Absolute quantitation of individual SARS-CoV-2 RNA molecules provides a new paradigm for infection dynamics and variant differences.
Absolute quantitation of individual SARS-CoV-2 RNA molecules provides a new paradigm for infection dynamics and variant differences.
复制标题
对单个SARS-CoV-2RNA分子的绝对定量为研究感染动力学和变异差异提供了新的范例。
DOI：
10.7554/elife.74153
复制
发表时间：
2022-01-20
期刊：
影响因子：
7.7
通讯作者：
Davis I
中科院分区：
文献类型：
--
作者：
Lee JY;Wing PAC;Gala DS;Noerenberg M;Järvelin AI;Titlow J;Zhuang X;Palmalux N;Iselin L;Thompson MK;Parton RM;Prange-Barczynska M;Wainman A;Salguero FJ;Bishop T;Agranoff D;James W;Castello A;McKeating JA;Davis I
尽管全球对SARS-CoV-2进行了前所未有的研究，但对早期复制事件仍然知之甚少。鉴于传播增加的新出现的病毒变异的临床重要性，迫切需要了解病毒复制和转录的早期阶段。我们使用单分子荧光原位杂交（smFISH）以95%的检测效率定量正义RNA基因组，同时可视化负义基因组，亚基因组RNA和病毒蛋白。我们对病毒RNA和复制工厂的绝对定量显示，SARS-CoV-2基因组RNA在进入后是长寿命的，这表明它避免了细胞核酸酶的降解。此外，我们观察到SARS-CoV-2复制在细胞之间是高度可变的，只有一小部分细胞群体显示出高病毒RNA负荷。出乎意料的是，在英国首次发现的B.1.1.7变异株表现出比维多利亚株明显更慢的复制动力学，这表明一种新的机制有助于其具有重要临床意义的更高的传播性。
Despite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single-molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, subgenomic RNAs, and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications.