Antiviral and anti-inflammatory properties of novel anti-HIV candidate ABX464 promotes specifics RNA splicing while preserving cellular RNA integrity.

Purpose: To ensure that ABX464 acted specifically on HIV splicing and did not significantly or globally affect the splicing events of human genes, we used an assembly approach of HIV (YU2 strain) putative transcripts and human long non-coding sequences from paired-reads (2x75bp) captured on a NimbleGen SeqCap® EZ Developer Library (Roche/NimbleGen). Methods: Cells were infected with 80 ng of p24/106 cells of the YU-2 strain for 4 to 6 hours and then rinsed with PBS before medium renewal, followed by high-throughput RNAseq from custom SeqCap EZ capture libraries. Each raw dataset of the samples contained between 5 and 30 million paired-end reads (75 bp), with an average of approximately 12 million raw reads per sample. Results: The raw reads were then cleaned and assembled per library to generate contigs, giving an average of 930 contigs per sample for further analyses. Conclusions: Our results show that high-throughput analyses coupled with bioinformatics-specific tools offers a comprehensive and more accurate view of mRNA splicing within a cell. We used buffy coats from HIV-negative individuals were obtained from the local blood donation center, then human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (Histopaque, Sigma) gradient centrifugation. Cells were infected with 80 ng of p24/106 cells of the YU-2 strain for 4 to 6 hours and then rinsed with PBS before medium renewal.