNA) clones of CV-B3, CV-A10, CV-A6, CV-A16, EV-A71, and EV-D68 have been constructed, and the biological characteristics of the rescued viruses have been validated ( Liu et al., 2019 ; Ma et al., 2015 ; Yang et al., 2015 ; Pan et al., 2018 ; Wang et al., 2020 ).…”