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. Author manuscript; available in PMC: 2016 Dec 1.
Published in final edited form as: Virology. 2015 Sep 14;486:7–14. doi: 10.1016/j.virol.2015.08.027

RNAP II Processivity is a Limiting Step for HIV-1 Transcription Independent of Orientation to and Activity of Endogenous Neighboring Promoters

Katarzyna Kaczmarek Michaels 1,2, Frank Wolschendorf 3, Gillian M Schiralli Lester 4, Malini Natarajan 5, Olaf Kutsch 3, Andrew J Henderson 1,2,*
PMCID: PMC4679410  NIHMSID: NIHMS720228  PMID: 26379089

Abstract

Since HIV-1 has a propensity to integrate into actively expressed genes, transcriptional interference from neighboring host promoters has been proposed to contribute to the establishment and maintenance HIV-1 latency. To gain insights into how endogenous promoters influence HIV-1 transcription we utilized a set of inducible T cell lines and characterized whether there were correlations between expression of endogenous genes, provirus and long terminal repeat architecture. We show that neighboring promoters are active but have minimal impact on HIV-1 transcription, in particular, expression of the endogenous gene did not prevent expression of HIV-1 following induction of latent provirus. We also demonstrate that releasing paused RNAP II by diminishing negative elongation factor (NELF) is sufficient to reactivate transcriptionally repressed HIV-1 provirus regardless of the integration site and orientation of the provirus suggesting that NELF-mediated RNAP II pausing is a common mechanism of maintaining HIV-1 latency.

Keywords: HIV Latency, Transcription Interference, RNA Polymerase II Pausing, NELF

Introduction

In patients undergoing anti-retroviral therapy, a dynamic population of HIV-1 infected cells persists and is mobilized to contribute to robust viral replication and spread upon treatment interruption (Bruner et al., 2015; Dahabieh et al., 2015). Since these latently infected cells present a major challenge to curing HIV infection, there is interest in understanding the biochemical mechanisms responsible for establishing, maintaining and reactivating transcriptionally repressed HIV-1 provirus. Studies with cell lines and primary cells have suggested that multiple mechanisms are responsible for the transcriptional repression that promotes HIV-1 latency. Some of the mechanisms that have been implicated in limiting HIV-1 transcription and contributing to the establishment of latency include RNA polymerase II (RNAP II) pausing, chromatin structure, recruitment of transcriptional repressor complexes and silencing by non-coding RNAs (Mbonye and Karn, 2014; Schiralli Lester and Henderson, 2012).

With approximately 70% of HIV-1 integrated into introns of actively transcribed host genes (Ding et al., 2013; Han et al., 2004; Lenasi et al., 2008; Lewinski et al., 2005; Rezaei and Cameron, 2015; Shan et al., 2011; Sherrill-Mix et al., 2013) it has been proposed that neighboring promoters transcriptionally interfere with the HIV-1 long terminal repeat (LTR) to repress proviral transcription. Whether a neighboring promoter influences HIV transcription may depend on the orientation of the two promoters, displacement or competition for the transcriptional machinery and transcriptional activators, local chromatin organization, or collisions between two active RNAP II complexes (Greger et al., 1998; Lenasi et al., 2008). Although transcriptional interference has been implicated as a mechanism of HIV-1 latency (Gallastegui et al., 2011; Greger et al., 1998; Lenasi et al., 2008), whether there are common biochemical mechanisms by which a neighboring promoter represses HIV-1 provirus has not been addressed.

To investigate the influence of proximal host promoters on HIV-1 transcription, we examined the induction of HIV-1 in three different latently infected cell lines in which the integration sites have been mapped. We demonstrate that transcription of host genes does not exclude transcription from a proximally integrated HIV-1 LTR, and that a common regulatory check point shared by these cell lines regardless of orientation of the HIV-1 provirus is RNAP II processiveness.

Materials and methods

Cell Culture

Jurkat clone E6-1 was originally purchased from American Type Culture Collection (ATCC, Manassas, VA). CA5, BA1 and 11B10 are Jurkat derived lines that harbor a single repressed copy of NLENG, a recombinant HIV-1-GFP provirus. The selection strategy for these cell lines has been previously described (Duverger et al., 2009; Jones et al., 2007). Cells were propagated in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin (P/S), and 0.2 M L-glutamine. For some experiments cells were activated with 2 μg/mL PHA and 10 ng/mL PMA or 0.5 μM TSA and harvested 24 h post-stimulation.

