Development and characterization of a double-fluorescent HIV-1 reporter cellular model to tackle the Rev-dependent export pathway

The Rev-dependent nuclear export of unspliced and singly-spliced transcripts of human immunodeficiency virus type 1 (HIV-1) constitutes a critical yet poorly characterized post-transcriptional event essential for effective viral replication. In this study, we engineered a dual-fluorescent HIV-1-based cellular reporter system to elucidate the mechanisms underpinning Rev-dependent export. By generating multiple stably integrated inducible cellular clones, we ensured the expression of two distinct fluorescent proteins, mKO2, and ECFP, from unspliced (Rev dependent) and multiply spliced (Rev independent) HIV-1 transcripts, respectively. Utilizing flow cytometry, we performed quantitative analyses of dual-fluorescent cell populations. The developed tool enables precise assessment of the Rev-dependent export, and we validated it using known inhibitors of this pathway (leptomycin D), as well as targeted depletion of MATR3, an essential cofactor of Rev, and CRNKL1, a repressor of unspliced HIV-1 RNA export.IMPORTANCEThe developed dual-fluorescent reporter system represents a powerful and handy tool for the identification and characterization of novel molecular players involved in the Rev-dependent export pathway. This system not only holds promise for advancing our understanding of human immunodeficiency virus type 1 (HIV-1) biology but also serves as an invaluable platform for high-throughput drug screening aimed at targeting post-transcriptional HIV-1 RNA processes, particularly nuclear export. Consequently, this study offers significant implications for the development of novel therapeutic strategies to eradicate the virus.