Institut Cochin, INSERM, CNRS, Université de Paris, 75014, Paris, France.
Genomic Facility, Institut de Biologie de l'ENS (IBENS), Département de biologie, École normale supérieure, CNRS, INSERM, Université PSL, 75005, Paris, France.
Retrovirology. 2020 Aug 17;17(1):25. doi: 10.1186/s12977-020-00533-1.
Alternative splicing is a key step in Human Immunodeficiency Virus type 1 (HIV-1) replication that is tightly regulated both temporally and spatially. More than 50 different transcripts can be generated from a single HIV-1 unspliced pre-messenger RNA (pre-mRNA) and a balanced proportion of unspliced and spliced transcripts is critical for the production of infectious virions. Understanding the mechanisms involved in the regulation of viral RNA is therefore of potential therapeutic interest. However, monitoring the regulation of alternative splicing events at a transcriptome-wide level during cell infection is challenging. Here we used the long-read cDNA sequencing developed by Oxford Nanopore Technologies (ONT) to explore in a quantitative manner the complexity of the HIV-1 transcriptome regulation in infected primary CD4+ T cells.
ONT reads mapping to the viral genome proved sufficiently long to span all possible splice junctions, even distant ones, and to be assigned to a total of 150 exon combinations. Fifty-three viral RNA isoforms, including 14 new ones were further considered for quantification. Relative levels of viral RNAs determined by ONT sequencing showed a high degree of reproducibility, compared favourably to those produced in previous reports and highly correlated with quantitative PCR (qPCR) data. To get further insights into alternative splicing regulation, we then compiled quantifications of splice site (SS) usage and transcript levels to build "splice trees", a quantitative representation of the cascade of events leading to the different viral isoforms. This approach allowed visualizing the complete rewiring of SS usages upon perturbation of SS D2 and its impact on viral isoform levels. Furthermore, we produced the first dynamic picture of the cascade of events occurring between 12 and 24 h of viral infection. In particular, our data highlighted the importance of non-coding exons in viral RNA transcriptome regulation.
ONT sequencing is a convenient and reliable strategy that enabled us to grasp the dynamic of the early splicing events modulating the viral RNA landscape in HIV-1 infected cells.
选择性剪接是人类免疫缺陷病毒 1 型（HIV-1）复制的关键步骤，其在时间和空间上受到严格调控。从单个 HIV-1 未剪接的前信使 RNA（pre-mRNA）可以产生超过 50 种不同的转录本，未剪接和剪接转录本的平衡比例对于产生感染性病毒粒子至关重要。因此，了解参与病毒 RNA 调节的机制具有潜在的治疗意义。然而，在细胞感染过程中监测整个转录组水平的选择性剪接事件的调节具有挑战性。在这里，我们使用牛津纳米孔技术（ONT）开发的长读 cDNA 测序来定量探索感染原代 CD4+T 细胞时 HIV-1 转录组调节的复杂性。
ONT 读取映射到病毒基因组的长度足以跨越所有可能的剪接接头，甚至是远距离的剪接接头，并被分配到总共 150 个外显子组合。进一步考虑了总共 53 种病毒 RNA 异构体，包括 14 种新的异构体进行定量。ONT 测序确定的病毒 RNA 相对水平具有高度的重现性，与之前的报告相比具有优势，并且与定量 PCR（qPCR）数据高度相关。为了更深入地了解选择性剪接调节，我们然后编译了剪接位点（SS）使用和转录本水平的定量信息，以构建“剪接树”，这是导致不同病毒异构体的一系列事件的定量表示。这种方法可以可视化 SS D2 扰动时 SS 使用的完全重新布线及其对病毒异构体水平的影响。此外，我们首次动态描绘了病毒感染后 12 至 24 小时发生的事件级联。特别是，我们的数据强调了非编码外显子在病毒 RNA 转录组调节中的重要性。
ONT 测序是一种方便可靠的策略，使我们能够掌握调节 HIV-1 感染细胞中病毒 RNA 景观的早期剪接事件的动态。
