J. Craig Venter Institute, 4120 Capricorn Lane, La Jolla, CA 92037, USA.
J. Craig Venter Institute, 9605 Medical Center, Rockville, MD 20850, USA.
Viruses. 2021 Apr 1;13(4):603. doi: 10.3390/v13040603.
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.
辛德比斯病毒（SINV）是一种正链单链 RNA 病毒，可引起人类的轻度症状，通过蚊子叮咬传播。SINV 的反向遗传学具有许多意义，不仅在于理解甲病毒的传播、复制周期和病毒-宿主相互作用，还在于生物技术和生物医学应用。SINV 感染性颗粒的拯救通常是通过用 SINV 感染性 mRNA 基因组转染易感细胞（BHK-21）来实现的，该基因组是通过体外翻译（IVT）构建的 cDNA 产生的。该过程耗时、昂贵，并且严重依赖试剂质量。在这里，我们构建了一种新型的感染性 SINV cDNA 构建体，该构建体在酵母细胞中表达其基因组 RNA，受半乳糖诱导控制。使用来自该酵母的原生质球，我们建立了一种强大的聚乙二醇介导的酵母：BHK-21 融合方案来拯救感染性 SINV 颗粒。我们的方法省时省力，并且使用常见的实验室试剂进行 SINV 拯救。它可能是拯救大的单链 RNA 病毒（如 SARS-CoV-2）的有用工具。
