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.

Keywords: PEG-mediated fusion; Saccharomyces cerevisiae; Sindbis virus; alphavirus; frozen yeast spheroplasts; galactose induction; in vitro transfection; positive-sense single strand RNA virus; reverse genetics; transformation-associated recombination (TAR) cloning.

Figure 1

New yeast centromeric plasmids (YCps) for galactose inducible expression. (A) A schematic of three yeast centromeric plasmids. Not drawn to scale. See GenBank files in supplementary data for sequences. (B) Detailed sequence detail from Adapter 1 and 2. Yellow, left: Adapter 1, right: Adapter 2; Green: Yeast Translational Entry Site; Blue: Myc Tag; Red: Stop codon.

Figure 2

Yeast cytoplasmic elements are transferred to mammalian cells (BHK-21) via fusion. (A) An outline of yeast to mammalian cell fusion procedure (from sequence to fusion). * Colony PCR screening was used if transformation-associated recombination (TAR) cloning (ii) pathway was used. (B) Fluorescence micrographs of BHK-21 cells fused with yeast spheroplast prepared from induced yLDJIF9 (W303α transformed with pLDJIF15). yeCherry (pink) was expressed in yeast only. Pink BHK-21 cells (pointed out with an arrow) appear when the cytoplasmic element, including yeCherry, is transferred from yeast to BHK-21 cells via fusion.

Figure 3

Sindbis virus was rescued by yeast to mammalian cell fusion. (A) A schematic of pLDJIF15-SINV, a green fluorescent protein (GFP)-tagged infectious cDNA clone of Sindbis virus (SINV). See GenBank files in supplementary data for sequences. (B) Fluorescence micrographs of fusion experiments taken on day 2 and 3 post fusion experiments. yLDJIF21 (W303α transformed with pLDJIF15-SINV) was grown overnight in −TRP medium was used to inoculate −TRP medium (uninduced) or YPG medium (induced) for 5 h at 30 °C. Cytopathic Effect (CPE) was observed on day 3 post fusion experiment with galactose-induced yeast cells. Representative fields; scale bar: 500 µm. (C) Growth curve of SINV rescued from yeast-mammalian fusions. SINV viral titers from day 1 to 5 after fusion were measured by plaque assay. Rescue of SINV viral particle was observed from spheroplast prepared from galactose-induced yeast culture. (Day 1: 4.2 × 10³ pfu/mL; Day 2: 3.3 × 10⁸ pfu/mL; Day 3: 7.3 × 10⁸ pfu/mL; Day 4: 3.4 × 10⁸ pfu/mL; Day 5: 4 × 10⁷ pfu/mL). No SINV was detected from uninduced yeast culture. Induced: Left Y-axis (log scale). Uninduced: Right Y-axis (linear scale).