Transcriptional analysis of HIV-1 LTR in TZM-bl cells. (a) The LTR from prototype HIV-1 virus LAI is shown schematically with the various primer sets used to assess transcriptional activity across the LTR. (b) TZM-bl cells were transfected with either pTatDsRed (+Tat) or with the control pcDNA3.1(+) (−Tat). The cultures were collected 48 hours later and cellular mRNA collected and assessed using the various primer sets (1–4) to determine relative transcription across the 5′ LTR. Sets 1–4 are shown from either RT converted (+RT) or cellular mRNA alone (−RT).

Model for 362as-mediated transcriptional control of HIV-1 gene expression. (a) Once integrated into the genome of the target cell, the mobilization-competent vector can (b) express a small noncoding RNA which may then (c) interact directly with Ago-1 and localize to the elongating transcript (d) which contains the target loci for the small noncoding RNA. The small noncoding RNA and Ago-1 might also localize at the targeted site with DNMT3a, HDAC-1, and possibly Ezh2. The result of this complex localizing at the targeted promoter is histone, DNA methylation, and transcriptional gene silencing.

Mobilization-competent vector-mediated transcriptional silencing of HIV-1 LTR expression. (a) HIV-2 mobilization-competent vectors were generated to contain various small RNAs specifically targeted to the LTR/promoter regions of HIV-1b (Supplementary Materials and Methods).¹⁰ (b) Transcriptional silencing of LTR-expressed luciferase. TZM-bl cells were transfected with the respective pHIV2 vectors +/− Tat. The results from triplicate-treated cultures are shown with the standard deviations. (c) Nuclear run-on analysis of TZM-bl cells treated with either 362as or pHIV2 (control) +/− Tat. The results from triplicate RT-PCR reactions are shown with the standard errors of the mean. (d,e) Chromatin immunoprecipitation assays were performed on TZM-bl cells treated with the vectors in the presence of Tat for (d) Ago-1 and (e) H3K27me3. The results from triplicate-treated cultures are shown with the standard deviations.

The effects of vector treatment on HIV-1b expression. (a) Lentiviral vectors were co-transfected with the HIV-1 molecular clone HX10 in triplicate at varying ratios. Culture supernatants were collected, pooled together, and assayed by enzyme-linked immunosorbent assay for p24 expression. The effects of varied ratios of vector or virus on vector mobilization were determined. (b) Shed viral particles from the co-transfections were assessed by qRT-PCR for either virus or vector. Ratios of either (b) copies of vector/virus or (c) vector/virus ratios standardized to HIV-1 p24 pg/ml are shown. (d) Supernatants from 3:1 vector to virus ratio transfections were used to infect Jurkat cells, and a p24 analysis was carried out over the month-long serial passage. (e) Supernatants from day 28 of the serial passage were used to infect TZM-bl cells and luciferase expression determined 48 hours later. The results from triplicate-treated cultures are shown with the standard deviations. (f) Supernatants from day 28 post-serial passage were passaged to TZM-bl cells and a ChIP assay was performed for H3K27me3 or NF-κB (p65) enrichment, specifically at the targeted LTR. The results from a single experiment are shown.

362as treatment results in directed epigenetic changes at the HIV-1 LTR. (a) The 247as and 362as target loci are shown schematically with the target LTR. The 2 transcriptional start sites,³⁰ TAGAA or TATAA, and 3 NF-κB, Sp1 and Ap1 transcription factor-binding sites are also shown. (b) Treatment of TZM-bl cells with 362as results in a loss of NF-κB p50 and p65 at the targeted loci whereas the control-treated HIV-2 samples do not. (c) Treatment of TZM-bl cells with 362as results in a gain of SP1 and loss of NF-κB (p65) at the targeted loci relative to the control-treated HIV-2 samples. The results of a single experiment are shown. (d) Expression levels of various protein components known to be involved in RNAi and/or epigenetic gene regulation were suppressed by RNAi. Data represents the average knockdown from cells transfected in triplicate and then pooled to isolate total RNA. (e) DNMT3a, Ago-1 and HDAC-1 are required for 362as-mediated transcriptional silencing of LTR gene expression. The averages from triplicate-treated cultures are shown with the respective standard error of the means relative to the control-treated cultures for each treatment. (f) Treatment of cultures with 362as resulted in an enrichment of DNMT3a and HDAC-1 at the targeted LTR loci. Results from duplicate treated cultures are shown with the respective ranges. (g) Treatment of cultures with 362as results in an enrichment of DNA methylation at the targeted loci relative to control-treated cultures as determined by bisulfite sequence analysis from anti-Flag (first IP)-eluted samples shown in e.