A two-color haploid genetic screen identifies novel host factors involved in HIV-1 latency

To identify novel host factors as putative targets to reverse HIV-1 latency, we performed an insertional mutagenesis genetic screen in a latently HIV-1-infected pseudohaploid KBM7 cell line (Hap-Lat). Following mutagenesis, insertions were mapped to the genome, and bioinformatic analysis resulted in the identification of 69 candidate host genes involved in maintaining HIV-1 latency. Overall design: A latently HIV-1-infected pseudohaploid KBM7 cell line was generated according to a previously described strategy used in Jurkat cells (Jordan et al., 2003). Subsequently, haploid latent (Hap-Lat) cells harboring a latent integrated HIV-1-derived virus containing a green fluorescent protein (GFP) reporter were subjected to insertional mutagenesis using a gene-trap virus carrying an mCherry reporter. Instead of using a lethality-selecting scheme, our system relies on fluorescence-activated cell sorting (FACS) to select for reactivated cells, marked by elevated GFP expression resulting from insertional mutagenesis of genes essential for maintenance of HIV-1 latency. Cells expressing both GFP and mCherry were FACS sorted and expanded for multiple rounds, after which GT insertion sites were mapped and identified by inverse PCR and high-throughput sequencing.