Analysis of HIV-1 transcriptome in different cell models with nanopore sequencing [HIVTCD4_A2018]

To explore in depth and in a quantitative manner the complexity of the HIV-1 splicing landscape we used nanopore long-read cDNA (ONT) sequencing in NL4-3 HIV-1 infected primary CD4+ T cells and transfected/infected HeLa cells. Mean read lengths were between 1286 and 2626 nucleotides with maximum sizes of up to 9182 nucleotides, sufficiently long to span all possible splice junctions and to be assigned to a total of 229 exon combinations. Seventy isoforms were above the threshold of 5 copies in infected T cells and a core of 36 isoforms were found in common in infected T cells, infected HeLa cells and transfected HeLa cells. Quantification of both all viral isoforms as well as splice site (SS) usage were compiled to build “splice trees”, a quantitative representation of the splicing pathways leading to the different viral isoforms.  This approach allowed visualizing the complete rewiring of SS usages upon perturbation of SS D2, using over-expression of U1 D2UpEx snRNA in transfected HeLa cells and its impact on viral RNA expression. Furthermore, we produced the first dynamic picture of the cascade of events occuring between 12 and 24 hours of CD4+ T cells HIV-1 infection. In particular, our data highlighted the importance of non-coding exons in viral RNA transcriptome regulation. Altogether, our results show that ONT sequencing allows one to grasp the dynamic of splicing events modulating the viral RNA landscape in infected cells. To assess the ability of ONT sequencing to explore HIV-1 transcriptome, CD4+ T cells were isolated from 3 donors and infected with VSVg pseudotyped NL4-3 HIV-1. Total RNA was extracted 24h post-infection