Human embryonic kidney 293T cells (HEK293T) were purchased from ATCC and cultured in Dulbecco’s modified Eagle’s medium containing 10% FBS and P/S. Cells were incubated in a 37°C humidified incubator with 5% CO2.

Identification of Proviral Integration Sites

Briefly, the proviral integration sites were mapped using a modification of a previously published method (Han et al., 2004) that combined inverse and nested PCR strategies. Chromosomal DNA was digested with either PstI, SphI or SpeI. Purified digested DNA was then subjected to self-ligation by with T4 ligase. The ligation product was used as template in an inverse PCR reaction with primers FW068 (5′-GGTCAGCCAAAATTACCCTATAGTG-3′) and FW032 (5′-AGTAGCCTTGTGTGTGGTA GAT-3′), which bind in gag and the viral LTR, respectively, followed by a nested PCR using primer FW067 (5′-TGTTAAAAGAGACCATCAATGAG-3′) and FW033 (5′-TGGTGTGTA GTTCTGCCAATCA-3′). PCR products were purified and subjected to sequencing using primers from the nested PCR reaction. Only PCR products containing parts of the gag gene and/or the 5′ end of the viral LTR were considered for mapping proviral integration sites.

Flow Cytometry

Cells were washed with PBS and fixed with 2% paraformaldehyde. Fluorescence was measured using Becton Dickinson FACScan at the Flow Core Facility at Boston University Medical Center.

Transfections, Virus Generation and Infections

NELF shRNA and control scrambled shRNA (OriGene) were packaged by cotransfecting Tat, RSV-Rev, Gag/Pol and VSV-G into HEK293T cells using calcium phosphate as previously described (Natarajan et al., 2013). In addition, shRNA experiments were validated with siRNA cocktails purchased from Dharmacon. Viruses were collected 48 h post-transfection and filtered through a Puradisc 25 Syringe Filter with 0.45-μm Polyethersulfone membrane (Whatman). Jurkat cells were transduced by culturing with supernatants containing lentiviral constructs for 12–16 h.

Quantitative Real Time-PCR

RNA was prepared by resuspending cells in TRIzol (Life Technologies), and cDNA was generated using SuperScript II Reverse Transcriptase (Invitrogen) and random primers (Promega). GoTaq qPCR Master Mix (Promega) was used for quantitative real-time PCR reactions. Initiated HIV-1 transcripts (+1 to +40) were amplified using 5′-GGGTCTCTCTGGTTAGA-3′ and 5′-AGAGCTCCCAGGCTCA-3′ primers and elongated HIV-1 transcripts (+5396 to +5531) were amplified using 5′-GACTAGAGCCCTGGAAGCA-3′ and 5′-GCTTCTTCCTGCCATAGGAG-3′ primers as described previously (Natarajan et al., 2013). RBM12 transcripts (+9,746 to +9,944) were amplified using 5′-GGTGAACTGGGTGAGGCTTT-3′ and 5′-TACTGGCATTTGCTGGTGGT-3′ primers. HELZ transcripts (+84,209 to +95,267) were amplified using 5′-CCAGCTGCCGCCTGTGCTTA-3′ and 5′-CACTCCATGGCCTGGGC AGC-3′ primers. PDZD8 transcripts (+880 to +957) were amplified using 5′-AAGGCTGCGC TTGGTCTTTA-3′ and 5′-AAGTCGATCAGCGGGTCTTC-3′ primers. To amplify chimeric RBM12 transcripts RBM12 (+14,769F) 5′-AGTGCTCGTTGTGGACTGTAAT-3′ and HIV-1 LTR (−346R) 5′-AGTGCTCGTTGTGGACTGTAAT-3′ primers were used. To amplify chimeric PDZD8 and HELZ transcripts HIV-1 LTR (−102F) 5′-GACTTTCCGCTGGGGACTTTC-3′ and either PDZD8 (+36,250F) 5-′ACATTTGTGTCCTTGCTAATGGT-3′ or HELZ (+96,171) 5′-ACAGTGATACAGTGGGTTGCAT-3′ primers were used. β-actin mRNA was amplified using a QuantiTect primer assay (Qiagen). PCR was carried out for 45 cycles, and the relative expression was calculated using the ΔΔCt method (Livak and Schmittgen, 2001), normalizing specific amplification of the transcripts of interest to the β-actin control for each specific sample.

