Interactome of the HIV-1 proteome and human host RNA

The human immunodeficiency virus (HIV-1) is highly dependent on a variety of host factors. Beside proteins, host RNA molecules have been reported to aid HIV-1 replication and latency maintenance. Here we report the use of multiple workflows of native RNA immunoprecipitation and sequencing (nRIPseq) to determine direct host RNA interaction partners of all 18 HIV-1 (poly)proteins. We identified 1,727 HIV-1 protein – human RNA interactions in the Jurkat cell line and 1,558 interactions in SupT1 cells for a subset of proteins. We discovered different cellular pathways that seem to be used or controlled by HIV-1 on the RNA level: Tat binds for example mRNAs of proteins involved in the super elongation complex (AFF1-4, Cyclin-T1). Correlation of the interaction scores (based on binding abundancy) allowed to identify the highest confidence interactions, for which we performed a small-scale knockdown screen that led to the identification of three HIV-1 protein binding RNA interactors involved in HIV-1 replication (AFF2, H4C9 and RPLP0). Overall design: RIPseq was performed in batches of 3 HIV-1 protein IPs and 1 FLAG-tagged GFP background control IP (all in triplicate). For each HIV-1 protein IP also a background control IP was included in triplicate using 10 µg anti-mouse IgG rabbit IgG antibody (ThermoFisher, no. 11895905). For Streptavidin-based RIPseq a similar procedure was performed using 50 µL preconjugated MagStrep “type3” XT beads (iba, no. 2-4090-002) for IP and unconjugated MagStrep “type3” XT beads for preclearance and background control. This RIPseq series contains 4 data sets, assayA is Jurkat FLAG uninfected, assayB is Jurkat STREP uninfected, assayC is SupT1 FLAG uninfected, assayD is SupT1 FLAG HIV infected