Webb Isobel, Erdmann Maximillian, Milligan Rachel, Savage Megan, Matthews David A, Davidson Andrew D
School of Cellular and Molecular Medicine, Faculty of Health and Life Sciences, University of Bristol, Bristol BS8 1TD, UK.
J Gen Virol. 2025 Feb;106(2). doi: 10.1099/jgv.0.002072.
The SARS-CoV-2 genome encodes at least nine accessory proteins, including innate immune antagonist and putative viroporin ORF3a. ORF3a plays a role in many stages of the viral replication cycle, including immune modulation. We constructed two recombinant (r)SARS-CoV-2 viruses in which the ORF3a gene was replaced with mScarlet (mS) or mNeonGreen (mNG), denoted as rSARS-CoV-2-Δ3a-mS and rSARS-CoV-2-Δ3a-mNG, respectively. rSARS-CoV-2-Δ3a-mNG generated a fluorescent signal after infection in both A549-ACE-2-TMPRSS2 (AAT) and Vero-E6-TMPRSS2 (VTN) cells, unlike rSARS-CoV-2-Δ3a-mS. rSARS-CoV-2-Δ3a-mS mS protein could be detected immunologically in VTN but not AAT cells, indicating the expression of a non-fluorescent mS protein. The analysis of the viral transcriptomes in infected AAT cells by nanopore direct RNA sequencing (dRNAseq) revealed that the level of mS transcript was below the limit of detection in AAT cells. rSARS-CoV-2-Δ3a-mNG virus was found to be genetically stable in AAT and VTN cells, but rSARS-CoV-2-Δ3a-mS acquired partial deletions of the mS gene during sequential passaging in VTN cells, creating the virus rSARS-CoV-2-Δ3a-ΔmS. The mS deletion in VTN cells removes the chromophore coding sequence, and this may explain the presence of a non-fluorescent mS protein detected in VTN cells. The rSARS-CoV-2-Δ3a-mNG, rSARS-CoV-2-Δ3a-mS and rSARS-CoV-2-Δ3a-ΔmS viruses all replicated to a lower titre and produced smaller plaques than the parental rSARS-CoV-2-S-D614G. Interestingly, the rSARS-CoV-2-Δ3a-ΔmS virus produced higher virus titres and larger plaque sizes than rSARS-CoV-2-Δ3a-mS. This suggested that both the insertion of mS coding sequence and the deletion of ORF3a coding sequence contributed to attenuation. In comparison with rSARS-CoV-2, the rSARS-CoV-2-Δ3a-mS and rSARS-CoV-2-Δ3a-mNG viruses showed increased sensitivity to pre-treatment of cells with IFN-α but did not exhibit a dose-dependent increase in replication in the presence of the Janus kinase-signal transducer and activator of transcription signalling pathway inhibitor, ruxolitinib. In conclusion, the replacement of the ORF3a coding sequence with those of fluorescent reporter proteins attenuated the replication of SARS-CoV-2 and its ability to effectively evade the innate immune response .
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）基因组编码至少9种辅助蛋白，包括先天免疫拮抗剂和假定的病毒孔蛋白ORF3a。ORF3a在病毒复制周期的许多阶段发挥作用，包括免疫调节。我们构建了两种重组（r）SARS-CoV-2病毒，其中ORF3a基因分别被mScarlet（mS）或mNeonGreen（mNG）取代，分别表示为rSARS-CoV-2-Δ3a-mS和rSARS-CoV-2-Δ3a-mNG。与rSARS-CoV-2-Δ3a-mS不同，rSARS-CoV-2-Δ3a-mNG在感染A549-ACE-2-TMPRSS2（AAT）细胞和Vero-E6-TMPRSS2（VTN）细胞后均产生荧光信号。rSARS-CoV-2-Δ3a-mS的mS蛋白可以在VTN细胞中通过免疫方法检测到，但在AAT细胞中检测不到，这表明存在非荧光mS蛋白。通过纳米孔直接RNA测序（dRNAseq）分析感染的AAT细胞中的病毒转录组，发现AAT细胞中mS转录本水平低于检测限。发现rSARS-CoV-2-Δ3a-mNG病毒在AAT和VTN细胞中基因稳定，但rSARS-CoV-2-Δ3a-mS在VTN细胞中连续传代期间获得了mS基因的部分缺失，产生了病毒rSARS-CoV-2-Δ3a-ΔmS。VTN细胞中的mS缺失去除了发色团编码序列，这可能解释了在VTN细胞中检测到的非荧光mS蛋白的存在。rSARS-CoV-2-Δ3a-mNG、rSARS-CoV-2-Δ3a-mS和rSARS-CoV-2-Δ3a-ΔmS病毒的复制滴度均低于亲本rSARS-CoV-2-S-D614G，且产生的噬斑更小。有趣的是，rSARS-CoV-2-Δ3a-ΔmS病毒产生的病毒滴度高于rSARS-CoV-2-Δ3a-mS，噬斑尺寸也更大。这表明mS编码序列的插入和ORF3a编码序列的缺失均导致病毒减毒。与SARS-CoV-2相比，rSARS-CoV-2-Δ3a-mS和rSARS-CoV-2-Δ3a-mNG病毒对用IFN-α预处理细胞表现出更高的敏感性，但在存在Janus激酶-信号转导和转录激活因子信号通路抑制剂鲁索替尼的情况下，复制没有呈现剂量依赖性增加。总之，用荧光报告蛋白的编码序列替换ORF3a编码序列会减弱SARS-CoV-2的复制及其有效逃避先天免疫反应的能力。
