DataSheet_2_Long-Read RNA Sequencing Identifies Polyadenylation Elongation and Differential Transcript Usage of Host Transcripts During SARS-CoV-2 In Vitro Infection.zip

Better methods to interrogate host-pathogen interactions during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections are imperative to help understand and prevent this disease. Here we implemented RNA-sequencing (RNA-seq) using Oxford Nanopore Technologies (ONT) long-reads to measure differential host gene expression, transcript polyadenylation and isoform usage within various epithelial cell lines permissive and non-permissive for SARS-CoV-2 infection. SARS-CoV-2-infected and mock-infected Vero (African green monkey kidney epithelial cells), Calu-3 (human lung adenocarcinoma epithelial cells), Caco-2 (human colorectal adenocarcinoma epithelial cells) and A549 (human lung carcinoma epithelial cells) were analyzed over time (0, 2, 24, 48 hours). Differential polyadenylation was found to occur in both infected Calu-3 and Vero cells during a late time point (48 hpi), with Gene Ontology (GO) terms such as viral transcription and translation shown to be significantly enriched in Calu-3 data. Poly(A) tails showed increased lengths in the majority of the differentially polyadenylated transcripts in Calu-3 and Vero cell lines (up to ~101 nt in mean poly(A) length, padj = 0.029). Of these genes, ribosomal protein genes such as RPS4X and RPS6 also showed downregulation in expression levels, suggesting the importance of ribosomal protein genes during infection. Furthermore, differential transcript usage was identified in Caco-2, Calu-3 and Vero cells, including transcripts of genes such as GSDMB and KPNA2, which have previously been implicated in SARS-CoV-2 infections. Overall, these results highlight the potential role of differential polyadenylation and transcript usage in host immune response or viral manipulation of host mechanisms during infection, and therefore, showcase the value of long-read sequencing in identifying less-explored host responses to disease.

深入解析严重急性呼吸综合征冠状病毒2型（Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2）感染过程中的宿主-病原体相互作用，是理解并防控该疾病的关键需求，因此亟需开发更优的研究方法。本研究采用牛津纳米孔技术（Oxford Nanopore Technologies, ONT）长读长RNA测序（RNA-sequencing, RNA-seq），对SARS-CoV-2感染与模拟感染的多株上皮细胞系中的宿主差异基因表达、转录本多聚腺苷酸化及异构体使用情况进行定量检测，这些细胞系涵盖对SARS-CoV-2感染具有允许性与非允许性的细胞类型。本研究对感染组与模拟感染组的Vero细胞（非洲绿猴肾上皮细胞）、Calu-3细胞（人肺腺癌上皮细胞）、Caco-2细胞（人结直肠腺癌上皮细胞）及A549细胞（人肺癌上皮细胞）在0、2、24、48小时四个时间点的样本进行了测序分析。结果显示，在感染后期（感染后48小时，48 hpi），Calu-3与Vero细胞中均发生了差异多聚腺苷酸化现象；且Calu-3细胞的差异多聚腺苷酸化基因显著富集于病毒转录与翻译等基因本体论（Gene Ontology, GO）条目。在Calu-3与Vero细胞的多数差异多聚腺苷酸化转录本中，poly(A)尾长度均显著增加，平均poly(A)长度最高可达约101 nt，校正P值（padj）=0.029。其中，RPS4X、RPS6等核糖体蛋白基因的表达水平亦出现下调，提示核糖体蛋白基因在SARS-CoV-2感染过程中发挥重要作用。此外，本研究在Caco-2、Calu-3及Vero细胞中鉴定到差异转录本使用事件，包括此前已被报道与SARS-CoV-2感染相关的GSDMB、KPNA2等基因的转录本。整体而言，本研究结果凸显了差异多聚腺苷酸化与转录本异构体使用在宿主免疫应答或病毒操纵宿主感染机制中的潜在功能，同时彰显了长读长测序在识别尚未被充分探索的宿主疾病应答方面的应用价值。