HIV Nef Mediated Transcription in Primary CD4+ T cells. Homo sapiens

Our study investigates the role of the Nef protein on cellular gene transcription in infected primary CD4+ T cells with the goal of understanding how HIV Nef promotes viral replication and subsequent cellular pathology. To assess the effect of Nef on gene expression, RNA sequencing was done on 6 samples. In 3 control samples, we infected primary human CD4+ T cells with the wild-type HIV genome (NL4-3 Vpr mCD24 Δ-Env Wt-Nef). In the 3 experimental samples, we infected additional cells with a Nef-deleted variant of HIV (NL4-3 Vpr mCD24 Δ-Env Δ-Nef). The NL4-3 genome was introduced into the CD4+ T-cells through a pseudovirus infection. After 24 hours of infection, cells containing the HIV genome were then enriched through positive selection using anti-mCD24 microbeads. The mRNA was isolated using Trizol and subsequently reverse transcribed into complementary DNA. This cDNA seqments for each sample were sequence using High Throughput DNA Sequencing. Cells: 293 T cells were used to make pseudovirus containing vesicular stomatitis virus envelope glycoprotein G (VSV-G) and primary CD4 T cells were used for one round virus infection. Viruses: NL4-3 Vpr mCD24 Δ-Env Wt-Nef and NL4-3 Vpr mCD24 Δ-Env Δ-Nef. Note that both viruses express mouse CD24 or heat stable antigen (HSAg) in place of Vpr as well as are unable to express envelope (Env) due to deletion in envelope gene. However, the former one expresses functional Nef (wt Nef) and the latter one does not (Δ-Nef) Pseudovirus production: Pseudovirus was produced by co-transfection of 293T cells (5 x 106 293T cells that had been seeded in a T75 tissue culture flask 24 hours previously) with a pNL4-3Vpr-CD24ΔEnvWt-Nef or a pNL4-3Vpr-CD24ΔEnvΔ-Nef plasmid (15µg) and the vesicular stomatitis virus envelope glycoprotein G expression vector pHCMVG (5µg) using BioT transfection reagent following the the manufacturer’s protocol (Bioland, Paramount, CA). Supernatants were obtained 24 and 48 hours after transfection, passed through a 0.45µm filter, and concentrated by ultracentrifugation (26,000 rpm for 90 minutes at 4˚C, SW28 rotor, Beckman Coulter, Fullerton, CA). Aliquots containing approximately 50 ng HIV-1 p24 antigen in 50 µl were frozen at -80˚C until use. Concentration of antigen in the aliquots were measured by p24 ELISA kit (XpressBio, Frederick, MD). CD4 T cells preparation: PBMCs from a healthy donor were prepared by by Ficoll separation and cultured in the presence of bispecific monoclonal antibody CD3:CD8 (NIH Reagent program #12277). Briefly, PBMCs were resuspended at a concentration of 1.5X106 cells/ml in R10-50 and 1 ml of cell suspension transferred into each well of 24-well plate. After adding, bispecific antibody (final concentration 0.5 µg/ml), cell were cultured for 6 days at 37 0C incubator in the presence of 5% CO2. Cells were about 97% CD4 T cell positive when analyzed by flow cytometer on day 6 using anti-CD4 antibody (Biolegend, San Diego, CA; Cat#301408Total RNA Isolation from the magnetic beads separated cells: Separated cells were washed two times with PBS and then used for total RNA isolation using Trizol kit following the protocol of supplier (ThermoFisher Scientific, Grand Island, NY). After air drying, RNA pellets were resuspended in 100 µl of DNase RNase free water containing 100 units of RNase inhibitor (ThermoFisher Scientific, Grand Island, NY). The purity and concentration of RNA was measured by NanoDrop 1000 (ThermoFisher Scientific, Grand Island, NY). Sequencing Information: * instrument_model: Illumina HiSeq 5000 * library_source: Transcriptomic * library_strategy: RNA-Seq * library_selection: RT-PCR * library_layout: paired * Platform: Illumina These data were produced through RNA-Seq for the Next Generation, a project of the DNA Learning Center of Cold Spring Harbor Laboratory supported by the National Science Foundation under Grant Number DUE #1323522