ChIP-qPCR

Chromatin immunoprecpitations were performed as previously described (Natarajan et al., 2013). Antibodies used were as follows: anti-NELF-D (Proteintech), anti-RNAP II (Santa Cruz Biotechnology), anti-AcH3 (Upstate Biotechnology) and rabbit IgG (Upstate Biotechnology). Quantitative real-time PCR analysis was carried out using SYBR green reagents and the primers 5′-TGCTTTTTGCCTGTACTGGGTCTC-3′ and 5′-GCACACACTACTTGAAGCACTCAAG-3′, which amplify the −14 to +113 region of HIV-1 LTR.

Immunoblot Analysis

Whole-cell lysates were prepared by washing cells with cold PBS and lysing with buffer containing 10 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1.0 mM EDTA (pH 8.0), 2.0 mM sodium vanadate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 1% Triton X-100, 1.0 mM phenylmethylsulfonyl fluoride, and protease inhibitor mixture III (Calbiochem). Protein was measured using the BSA assay (Pierce). Samples were heated for 5 min at 100 °C before loading onto a 10% SDS-PAGE gel. Proteins were transferred from the PAGE to a polyvinylidene difluoride membrane (Millipore) by electroblotting. Antibodies used were as follows: anti-AcH3 (Upstate Biotechnology), anti-H3 (Abcam), anti-NELF-B (kind gift from Dr. Rong Li (University of Virginia)), anti-β-actin (Sigma-Aldrich) and anti-HIV-1 p24 (3537; NIH AIDS Research and Reference Reagent Program).

Statistical analysis

Statistical analysis was carried out using Student t test. A two-tailed distribution was performed on paired samples. Values of <0.01 were considered significant.

Results

Characterization of proviral integration sites in HIV-1 inducible cell lines

To gain insights into mechanisms by which neighboring endogenous promoters influence HIV-1 transcription, we utilized three previously characterized T cell lines which harbor latent HIV-1-GFP provirus, CA5, BA1, and 11B10 (Duverger et al., 2009; Jones et al., 2007). The proviral integration sites were mapped and sequenced for these three lines and the orientation of the provirus relative to the endogenous genes were determined (Fig. 1). CA5 cells have provirus integrated in the exon of RNA binding motif protein 12 (RBM12) in a parallel orientation (Fig. 1). RBM12 has been implicated in meibomian cell carcinoma (Kumar et al., 2007) and shares a promoter and 5′exon with copine I (CPNE1) (Yang et al., 2008). The function of RBM12 remains unidentified, while CPNE1 plays role in cell-cycle and proliferation (Skawran et al., 2008). In BA1 cells, HIV-1 is integrated in the intron of PDZ domain containing 8 (PDZD8) in a convergent orientation (Fig. 1). PDZD8 is a cytoskeleton-regulating protein which was recently shown to bind HIV-1 Gag, stabilizing capsid and enhancing HIV reverse transcription (Guth and Sodroski, 2014; Henning et al., 2010). HIV-1 is inserted in the intron of HELZ in a convergent orientation in the 11B10 cells (Fig. 1). HELZ is a zinc-finger containing RNA-helicase important for global translational initiation (Hasgall et al., 2011). We examined 17 cell lines and did not find any examples of the HIV-1 LTR integrating in a divergent orientation relative to an endogenous promoter.

Fig. 1. Proviral Integration Sites in CA5, BA1 and 11B10.

Fig. 1

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Summary and schematic of HIV-1 provirus integration sites relative to neighboring host promoter for latently infected T cell lines. Sequencing is described in Materials and methods. The lengths of the endogenous host gene, as well as its position on chromosome and integration site of provirus are indicated.

