Transcriptomic analysis of Hsp90 inhibitor (Hsp90i)-treated HIV-infected pediatric tonsillar mononuclear cells

Purpose: Hsp90 is a host chaperone protein that regulates cellular homeostasis by interacting with a diverse clientele of cellular proteins. Several of these proteins are involved in cellular pathways that play crucial roles in facilitating HIV replication. Identification of key cellular genes that are utilized by HIV for their replication, and whose expression is altered by Hsp90i, will lead to the development of targeted HIV therapeutics, which will ultimately be utilized for HIV cure. Here we characterized the key cellular genes altered upon Hsp90i treatment of HIV-infected pediatric tonsillar mononuclear cells. Methods: Discarded tonsil tissues from 8 independent donors <15 years of age were obtained through Duke University School of Medicine Biorepository and Precision Pathology Center (BRPC), during routine tonsillectomy. Tonsil tissues were washed and minced in RPMI 1640 (Gibco), containing 1X antibiotic/antimycotic mixture (Gibco), 50μg/ml Gentamicin (Gibco), 10μg/ml Cephalothin (Sigma-Aldrich) and 5μg/ml Fluconazole (Sigma-Aldrich), and tonsillar mononuclear cells (TMCs) were isolated by filtering tonsillar tissue pieces through a 70-μm mesh filter, followed by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare, Waukesha, WI). 5x105 TMCs were infected with 250ng p24 equivalent of the cell-free CCR5-tropic HIV-JRFL virus. After 24hr, virus- infected cells were treated with either 0.1% DMSO or 0.1μM Hsp90i, daily. After 96hrs, total cellular RNA was extracted from TMCs using RNAeasy Mini kit (Qiagen). Total RNA quality and quantity was assessed using the Agilent Bionalyzer 2100 and the Qubit fluorometer, respectively. Poly(A) mRNA capture and construction of stranded mRNA-seq libraries from intact total RNA (RIN >7) was achieved using the commercially available KAPA Stranded mRNA-Seq Kit. In brief, mRNA transcripts were first captured from 200ng of total RNA using magnetic oligo-dT beads, fragmented using heat and magnesium, and reverse transcribed to produce dscDNA. Illumina sequencing adapters were then ligated to the dscDNA fragments and amplified to produce the final RNA-seq library. Libraries were indexed using dual molecular indices allowing for multiple libraries to be pooled and sequenced on the same sequencing lane on a HiSeq 4000 Illumina sequencing platform. Before pooling and sequencing, fragment length distribution and library quality was assessed on a 2100 Bioanalyzer using the High Sensitivity DNA Kit (Agilent Technologies). All libraries were then pooled in equimolar ratio and sequenced. Multiplexing 8 libraries on one lane of an Illumina HiSeq 4000 flow cell yielded about 43 million 50bp sequences per sample. Once generated, sequence data was de-multiplexed and Fastq files generated using Bcl2Fastq conversion software provided by Illumina. Conclusion: This study identified differentially expressed genes between DMSO-treated and Hsp90i-treated HIV-infected pediatric tonsillar mononuclear cells.