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

Lin Ding,John I Glass,David M Brown

doi:10.3390/v13040603

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.

Highlights

Sindbis virus (SINV), a positive-sense single stranded RNA virus that causes mild symptoms in humans, is transmitted by mosquito bites
Our result suggested that the adaptors did not interfere with the galactose induction with the expression
SINV-based vector systems are routinely used as expression vectors for heterologous gene expression and infectious particles production [39,46]

Summary

Introduction

Sindbis virus (SINV), a positive-sense single stranded RNA virus that causes mild symptoms in humans, is transmitted by mosquito bites. 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). We constructed a novel infectious SINV cDNA construct that expresses its genomic RNA in yeast cells controlled by galactose induction. The SINV genome, a 11.7 kb positive-sense single-stranded RNA, is encapsulated by capsid protein and packaged into an envelope derived from the host cell plasma membrane containing viral glycoproteins [5]. SINV particles were rescued by transfection of the resulting mRNA in the BHK-21 cells. SINV mRNA-based reverse genetics helped advance research on many positive stranded RNA viruses and is used as a core component of RNA virus-based vectors [8]. Many SINV strains were developed over the years [15,16], among which a nonvirulent recombinant SINV strain, dsTE12Q, was made by changing the 55th amino acid residue of

Methods

Results

Conclusion