We examined the expression of these genes in the T cell lines using quantitative real time PCR to determine their normal expression pattern and whether HIV-1 might alter their expression. All three genes are expressed in Jurkat cells and the process of HIV-1 infection and selection of the cells, in general, only had a modest impact on the baseline expression of the host genes in which the virus integrated into (Fig. 2A). Specifically, RMB12 expression was not affected in any of the cell lines including CA5, the cell line in which the provirus is integrated into this gene (Fig 2). Integration of provirus into PDZD8 and HELZ genes increased their expression; PDZD8 was enhanced by 3 fold in BA1 cells and HELZ was expressed 3.5 fold more in 11B10 (Fig 2A). Since increased expression of PDZD8 and HELZ was not observed in all cell lines, this may reflect functional interactions between the endogenous promoters and the integrated HIV-1 proviral LTRs. We also assessed the expression of these genes following T cell activation. Cells lines were induced by treating with PMA + PHA for 24 h and mRNAs were measured by real-time PCR. Consistent with previous reports (Duverger et al., 2009), PMA + PHA induced robust HIV-1 expression in all three lines (Fig 2C). The ability to strongly induce HIV-1 transcription in these cell lines was not influenced by the orientation of the provirus relative to the endogenous gene since all three lines robustly expressed HIV-1 (Fig 2C). None of the three genes were inducible in any of the cell lines (Fig 2A). Based on endogenous levels of RBM12, PDZD8 and HELZ in Jurkat cells and cell lines in which HIV-1 provirus is not integrated into the host gene HIV-1 infection itself did not affect host gene expression (Fig. 2A). Furthermore, with the exception of 11B10 in which the HELZ gene was decreased by greater than 90% following PMA + PHA treatment, induction of HIV-1 expression did not require significant repression of the genes which harbored the provirus (Fig 2A) suggesting that expression of the endogenous gene does not necessarily exclude expression of HIV-1. To assure that integration did not inactivate the endogenous promoters we used RT-quantitative PCR to detect RNAs that included HIV and host RNA sequences. These chimeric transcripts were detected in all three cell lines.

Fig. 2. Activation of Latent Cells Results in Altered Host Gene Expression.

Fig. 2

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Uninfected Jurkat T cells as well as CA5, BA1 and 11B10 cells were treated with 2 μg/mL PHA and 10 ng/mL PMA for 24 h. A) Expression of the neighboring host gene mRNAs was measured by qRT-PCR and normalized to β-actin. B) Expression of chimeric RNA was amplified by RT-PCR and PCR products were visualized on 1% agarose gel. C) Expression of elongated HIV-1 mRNAs was measured by qRT-PCR and normalized to β-actin. These data are from a single experiment performed in triplicate and are representative of three independent experiments. Bars show average values ±SD, n=3. *p < 0.05, **p < 0.01 and ***p < 0.001 (Student’s t test).

HIV-1 transcription in latently infected cell lines

To explore the molecular mechanisms that are contributing to the repression of HIV-1 provirus integrated into actively expressed endogenous genes we initially inhibited histone deacetylases (HDACs) to determine whether this induces HIV-1 expression in the cell lines. HDACs, by regulating chromatin organization through removing acetyl groups from histones, have been implicated as a primary mechanism that limits HIV-1 transcription and contributes to HIV-1 latency. Cells were treated with PMA + PHA or Trichostatin A (TSA), a general inhibitor of class I and II HDACs and a potent inducer of HIV-1 transcription (Van Lint et al., 1996). We examined the induction of HIV-1 transcription by monitoring GFP expression via flow cytometry in the different cell lines (Fig. 3). PMA+PHA treatment induced HIV-1 expression in more than 65% cells as measured by GFP expression (Fig. 3B). Overall, treatment with TSA was not as effective as treatment with PMA + PHA at inducing HIV-1 expression (Fig. 3B). TSA treatment differentially reactivated HIV-1 and a robust induction of HIV-1 expression following TSA treatment was only observed in 11B10 cells (Fig. 3). Induction of HIV-1 in BA1 and CA5 cells by TSA was 70% less efficient than induction seen with PMA + PHA treatment (Fig. 3B). We confirmed that all three cell lines were responsive to TSA by measuring global changes in acetylated histone H3 (AcH3) by immunoblots (Fig. 3C). Since these lines represent cells that have proviruses integrated in converse (BA1, 11B10) and parallel (CA5) orientations the integration orientation does not impose a specific sensitivity to HDAC inhibitors. Overall, these data suggest that chromatin organization is not a general mechanism of repression in these cell lines which is consistent with HIV-1 integrating into transcriptionally active genes with an open chromatin structure.

