Stimulation of Tat-independent transcriptional processivity from the HIV-1 LTR promoter by matrix attachment regions

Abstract

The chromatin environment and the sites of integration in the host genome are critical determinants of human immunodeficiency virus (HIV) transcription and replication. Depending on the chromosomal location of provirus integration within the genome, HIV-1 long terminal repeat (LTR)-mediated transcription may vary from 0- to 70-fold. Cis-elements such as topoisomerase II cleavage sites, Alu repeats and matrix attachment regions (MARs) are thought to be targets for retroviral integration. Here we show that a novel MAR sequence from the T-cell receptor beta locus (MARbeta) and the IgH MAR mediate transcriptional augmentation when placed upstream of the HIV-1 LTR promoter. The effect of transcriptional augmentation is seen in both transient and stable transfection, indicating its effect even upon integration in the genome. MAR-mediated transcriptional elevation is independent of Tat, and occurs synergistically in the presence of Tat. Further, we show that MAR-mediated transcriptional elevation is specific to the HIV-1 LTR and the Moloney murine leukemia virus LTR promoter. In a transient transfection assay using over-expressed IkappaB, the inhibitor of NF-kappaB, we show that MAR-induced processive transcription is NF-kappaB dependent, signifying the role of local enhancers within the LTR promoter. Furthermore, by RNase protection experiments using proximal and distal probes, we show that MAR-mediated transcriptional upregulation is more prominent at the distal rather than the proximal end, thus indicating the potential role of MARs in promoting elongation.

Figure 1

Schematic representation of the 1.5 kb HIV-1 LTR together with GFP-Luc as the reporter system. (A) L1 and L2 primers used for amplification are indicated by arrows. To make the short LTR (sLTR), the HindIII site was used as depicted. Hatched and solid boxes show SV40 and Vβ13 promoters, respectively. (B) Sequences of IgH and MARβ used in the studies. Underlined bold letters show the base unpairing region within the MAR sequences.

Figure 2

MAR-mediated enhancement of transcription through the LTR promoter. (A) FACS analysis of the GFP-positive 293 cells. One million 293 cells were seeded on a 30 mm plate and transiently transfected with the plasmid constructs expressing GFP under the influence of the LTR and MARβ-LTR promoters. A 2 µg aliquot of LTR-GFP and MARβ-LTR-GFP plasmid DNAs was transfected. In the case of the presence of Tat protein, 1 µg of pcDNA-Tat was co-transfected. The photographs were taken after 40 h using an inverted microscope (Olympus) under UV fluorescence. (B) Similarly, FACS analysis was performed from the same cells and the percentage of GFP-positive cells was plotted using a Sigma plot. (C and D) 293 cells were seeded as described above and transiently transfected with LTR-Luc, MARβ-LTR-Luc and IgH-LTR-Luc at 2 µg/well. In the case of Tat, 1 µg of pcDNA-Tat was co-transfected. Relative luciferase activities were calculated 40 h post-transcription by loading an equal amount of protein (50 µg). (E) 293 cells were seeded as described above and transiently transfected with TARδ and MARβ-TARδ at 2 µg/well. In the case of pcDNA-Tat, 1 µg of the plasmid was co-transfected. Relative lucierase activity was calculated 40 h after transfection. A 50 µg aliquot of protein was assayed for luciferase assay.

