Direct RNA sequencing reveals m6A modifications and isoform changes in SARS-CoV-2-infected HEK cells.

Access microbiology Pub Date : 2025-09-17 eCollection Date: 2025-01-01 DOI:10.1099/acmi.0.001019.v3
Ilhan Cem Duru, Zlatka Plavec, Anne Ylinen, Pia Laine, Martyn James, Lotta Riihimäki, Sarah J Butcher, Maria Anastasina, Petri Auvinen
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Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection triggers complex host responses, including alterations in RNA transcription and modification. Understanding these changes is crucial for elucidating viral pathogenesis and identifying potential therapeutic targets. We used direct RNA sequencing to comprehensively profile the transcriptomic and epitranscriptomic landscapes of human HEK-AT cells infected with SARS-CoV-2 at 8 h post-infection, compared to mock controls. We analysed viral and host transcriptomes, focusing on gene and transcript expression, isoform usage and RNA m6A modifications. Viral RNA sequencing reads showed 3' end-biassed coverage indicative of subgenomic RNA synthesis, with high expression of N gene subgenomic RNA reads. Sixteen m6A modification sites were consistently identified in the viral genome, primarily within the ORF1ab and S genes. In the human transcriptome, we found 254 positions with significantly altered m6A modification rates, with 119 showing decreased modification and 135 showing increased modification in infected cells. Genes with decreased m6A modifications were enriched in the neurotrophin signalling pathway. Transcript-level analysis identified 19 upregulated and 12 downregulated transcripts. Notably, transcript discovery and quantification revealed a novel isoform of the HIST1H2BK gene, which was significantly more expressed in infected cells compared to mock controls. Isoform switching analysis revealed 24 significant switches involving 21 genes, implicating mitochondrial reprogramming and immune-related pathways. In conclusion, this study provides a detailed, direct RNA sequencing-based characterization of host-virus RNA interactions, revealing key insights into SARS-CoV-2 infection mechanisms and potential therapeutic targets.

直接RNA测序揭示了sars - cov -2感染的HEK细胞中m6A的修饰和异构体的变化。
严重急性呼吸综合征冠状病毒2 (SARS-CoV-2)感染引发复杂的宿主反应,包括RNA转录和修饰的改变。了解这些变化对于阐明病毒发病机制和确定潜在的治疗靶点至关重要。与模拟对照组相比,我们使用直接RNA测序技术全面分析了感染SARS-CoV-2的人HEK-AT细胞在感染后8小时的转录组学和表转录组学景观。我们分析了病毒和宿主转录组,重点关注基因和转录物表达、异构体使用和RNA m6A修饰。病毒RNA测序reads显示3'端偏覆盖,表明亚基因组RNA合成,高表达N基因亚基因组RNA reads。在病毒基因组中一致鉴定出16个m6A修饰位点,主要在ORF1ab和S基因中。在人类转录组中,我们发现254个位置的m6A修饰率显著改变,其中119个位置的修饰率降低,135个位置的修饰率增加。m6A修饰减少的基因在神经营养因子信号通路中富集。转录水平分析鉴定出19个上调转录本和12个下调转录本。值得注意的是,转录物发现和定量发现了一种新的HIST1H2BK基因亚型,与模拟对照相比,该基因在感染细胞中的表达明显增加。同种异构体开关分析揭示了涉及21个基因的24个重要开关,涉及线粒体重编程和免疫相关途径。总之,本研究提供了基于RNA测序的宿主-病毒RNA相互作用的详细、直接的表征,揭示了SARS-CoV-2感染机制和潜在治疗靶点的关键见解。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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