Fig. 3. Provirus Integration Site Affects Reactivation Rate of Latent HIV-1.

Fig. 3

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Cell lines were treated with 2μg/mL PHA and 10 ng/mL PMA or 0.5 μM TSA for 24 h. A) GFP expression was measured by flow cytometry. B) Data represent percentage of GFP-positive cells. C) Immnoblots of lysates from DMSO- and TSA-treated cells for AcH3. H3 served as loading control. These data are from a single experiment and are representative of three independent experiments.

RNAP II pausing has been suggested to be a critical checkpoint for HIV-1 transcription and we examined if transcriptional elongation was a limiting step in these latent cell lines. RNAP II pausing is characterized by accumulation of RNAP II and NELF, which is necessary for pausing, at the transcriptionally repressed LTR (Jadlowsky et al., 2014; Peterlin and Price, 2006; Zhang et al., 2007). We performed chromatin immunoprecipitation assays (ChIPs) to assess whether RNAP II and NELF were associated with the integrated proviruses. RNAP II and NELF were detected at the HIV-1 LTR consistent with RNAP II pausing (Fig. 4). Furthermore, we observed the presence of AcH3 at the LTR in CA5 and BA1 cells (Fig. 4) suggesting open chromatin in these cells which is also consistent with the minimal proviral induction seen in TSA treated cells (Fig. 3). 11B10 cells had low levels of AcH3 at the 5′ LTR (Fig. 4), which could explain why 11B10 cells were more sensitive to TSA treatment (Fig. 3). RNAP II pausing is associated with the accumulation of initiated HIV-1 mRNAs relative to full length HIV-1 mRNAs (Natarajan et al., 2013). We used quantitative real time (RT) PCR to determine the ratio of initiated to elongated HIV-1 mRNA (Natarajan et al., 2013). In all three cell lines, the ratio of initiated transcripts was 3–10 fold higher than elongated HIV-1 mRNA prior to cell activation (Fig. 5). Upon treatment with PMA + PHA, we observed robust HIV-1 transcription and a shift in the ratio of initiated to elongated transcripts that approached 1 suggesting a release in paused RNAP II (Fig. 5).

Fig. 4. RNAP II and NELF Bind to Repressed HIV-1 LTR.

Fig. 4

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ChIP assays were performed to examine RNAP II and NELF binding to the HIV-1 LTR as well as acetylation of histone H3. These data are from a single experiment performed in triplicate and are representative of three independent experiments. Bars show average values ±SD, n=3. *p < 0.05 and **p < 0.01 (Student’s t test).

Fig. 5. RNAP II Pausing Occurs in Latent Cell Lines.

Fig. 5

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Latently HIV-1-infected T cell lines were treated with 2μg/mL PHA and 10ng/mL PMA for 24 h. Expression of initiated and elongated HIV-1 was measured by qRT-PCR using primers described in Methods. These data are from a single experiment performed in triplicate and are representative of three independent experiments.

These above data suggest that RNAP II may be a common checkpoint limiting HIV-1 transcription in the different cell lines regardless of integration sites and orientation. It has been previously demonstrated by our group and others that targeting NELF releases paused RNAP II from the HIV-1 promoter and induces HIV-1 transcription (Jadlowsky et al., 2014; Natarajan et al., 2013; Zhang et al., 2007). To formally test if RNAP II pausing is limiting HIV-1 in these cell lines we used a lentiviral vector expressing sh-NELF-B to knockdown NELF and determine if this is sufficient for activating HIV-1 transcription (Fig. 6). Despite our inability to efficiently knock down NELF (Fig. 6C), which was hampered by toxicity, reflecting the importance of this complex in regulating general transcription, knocking down NELF-B in the absence of any additional treatment significantly induced HIV-1 transcription in all three lines (Fig. 6A–C). For example, even a depletion of 19%, as determined by densitometry, of NELF-B induced HIV-1 expression by 9-fold in BA1 cells (Fig. 6). Similar results were seen using siRNAs rather than shRNA (data not shown). These data support the conclusion that RNAP II pausing limits HIV-1 transcription independent of orientation of the HIV-1 provirus and expression of the endogenous gene and suggest that transcriptional elongation may be a general mechanism that contributes to the maintenance of HIV-1 latency.