Figure 2

MAR-mediated enhancement of transcription through the LTR promoter. (A) FACS analysis of the GFP-positive 293 cells. One million 293 cells were seeded on a 30 mm plate and transiently transfected with the plasmid constructs expressing GFP under the influence of the LTR and MARβ-LTR promoters. A 2 µg aliquot of LTR-GFP and MARβ-LTR-GFP plasmid DNAs was transfected. In the case of the presence of Tat protein, 1 µg of pcDNA-Tat was co-transfected. The photographs were taken after 40 h using an inverted microscope (Olympus) under UV fluorescence. (B) Similarly, FACS analysis was performed from the same cells and the percentage of GFP-positive cells was plotted using a Sigma plot. (C and D) 293 cells were seeded as described above and transiently transfected with LTR-Luc, MARβ-LTR-Luc and IgH-LTR-Luc at 2 µg/well. In the case of Tat, 1 µg of pcDNA-Tat was co-transfected. Relative luciferase activities were calculated 40 h post-transcription by loading an equal amount of protein (50 µg). (E) 293 cells were seeded as described above and transiently transfected with TARδ and MARβ-TARδ at 2 µg/well. In the case of pcDNA-Tat, 1 µg of the plasmid was co-transfected. Relative lucierase activity was calculated 40 h after transfection. A 50 µg aliquot of protein was assayed for luciferase assay.

Figure 3

MAR-mediated enhancement of transcription occurs at a distance. 293 cells were seeded as described above. Briefly, cells were transiently transfected with sLTR-Luc, MARβ-sLTR-Luc and IgH-sLTR-Luc. The experiment was performed in the presence and absence of HIV Tat. Relative light units were calculated 40 h post-transfection by loading an equal amount of protein (50 µg).

Figure 4

MAR-mediated transcriptional enhancement is specific for the HIV LTR promoter. 293 cells were seeded as described previously and transiently transfected with LTR-Luc, MARβ-LTR-Luc, SV40-Luc, MARβ-SV40-Luc, Vβ13-Luc, MARβ-Vβ13-Luc, MoMuLV LTR and MARβ-MoMuLV LTR-Luc plasmids at 2 µg/well. Luciferase activity was calculated 40 h after transfection by loading an equal amount of protein (50 µg).

Figure 5

MAR-mediated enhancement of transcription by elongation. 293 cells were seeded as described previously and transiently transfected with LTR-Luc, MARβ LTR-Luc and IgH LTR-Luc plasmids at 2 µg/well. Transfections in the presence of Tat were performed by co-transfecting 1 µg of pcDNA-Tat. Total RNA was isolated using TRIZOL (Sigma), 40 h after transfection. (A) The proximal probe overlapping 300 bases from the +1 start site of the LTR was in vitro transcribed from a linearized template (NC-1 digested by XmnI) by T7 RNA polymerase. (B) For the distal probe, overlapping 213 bp from +1267 to +1480, in vitro transcription was carried out using a linearized template (XmnI-digested NC1) and T3 polymerase. A 10 µg aliquot of total RNA was analyzed for hybridization reaction by using 10 000 c.p.m. of probe RNA. Protected transcripts after digestion with RNase A and RNase T1 were analyzed on a 6% urea–polyacrylamide gel.

Figure 6

Effect of MAR on LTR-mediated transcription upon stable integration. CHO cells were seeded at a rate of 1 × 10⁶ cells per 6-well plate and transfected by LTR-GFP and MARβ-LTR-GFP at 2 µg/well. Transfected cells were stably selected using 800 µg/ml neomycin. A single cell population was obtained and grown. (A) Photographs of stably transfected LTR-GFP and MARβ-LTR-GFP cells were taken by an inverted microscope (Olympus) under UV fluorescence together with the bright field images. (B) Genomic DNA was prepared from LTR-GFP and MARβ-LTR-GFP stable cell lines using DNA-Zol reagent. A 20 µg aliquot of genomic DNA was digested with HindIII and BamHI. A 1 kb LTR probe core was used for hybridization with 10 000 c.p.m.

Figure 7

Transcriptional inhibition from LTR and MAR-LTR promoters by IκB in a dose-dependent manner. (A and B) 293 cells were seeded as described previously and transiently transfected with LTR-Luc, MARβ-LTR-Luc, IgH-LTR-Luc, MoMuLV LTR and MARβ-MoMuLV LTR plasmids at 2 µg/well. IκB plasmid was co-transfected at concentrations of 1 and 3 µg. Decreases in the relative light units were calculated 40 h after transfection by loading an equal amount of protein (50 µg).