DataSheet_1_Long-Read RNA Sequencing Identifies Polyadenylation Elongation and Differential Transcript Usage of Host Transcripts During SARS-CoV-2 In Vitro Infection.xlsx

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（SARS-CoV-2）感染过程中的宿主-病原体互作机制，对于理解该疾病并制定防控策略至关重要。本研究采用牛津纳米孔技术（ONT）长读长RNA测序（RNA-seq），对SARS-CoV-2易感和非易感的多种上皮细胞系中的宿主基因差异表达、转录本多聚腺苷酸化及转录本使用情况进行定量分析。我们分别在0、2、24、48小时四个时间点，对SARS-CoV-2感染及假感染的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）与A549细胞（人肺癌上皮细胞）开展了测序分析。研究发现，在感染后期（感染后48小时，48 hpi），Calu-3细胞与Vero细胞中均出现了差异多聚腺苷酸化现象；在Calu-3细胞的测序数据中，病毒转录、翻译等基因本体（GO）术语显著富集。在Calu-3和Vero细胞的差异多聚腺苷酸化转录本中，绝大多数的poly(A)尾长度均有所增加，平均poly(A)长度最高可达约101 nt，校正后P值（padj）为0.029。其中，RPS4X、RPS6等核糖体蛋白基因的表达水平也出现下调，提示核糖体蛋白基因在病毒感染过程中发挥重要作用。此外，我们在Caco-2、Calu-3及Vero细胞中均鉴定到了差异转录本使用事件，包括此前被报道与SARS-CoV-2感染相关的GSDMB、KPNA2等基因的转录本。综上，本研究结果表明，差异多聚腺苷酸化与差异转录本使用可能在宿主免疫应答或病毒操控宿主通路的过程中发挥潜在作用，同时也凸显了长读长测序在解析尚未被充分探索的宿主疾病应答机制中的应用价值。