Total RNA-seq analysis of WT and Gag chimera HIV-1 virions

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 total RNA-seq to identify the RNAs that are packaged by WT Gag and Gag chimeras in HIV-1 virions. Overall design: 293T cells in 6-well dishes were transfected with PR- NL4-3-derived proviral plasmids expressing the indicated Gag. Virions were harvested from cell culture supernatants 2 days post transfection and pelleted on a sucrose cushion. Total RNA was extracted from virions using TRIzol following manufacturer's protocol. RNA was subsequently prepared for sequencing using Illumina Truseq stranded mRNA kit and Nextseq500 platform.