Fig. 6. NELF Limits HIV-1 Transcription in Inducible Cells.

Fig. 6

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Cell lines were transduced with sh-Ctrl Vector or sh-NELF-B specific lentivirus. A, B) 72 h post-transduction GFP expression was measured by flow cytometry. C) The knockdown of NELF-B and induction of HIV-1 was validated by immunoblots for NELF and p24 Gag. These data are from a single experiment and are representative of three independent experiments.

Discussion

Since HIV-1 has a propensity to integrate into transcriptionally active genes, transcriptional interference in which a neighboring promoter represses HIV-1 LTR activity has been suggested as a common pathway for the establishment and maintenance of proviral latency. Although several possible mechanisms have been suggested to be responsible for transcriptional interference such as RNAP II collisions, competition for key transcription factors and recruitment of chromatin remodeling factors (Gallastegui et al., 2011; Lenasi et al., 2008; Schiralli Lester and Henderson, 2012), whether there are common biochemical processes that lead to the establishment of latently infected cells is controversial. In this study we examined three different cell lines which had a single inducible HIV-1 provirus integrated into actively transcribed host genes to gain insights into the biochemical processes responsible for transcriptional interference. We show that induction of HIV-1 expression was not incompatible with transcription of the host gene and that RNAP II pausing was a checkpoint in all three lines regardless of the orientation of the HIV-1 LTR to the host gene promoter.

The three lines examined included proviral integrations either in a parallel or convergent orientation. Although 17 lines have been examined for orientation of provirus integration, we have not observed latently infected cell lines with HIV-1 provirus in a divergent orientation. The endogenous genes in these lines were all expressed and repression of the endogenous gene following cell activation was not a general requirement for inducible HIV-1 transcription. This observation would suggest that upstream host promoters in general are not over-powering or disrupting the transcriptional machinery of the integrated LTR and that active transcription is not incompatible with HIV-1 expression. These results appear to differ from previous studies using cell line models of latency, which suggest HIV-1 transcriptional activation requires downregulation of the neighboring host gene (Lenasi et al., 2008); however, they are consistent with recent experiments using dual reporter viruses, in which one marker is driven by the HIV-1 LTR and a second marker is regulated by a constitutive promoter, where double-positive cells were observed (Calvanese et al., 2013; Dahabieh et al., 2013). Discrepancies between these studies may reflect differences between cell lines, mediators used to activate cells or reporter viruses and warrant further investigation of mechanisms of transcriptional interference.

Chromatin does not appear to be the primary mechanism repressing HIV-1 in these cell lines since two of the three lines are only partially responsive to treatment with the HDAC inhibitor TSA. In addition, we observe the presence of acetylated histone H3 associated with the HIV-1 5′ LTR prior to activation in CA5 and BA1 cells. Our results would also be consistent with these proviruses being integrated into sites that are transcriptionally active and thus being in an open chromatin state. It is tempting to speculate that integration of the HIV-1 provirus into transcriptionally active genes rather than untranscribed DNA may actually facilitate reactivation by assisting with the clearing of the LTR of repressive or chromatin-remodeling factors, and thus assuring open chromatin structure (Gallastegui et al., 2011; Lenasi et al., 2008; Marini et al., 2015). Furthermore, the bias of HIV integrating into transcriptionally active genes and the potential modest influence of chromatin in regulating provirus transcription may in part explain why HDAC inhibitors target only a subset of latently infected cells and their limited success in clinical trials (Siliciano and Greene, 2011).

