Analysis of monocyte-derived dendritic cell transcriptome upon HIV-1 challenge and effect of MDA5 knockdown

The purpose of this study is to characterize the changes in global transcriptome of monocyte-derived dendritic cells (MDDCs) upon HIV-1 challenge, and examine the effect of MDA5 knockdown on MDDC transcriptome in the presence and absence of HIV-1 challenge. For this study, human CD14+ monocytes were enriched from peripheral blood mononuclear cells (PBMCs) and transduced with lentivectors encoding puromycin acetyltransferase and a short hairpin RNA (shRNA) targeting either control luciferase (Luc) or MDA5. Immediately following challenge with the knockdown vector, monocytes were placed in culture with IL-4 and GM-CSF to promote differentiation into MDDCs. Three days after transduction, puromycin was added to the medium to select for cells that had been transduced. On day seven, the resulting immature MDDCs were either challenged with a single-cycle HIV-1 vector or left untreated for 48 hours. RNA isolated from these samples was used to generate poly(A)-selected RNA-seq libraries. Analyses of these samples revealed that HIV-1 challenge in MDDCs induces a robust type-1 interferon response, whereas MDA5 suppression significantly limits this innate immune response to HIV-1. Overall design: mRNA profiles of MDDCs, isolated from 3 different donors, that are transduced with shRNA lentivectors targeting either luciferase or MDA5, and subsequently either challenged with HIV-1 for 48 hours or left uninfected. RNA-seq libraries were generated via poly(A)-enrichment.