RNA-Seq and RNC-Seq of HeLa cells stably expressing NDV NP protein

The oncolytic potential of NDV has gained significant attention in the context of clinical trials. After systematically comparing the oncolytic activities of different NDV subtypes,we found that all subtypes, except for highly pathogenic genotype VII NDVs, display remarkable infectivity on tumor cell lines.A single amino acid mutation (F450L) in the NP protein at position 450 leads to an approximate 1000000-fold increase in the TCID50 value of genotype VII NDVs on tumor cells.Mechanistic inquiries unveiled that the amino acid at position 450 of the NP protein governs preferential translation of NDV mRNA.The preferential translation that regulates viral mRNA translation is often accompanied by modulation of the host translation machinery. Therefore, we established HeLa cell lines stably expressing NP protein from different NDV strains. Ribosome-bound mRNA was enriched to perform Ribosome Nascent-chain Complex sequencing(RNC-seq), while total cellular mRNA was enriched to conducted RNA-Seq . Untreated cells were used as controls. These experiments aimed to investigate the impact of NP on the host translation system. We generated HeLa cell lines that stably expressed the NP protein from different NDV strains using a lentiviral packaging system. The NP protein derived from the oncolytic Herts/33 strain was designated as HNP, while the NP protein from the non-oncolytic I4 strain was referred to as INP. Subsequently, ribosomes were isolated, and the RNA bound to the ribosomes was extracted from the ribosomal fractions for Ribosome Nascent-chain Complex sequencing (RNC-seq). Total RNA was used for RNA-Seq analysis, serving as a control for changes in RNA abundance. The key distinction between RNC-Seq and RNA-Seq lies in the source of RNA, while the subsequent library preparation and sequencing methods were consistent.