Recently, it has been suggested that integration of HIV-1 impacts the expression of the endogenous genes which potentially contribute to persistence and expansion of HIV-1 infected cells (Ikeda et al., 2007; Maldarelli et al., 2014). For example, integration of provirus into introns of BACH2 and MKL2 in the same transcriptional orientation correlated with clonal expansion of infected cells (Ikeda et al., 2007; Maldarelli et al., 2014). We observed that integration of provirus resulted in modest increases in the expression of the endogenous genes, PDZD8 and HELZ, in the BA1 and 11B10 cell lines, respectively. In both of these cell lines provirus was integrated in a convergent orientation and, although we did not explore the mechanism, it is possible that the HIV LTR in the converse orientation is altering the local chromatin environment to promote targeted host gene expression. PDZD8, a moesin-interacting protein that regulates cytoskeleton organization, has been shown to influence herpes simplex virus type 1 and HIV-1 infections (Guth and Sodroski, 2014; Henning et al., 2010). For HIV-1, PDZD8 enhances infection by interacting with Gag to stabilize HIV-1 capsid and modulating the uncoating process during reverse transcription (Guth and Sodroski, 2014; Henning et al., 2010). HELZ is a zinc-finger containing RNA-helicase important for global translational initiation (Hasgall et al., 2011). Targeting of these genes does not appear to present any significant growth advantages and integration into these sites was not over-represented during the generation of cell lines. However, we cannot rule out the possibility that there are intrinsic properties that led to selective advantages for these cell lines during the cloning process. Regardless, these lines still provide a useful experimental system to explore how HIV-1 behaves in the context of a transcriptionally active unit.

RNAP II pausing has been implicated as a mechanisms for transcriptional interference of coliphage λ and the tandem promoters that encode the mouse gene FPGS (Palmer et al., 2009). Furthermore, previous studies have indicated that RNAP II pausing is a key checkpoint that limits HIV-1 transcription in cell line models of latency as well as primary CD4+ T cells (Feinberg et al. 1991; Kao et al. 1987; Laspia et al. 1989; Natarajan et al. 2013; Jadlowsky et al. 2014). Our results demonstrating an abundance of initiated transcripts relative to fully transcribed HIV-1 mRNA and an accumulation of RNAP II and NELF at HIV-1 LTR prior to induction of the HIV-1 provirus are consistent with a model in which pausing contributes to transcriptional interference of the HIV-1 LTR. Most importantly, knocking down the NELF complex which is necessary for RNAP II pausing (Adelman and Lis, 2012) is sufficient to induce HIV-1 transcription in all three cell lines suggesting that RNAP II pausing is contributing to HIV-1 transcriptional repression in these cell lines.

Taken together, based on the analysis of several cell lines that harbored inducible latent HIV-1, the orientation of HIV-1 integration has minimal impact on the repression and reactivation of HIV-1 provirus. The latently infected T cell lines could all strongly induce HIV-1 transcription independent of the expression of the different host genes the provirus was integrated into. Therefore, HIV-1 transcription is not necessarily incompatible with an active upstream endogenous promoter. More importantly, although it is unclear as to whether common pathways are responsible for initially repressing HIV-1 transcription, there is a convergence of repressive mechanisms on RNAP II processivity suggesting that this is a common limiting step that maintains HIV-1 latency in multiple cellular contexts including primary cells (Kaczmarek Michaels et al., 2015; Natarajan et al., 2013). Exploring agents that modulate RNAP II processiveness will potentially identify novel therapeutics against latently infected cells.

HIGHLIGHTS.

  • The ability of endogenous genes to influence HIV provirus expression was examined.

  • Neighboring promoters had minimal impact on HIV transcription.

  • Endogenous gene expression did not prevent HIV transcription.

  • Releasing RNAP II reactivated latent HIV provirus regardless of the integration sites.

Acknowledgments

Parts of the work were performed in the UAB CFAR BSL-3 facilities and by the UAB CFAR Flow Cytometry Core/Joint UAB Flow Cytometry Core, which are funded by NIH/NIAID P30 AI027767 and by NIH 5P30 AR048311 and the Flow Core Facility at Boston University Medical Center. This work was funded in part by NIH grants R01-AI104499 and R21-AI116188 to OK and R01-AI097117 and R56-DE023950 to AJH.

Footnotes

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