Taha Taha Y, Ezzatpour Shahrzad, Hayashi Jennifer M, Ye Chengjin, Zapatero-Belinchón Francisco J, Rosecrans Julia A, Kimmerly Gabriella R, Chen Irene P, Walcott Keith, Kurianowicz Anna, Jorgens Danielle M, Chaplin Natalie R, Choi Annette, Buchholz David W, Sahler Julie, Hilt Zachary T, Imbiakha Brian, Vagi-Szmola Cecilia, Montano Mauricio, Stevenson Erica, Gordon Martin, Swaney Danielle L, Krogan Nevan J, Whittaker Gary R, Martinez-Sobrido Luis, Aguilar Hector C, Ott Melanie
Gladstone Institutes, San Francisco, California, United States of America.
Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, California, United States of America.
PLoS Pathog. 2025 Mar 31;21(3):e1013020. doi: 10.1371/journal.ppat.1013020. eCollection 2025 Mar.
The COVID-19 pandemic has been driven by SARS-CoV-2 variants with enhanced transmission and immune escape. Apart from extensive evolution in the Spike protein, non-Spike mutations are accumulating across the entire viral genome and their functional impact is not well understood. To address the contribution of these mutations, we reconstructed genomes of recent Omicron variants with disabled Spike expression (replicons) to systematically compare their RNA replication capabilities independently from Spike. We also used a single reference replicon and complemented it with various Omicron variant Spike proteins to quantify viral entry capabilities in single-round infection assays. Viral entry and RNA replication were negatively correlated, suggesting that as variants evolve reduced entry functions under growing immune pressure on Spike, RNA replication increases as a compensatory mechanism. We identified multiple mutations across the viral genome that enhanced viral RNA replication. NSP6 emerged as a hotspot with a distinct L260F mutation independently arising in the BQ.1.1 and XBB.1.16 variants. Using mutant and revertant NSP6 viral clones, the L260F mutation was validated to enhance viral replication in cells and increase pathogenesis in mice. Notably, this mutation reduced host lipid droplet content by NSP6. Collectively, a systematic analysis of RNA replication of recent Omicron variants defined NSP6's key role in viral RNA replication that provides insight into evolutionary trajectories of recent variants with possible therapeutic implications.
新冠疫情由具有增强传播力和免疫逃逸能力的严重急性呼吸综合征冠状病毒2（SARS-CoV-2）变体引发。除了刺突蛋白发生广泛进化外，整个病毒基因组都在积累非刺突突变，而它们的功能影响尚不清楚。为了研究这些突变的作用，我们构建了刺突蛋白表达缺失的近期奥密克戎变体基因组（复制子），以独立于刺突蛋白系统地比较它们的RNA复制能力。我们还使用了单个参考复制子，并在单轮感染试验中用各种奥密克戎变体刺突蛋白对其进行补充，以量化病毒进入能力。病毒进入和RNA复制呈负相关，这表明随着变体在对刺突蛋白不断增加的免疫压力下进化出降低的进入功能，RNA复制作为一种补偿机制而增加。我们在病毒基因组中鉴定出多个增强病毒RNA复制的突变。非结构蛋白6（NSP6）成为一个热点，在BQ.1.1和XBB.1.16变体中独立出现了一个独特的L260F突变。使用突变型和回复型NSP6病毒克隆，验证了L260F突变可增强病毒在细胞中的复制并增加小鼠的致病性。值得注意的是，该突变通过NSP6降低了宿主脂滴含量。总体而言，对近期奥密克戎变体RNA复制的系统分析确定了NSP6在病毒RNA复制中的关键作用，这为深入了解近期变体的进化轨迹提供了思路，并可能具有治疗意义。
