An upstream YY1 binding site on the HIV-1 LTR contributes to latent infection

Abstract

During HIV-1 infection a population of latently infected cells is established. This population is the major obstacle preventing total eradication of the virus from AIDS patients. HIV-1 latency is thought to arise by various mechanisms including repressive chromatin modifications. Transcription factors such as YY1 have been shown to facilitate repressive chromatin modifications by the recruitment of histone deacetylases. In this study, we identified a novel binding site for YY1 on the HIV-1 LTR, 120 nucleotides upstream of the transcription start site. We show that YY1 can bind to this site in vitro and in vivo and that binding to the LTR is dissociated upon T cell activation. Overexpression of YY1 causes an increase in the proportion of cells that produce latent infections. These observations, in combination with previous results, demonstrate that YY1 plays a prominent role in controlling the establishment and maintenance of latent HIV-1 provirus in unstimulated cells.

Figure 1. YY1 binds to the HIV-1 LTR near RBEIII.

Panel A: EMSA was performed with Jurkat nuclear extracts and radiolabeled RBEIII probe (core RBEIII site highlighted in red). Antibodies to TFII-I (lane 2), USF1 (lane 3), USF2 (lane 4), or YY1 (lane 5) were added to analyze the complex components. Free probe (FP) is shown in lane 6. Panel B: An extract from SF9 cells infected with a baculovirus expressing YY1 was separated on 12% SDS-PAGE and analyzed by immunoblotting with YY1 antibody (lane 2). Lane 1 contains a high molecular weight protein ladder. Panel C: EMSA was performed with recombinant YY1 produced by baculovirus (lanes 1 and 2) or Jurkat nuclear extracts (lane 4) with the radiolabeled RBEIII probe. The RBF2 complex in lane 4 is shown. YY1 antibody was added to the reaction in lane 2. Free probe is shown in lane 3. Panel D: (Top) EMSA was performed with recombinant YY1 and labeled RBEIII probe (lane 1), unlabeled competitor oligonucleotides were added to the reactions at 100-fold molar excess (lanes 2-10) (Bottom). Sequences of unlabeled competitor probes; 2, a known YY1 binding sequence oligonucleotide from the parvovirus promoter; 3, wild type RBEIII probe; 4-5, three nucleotide substitutions; 6-12, point substitutions of the RBEIII probe; 13 free probe (FP). Non-specific (NS) bands are also shown. Substitutions are indicated in lower case.

Figure 2. Mutations at RBEIII prevent binding of YY1 in Jurkat-tat cells.

Panel A: Schematic representation of the pTY-LAI-dsRed reporter mini virus. The 5’ LTR controls expression of a p24 capsid-dsRed fusion, while the E1Fα promoter constitutively expresses eGFP. Panel B: Representation of primers used for ChIP analysis of the upstream RBEIII region, enhancer region (ER), and RBEI/ transcription start site region. Panel C: A representative pool of Jurkat-tat cells infected with the wild type and RBEIII/YY1 mutant reporter virus were used for ChIP analysis with YY1 antibody. Top, the core RBEIII sequence is shown in red and substitutions in the RBEIII/YY1 mutant indicated in lowercase. Bottom, Representative pools of Jurkat-tat cells infected with the wild type reporter virus or mutant RBEIII cell lines were used for ChIP analysis with YY1 antibody, using the primer sets indicated in Panel B. Error bars represent the standard deviation. ** Represents P values ≤ 0.01.

Figure 3. YY1 dissociates from the HIV-1 LTR in stimulated Jurkat-tat cells.

Panel A: Flow analysis of the untreated control (left) and PMA (right) induced Jurkat-tat cells bearing integrated pTY-LAI-dsRed reporter mini-virus. Expression of eGFP is indicated on the x-axis and dsRed expression on the y-axis. Panel B: Representative clones of Jurkat-tat cells infected with the wild type reporter virus induced with PMA or untreated (control) were used for ChIP analysis with YY1 antibody, using the primers indicated in Figure 2B. Panel C: Immunoblots of control and PMA induced Jurkat-tat cell lysates with YY1 antibody. Panel D: Immunoblot quantification of control and PMA induced cells using the Odyssey Infrared Imaging System. The y-axis represents the amount of YY1 divided by the amount of GAPDH normalized to one. The error bars show the standard deviation. * Represents P values ≤ 0.05, ** represents P values ≤ 0.01, *** represents P values ≤ 0.001.

Figure 4. YY1 is not bound to actively transcribed HIV-1 LTR in unstimulated Jurkat-tat cells.

Panel A: A representative population of Jurkat-tat cells infected with the wild type reporter virus was sorted using by FACS and populations of cells expressing only eGFP (green) or both eGFP and dsRed (red) were collected and expanded in culture. Panel B: ChIP analysis was performed on the expanded populations of Jurkat-tat cells expressing eGFP (green) or both eGFP and dsRed (red) using YY1 antibodies and the primer sets as described in Figure 2B. * Represents P values ≤ 0.05, ** represents P values ≤ 0.01, *** represents P values ≤ 0.001.

Figure 5. TFII-I binds to RBEIII constitutively.

Panel A: A representative pool of Jurkat-tat cells infected with the wild type (solid bars) and RBEIII/YY1 mutant (open bars) reporter virus were used for ChIP analysis with TFII-I antibody. Panel B: A representative clone of Jurkat-tat cells infected with the wild type reporter virus was induced with PMA or left untreated (control) and used for ChIP analysis with α-TFII-I antibodies, and the primers specific for RBEIII, the enhancer region (ER) and the RBEI site. * Represents P values ≤ 0.05, ** represents P values ≤ 0.01.

Figure 6. YY1 overexpression increases the proportion of virus that maintains latent infection.

Panel A: Schematic representation of expression constructs: wild type YY1 (YY1), a YY1 deletion mutant lacking the HDAC1 interaction region (g/a/k) and vector control (pEFlag) used to produce stable cell lines. Indicated are a histidine rich region (His), the glycine/alanine (GA) and glycine/lysine (GK) rich regions, and the zinc finger domains (ZF) DNA binding domain. Panel B: Immunoblot of cell extracts from stable lines transfected with the YY1 (lane 1), g/a/k YY1 mutant (lane 2) and vector control (lane 3) with α-Flag antibody. Panel C: ChIP was performed on pools of stably transfected Jurkat-tat cells overexpressing YY1, the g/a/k YY1 mutant or the vector control, infected with the wild type reporter virus using α-Flag antibody, and the primer sets as described in Figure 2B. Panel D: Stably transfected Jurkat-tat cells overexpressing YY1, the g/a/k YY1 mutant or the vector control were infected with the wild type pTY-LAI-dsRed reporter virus construct. Flow analysis was performed every 24 hours for 4 days and every week for a month post infection. The % active infection was calculated by dividing the number of cells with active HIV-1 LTR (eGFP and dsRed expressing cells) by the number of infected cells (total eGFP expressing cells).

Figure 7. Multiple transcription factors, including YY1, directly bind near the conserved RBEIII element.

The RBEIII sequence (red) and flanking sequences are shown, indicating the positions bound by USF1/2, TFII-I and YY1. Sequence variations observed in HIV-1 subtypes LAI-B (pTY-LAI-dsRed), LAI-A, LAI-C and LAI-E are indicated. A binding site for AP1 identified in subtypes A and C is indicated [34].