Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, Texas, USA.
Institute for Human Infection and Immunity, The University of Texas Medical Branch, Galveston, Texas, USA.
J Virol. 2024 Sep 17;98(9):e0090024. doi: 10.1128/jvi.00900-24. Epub 2024 Aug 28.
SARS-CoV-2 belongs to the family and carries a single-stranded positive-sense RNA genome. During coronavirus (CoV) replication, defective or defective interfering RNAs that lack a large portion of the genome often emerge. These defective RNAs typically carry the necessary RNA elements that are required for replication and packaging. We identified the minimum requirement of the 5' proximal region necessary for viral RNA replication by using artificially generated SARS-CoV-2 minigenomes. The minigenomes consist of the 5'-proximal region, an open reading frame (ORF) that encodes a fusion protein consisting of the N-terminal of viral NSP1 and a reporter gene, and the 3' untranslated region of the SARS-CoV-2 genome. We used a modified SARS-CoV-2 variant to support replication of the minigenomes. A minigenome carrying the 5' proximal 634 nucleotides replicated, whereas those carrying shorter than 634 nucleotides did not, demonstrating that the entire 265 nt-long 5' untranslated region and N-terminal portion of the NSP1 coding region are required for the minigenome replication. Minigenome RNAs carrying a specific amino acid substitution or frame shift insertions in the partial NSP1 coding sequence abrogated minigenome replication. Introduction of synonymous mutations in the minigenome RNAs also affected the replication efficiency of the minigenomes. These data suggest that the expression of the N-terminal portion of NSP1 and the primary sequence of the 5' proximal 634 nucleotides are important for minigenome replication.IMPORTANCESARS-CoV-2, the causative agent of COVID-19, is highly transmissible and continues to have a significant impact on public health and the global economy. While several vaccines mitigate the severe consequences of SARS-CoV-2 infection, mutant viruses with reduced reactivity to current vaccines continue to emerge and circulate. This study aimed to identify the minimal 5' proximal region of SARS-CoV-2 genomic RNA required for SARS-CoV-2 defective RNA replication and investigate the importance of an ORF encoded in these defective RNAs. Identifying cis-acting replication signals of SARS-CoV-2 genomic RNA is critical for the development of antivirals that target these signals. Additionally, replication-competent defective RNAs can serve as therapeutic reagents to interfere with SARS-CoV-2 replication. Our findings provide valuable insights into the mechanisms of SARS-CoV-2 RNA replication and the development of reagents that suppress SARS-CoV-2 replication.
SARS-CoV-2 属于 冠状病毒科，携带单链正链 RNA 基因组。在冠状病毒（CoV）复制过程中，经常会出现缺少基因组大部分区域的缺陷或缺陷干扰 RNA。这些缺陷 RNA 通常携带复制和包装所需的必要 RNA 元件。我们通过使用人工生成的 SARS-CoV-2 小基因组来确定病毒 RNA 复制所必需的 5'近端区域的最小要求。小基因组由 5'-近端区域、编码由病毒 NSP1 的 N 端和报告基因组成的融合蛋白的开放阅读框 (ORF) 和 SARS-CoV-2 基因组的 3'非翻译区组成。我们使用改良的 SARS-CoV-2 变体来支持小基因组的复制。携带 5'近端 634 个核苷酸的小基因组进行复制，而携带短于 634 个核苷酸的小基因组则不进行复制，这表明全长 265 个核苷酸的 5'非翻译区和 NSP1 编码区的 N 端部分对于小基因组的复制是必需的。在部分 NSP1 编码序列中带有特定氨基酸取代或框移插入的小基因组 RNA 会阻止小基因组的复制。小基因组 RNA 中的同义突变的引入也会影响小基因组的复制效率。这些数据表明，NSP1 的 N 端部分的表达和 5'近端 634 个核苷酸的一级序列对于小基因组的复制很重要。
导致 COVID-19 的 SARS-CoV-2 具有高度传染性，继续对公共卫生和全球经济产生重大影响。虽然有几种疫苗减轻了 SARS-CoV-2 感染的严重后果，但对当前疫苗反应降低的突变病毒继续出现并传播。本研究旨在确定 SARS-CoV-2 基因组 RNA 中用于 SARS-CoV-2 缺陷 RNA 复制的最小 5'近端区域，并研究这些缺陷 RNA 中编码的 ORF 的重要性。鉴定 SARS-CoV-2 基因组 RNA 的顺式作用复制信号对于开发针对这些信号的抗病毒药物至关重要。此外，具有复制能力的缺陷 RNA 可以作为治疗试剂来干扰 SARS-CoV-2 的复制。我们的研究结果为 SARS-CoV-2 RNA 复制的机制以及抑制 SARS-CoV-2 复制的试剂的开发提供了有价值的见解。
