在人类细胞中,RNA复制独立,DNA线性化依赖于来自SARS-CoV-2复制体编码DNA的记者基因的表达
Ronja Friedhoff1, Ghada Elfayres1, Natacha Mérindol2
1Department of Medical Biology, Université du Québec à Trois-Rivières, Trois-Rivières, Québec, Canada.
PloS one
|August 16, 2024
概括
研究人员发现了一种新方法,可以在细胞中以DNA为基础的SARS-CoV-2复制体表达基因,而不需要RNA转录或病毒复制. 这种DNA转染方法为开发RNA复制子应用提供了一种新的方法.
科学领域:
- 病毒学 病毒学
- 分子生物学分子生物学
- 基因工程是一种基因工程.
背景情况:
- RNA复制体是用于基因表达和复制的病毒遗传结构.
- 目前的方法涉及体外RNA转录和电穿孔,这可能是低效的.
- 在DNA转染后在细胞内产生复制体是一种可取的替代方案.
研究的目的:
- 为了研究将基因从基于DNA的SARS-CoV-2复制体转移到哺乳动物细胞后表达基因的可行性.
- 阐明DNA复制体基因表达的机制,特别是它对T7RNA聚合酶和病毒复制的依赖.
主要方法:
- 在T7促进器下构建了一个编码SARS-CoV-2复制体的细菌人造染色体 (BAC) DNA.
- 表达T7RNA聚合酶 (T7RNAP) 的HEK293T细胞与BACDNA进行了移植.
- 分析了记者基因表达 (GFP, luciferase),以及DNA线性化,T7 RNAP,remdesivir和SARS-CoV-2 N蛋白的影响.
主要成果:
- 从转染的BACDNA中观察到可复制的,尽管很低的记者蛋白的表达.
- 表达需要BACDNA的线性化,并且独立于T7RNAP和病毒RNA自我复制发生.
- 雷梅西维尔治疗没有影响基因表达,进一步表明缺乏自我复制.
- SARS-CoV-2 N 蛋白质增强了来自RNA复制体的表达,但不是来自DNA复制体的BAC.
结论:
- 编码冠状病毒复制体的大型DNA的转染可以通过未知的机制导致可复制的基因表达.
- 这种基于DNA的表达途径绕过了对T7RNA聚合酶和RNA自我复制的需求.
- 这些发现表明了基于DNA的RNA复制器应用的新方法,为未来的研究提供了宝贵的见解.
更多相关视频
08:41Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2
Published on: November 5, 2021
2.7K
09:35Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle
Published on: February 1, 2017
13.2K
相关概念视频
02:11Reporter Genes
11.3K
Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
11.3K
02:38S-Cdk Initiates DNA Replication
4.7K
The cell cycle is a series of events leading to DNA duplication followed by the division of cell content to form two daughter cells. The cell cycle progresses in four stages—the cell increases in size (gap 1 or G1-phase), duplicates its DNA (synthesis or S-phase), prepares to divide (gap 2 or G2-phase), and divides (mitosis or M-phase).
Two states at the origin of replication
In eukaryotes, the initiation of replication occurs at many sites on the chromosomes, called the origins of...
Two states at the origin of replication
In eukaryotes, the initiation of replication occurs at many sites on the chromosomes, called the origins of...
4.7K
02:28Leaky Scanning
5.1K
During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA. Marilyn Kozak discovered that the sequence RCCAUGG (where R...
5.1K