Direct RNA sequencing of respiratory syncytial virus infected human cells generates a detailed overview of RSV polycistronic mRNA and transcript abundance.
Direct RNA sequencing of respiratory syncytial virus infected human cells generates a detailed overview of RSV polycistronic mRNA and transcript abundance.
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DOI：
10.1371/journal.pone.0276697
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发表时间：
2022
期刊：
影响因子：
3.7
通讯作者：
中科院分区：
文献类型：
--
作者：
为了表征呼吸道合胞病毒 (RSV) 感染过程中产生的病毒 mRNA 转录物的种类，将人成纤维细胞样 MRC-5 肺细胞用 A 亚组 RSV 感染 6、16 和 24 小时。此外，我们还对感染后 48 小时受感染的 Calu-3 肺上皮细胞中的病毒转录组进行了表征。收获总 RNA，并使用 Oxford 纳米孔装置通过直接 RNA 测序来富集和测序多聚腺苷酸化 mRNA。该平台产生了超过 450,000 个直接 mRNA 转录本读数，这些读数被映射到病毒基因组，并使用我们内部以 ORF 为中心的管道进行分析以确定病毒基因的相对 mRNA 水平。我们检查了生成的多顺反子通读 mRNA 的频率，并评估了每组转录物的聚腺苷酸尾的长度。正如 RSV 基因转录模型所预测的那样，我们发现整个病毒基因组中的基因转录本丰度普遍但非线性下降。然而，转录本丰度的下降并不均匀。病毒聚合酶产生的聚腺苷酸尾的长度与宿主聚腺苷酸化机器产生的长度相似，并且随着感染的进展，大多数转录物的长度普遍下降。最后，我们观察到，与 NS1、NS2、P、M、F 和 M2 相比，两种细胞系中 N、SH 和 G 转录本的稳态丰度（具有小于 20 个核苷酸的非常短的多聚腺苷酸尾）的转录本的稳态丰度较低，这可能反映了细胞系内和细胞系之间 mRNA 稳定性和/或翻译率的差异。
To characterize species of viral mRNA transcripts generated during respiratory syncytial virus (RSV) infection, human fibroblast-like MRC-5 lung cells were infected with subgroup A RSV for 6, 16 and 24 hours. In addition, we characterised the viral transcriptome in infected Calu-3 lung epithelial cells at 48 hours post infection. Total RNA was harvested and polyadenylated mRNA was enriched and sequenced by direct RNA sequencing using an Oxford nanopore device. This platform yielded over 450,000 direct mRNA transcript reads which were mapped to the viral genome and analysed to determine the relative mRNA levels of viral genes using our in-house ORF-centric pipeline. We examined the frequency of polycistronic readthrough mRNAs were generated and assessed the length of the polyadenylated tails for each group of transcripts. We show a general but non-linear decline in gene transcript abundance across the viral genome, as predicted by the model of RSV gene transcription. However, the decline in transcript abundance is not uniform. The polyadenylate tails generated by the viral polymerase are similar in length to those generated by the host polyadenylation machinery and broadly declined in length for most transcripts as the infection progressed. Finally, we observed that the steady state abundance of transcripts with very short polyadenylate tails less than 20 nucleotides is less for N, SH and G transcripts in both cell lines compared to NS1, NS2, P, M, F and M2 which may reflect differences in mRNA stability and/or translation rates within and between the cell lines.
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影响因子：
3.7
作者：
Eshaghi A;Duvvuri VR;Lai R;Nadarajah JT;Li A;Patel SN;Low DE;Gubbay JB
通讯作者：
Gubbay JB
影响因子：
3.7
作者：
Delahaye C;Nicolas J
通讯作者：
Nicolas J
影响因子：
12.3
作者：
Almuqrin A;Davidson AD;Williamson MK;Lewis PA;Heesom KJ;Morris S;Gilbert SC;Matthews DA
通讯作者：
Matthews DA
影响因子：
5.4
作者：
Aljabr W;Touzelet O;Pollakis G;Wu W;Munday DC;Hughes M;Hertz-Fowler C;Kenny J;Fearns R;Barr JN;Matthews DA;Hiscox JA
通讯作者：
Hiscox JA
影响因子：
5.9
作者：
Donovan-Banfield, I'ah;Turnell, Andrew S.;Matthews, David A.
通讯作者：
Matthews, David A.