CLIP-seq analysis of WT and Gag chimera HIV-1 virions 1

To investigate how the major HIV-1 structural protein Gag packages viral genomes efficiently, we replaced the NC domain of Gag with RNA binding domains from heterologous cellular RNA binding proteins to generate Gag chimeras. We hypothesize that the Gag chimeras that preferentially bind to purine-rich RNA sequences will package viral genomes efficiently. We performed CLIP-seq to identify the RNA sequences bound by WT Gag and Gag chimeras in cells, at the plasma membrane, and in virions. Overall design: 293T cells in 10 cm dishes were transfected with PR- NL4-3-derived proviral plasmids expressing the indicated Gag. One day following transfection and 14-16 hours before cells and virions are harvested, 100 µM 4-thiouridine (4SU) or 6-thioguanosine (6SG) was added to the cell culture medium. For cell Gag CLIP and virion Gag CLIP, whole cells and virions were UV-crosslinked and lysed, respectively. For plasma membrane (PM) Gag CLIP, cells were UV irradiated and then fractionated via membrane flotation. Gag-RNA complexes were immunoprecipitated from whole cell lysates, virion lysates, and PM fraction. Gag-bound RNAs were subsequently extracted for CLIP-seq library construction. Sequencing was done on Nextseq500 platform.