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Involvement of histone methyltransferase GLP in HIV-1 latency through catalysis of H3K9 dimethylation
Virology 440 (2013) 182–189
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Involvement of histone methyltransferase GLP in HIV-1 latency through catalysis of H3K9 dimethylation Donglin Ding, Xiying Qu, Lin Li, Xin Zhou, Sijie Liu, Shiguan Lin, Pengfei Wang, Shaohui Liu, Chuijin Kong, Xiaohui Wang, Lin Liu, Huanzhang Zhu * State key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China
art ic l e i nf o
a b s t r a c t
Article history: Received 6 September 2012 Returned to author for revisions 27 December 2012 Accepted 20 February 2013 Available online 27 March 2013
Understanding the mechanism of HIV-1 latency is crucial to eradication of the viral reservoir in HIV-1infected individuals. However, the role of histone methyltransferase (HMT) G9a-like protein (GLP) in HIV-1 latency is still unclear. In the present work, we established four clonal cell lines containing HIV-1 vector. We found that the integration sites of most clonal cell lines favored active gene regions. However, we also observed hypomethylation of CpG of HIV 5′LTR in all four clonal cell lines. Additionally, 5′-deoxy5′-methylthioadenosine (MTA), a broad-spectrum histone methyltransferase inhibitor, was used to examine the role of histone methylation in HIV-1 latency. MTA was found to decrease the level of H3K9 dimethylation, causing reactivation of latent HIV-1 in C11 cells. GLP knockdown by small interfering RNA clearly induced HIV-1 LTR expression. Results suggest that GLP may play a significant role in the maintenance of HIV-1 latency by catalyzing dimethylation of H3K9. & 2013 Elsevier Inc. All rights reserved.
Keywords: HIV-1 latency Integration site CpG methylation H3K9 methylation GLP
Introduction Highly active antiretroviral therapy (HAART) can suppress HIV1 replication and cripple viral production to the point where viral levels become undetectable in HIV-1-infected individuals (Chun et al., 1999; Gulick et al., 1997; Hammer et al., 1997; Perelson et al., 1997). Despite the great advances that have been made in clinical antiretroviral therapy, interruption of HAART treatment is still closely associated with the recovery of HIV-1 replication and resumption of disease progression. This is because of the viral reservoir that HIV-1 can form in the human body (Chun et al., 2000; Finzi et al., 1997; Wong et al., 1997; Zhang et al., 2000). This reservoir consists of latently infected resting memory CD4 þ T lymphocytes into whose genomes HIV-1 proviruses have integrated (Loetscher et al., 1999; Stevenson, 2003). This latent HIV-1 reservoir can exist for long periods of time and so become a major obstacle to the eradication of HIV in HAART-treated patients. In latently infected cells, HIV-1 gene expression and viral production are inhibited after integration. Integration is a crucial step in the viral infection of CD4 þ T cells and one of the ways in which the virus avoids attack from the host immune system (Colin and Van Lint, 2009; Engelman et al., 1995; Englund et al., 1995; Lassen et al., 2004; Sakai et al., 1993). Because HIV DNA can integrate into the genome of the host cell, it is probable that
* Corresponding author. E-mail address: [email protected] (H. Zhu). 0042-6822/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.virol.2013.02.022
integration can take place in chromatin environments beneficial to latency (Trono et al., 2010). Some investigations of resting CD4 þ T cells purified from infected patients on HAART have suggested that HIV-1 integration acts in a non-random manner in vivo, preferring the intronic regions of active genes (Colin and Van Lint, 2009; Han et al., 2004; Wang et al., 2007). This may be because a transcriptional gene named LEDGF/p75 can induce HIV-1 DNA into the region of actively transcribed genes after interacting directly with viral integrase (Cherepanov et al., 2003; Lewinski et al., 2006; Meehan et al., 2009; Schröder et al., 2002; Vandegraaff et al., 2006). Other studies on the Jurkat cell model have reported that integration sites have many alphoid repeats rich in heterochromatin (Jordan et al., 2001; Jordan et al., 2003; Lenasi et al., 2008). Elucidating the characteristics of the integration may help us more clearly understand the mechanisms of HIV-1 latency. After HIV-1 integration into host genome, chromatin modifications in viral promoter of HIV-1 long terminal repeat (LTR) become involved in transcriptional regulations, including CpG methylation and histone methylation (Bednarik et al., 1987; Bednarik et al., 1990; Coiras et al., 2009; Gutekunst et al., 1993; Marban et al., 2007). One study on Jurkat clonal cell lines transfected by HIV-1 derived vector and primary resting memory T cells infected with HIV-1 showed that CpG methylation levels are universally low. Even after reactivation of latent HIV-1, these CpG islands retain hypomethylation (Pion et al., 2003). However, CpG methylation of HIV-1 5′LTR has been found to be selective and is considered an important epigenetic control in the maintenance of HIV-1 latency (Blazkova et al., 2009; Ishida et al., 2006). Observations made on
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the Jurkat cell model and primary lymphocytes latently infected with HIV-1 indicate that MBD2, a member of the methyl-CpG binding domain family of proteins, can specially bind methylated CpG islands and lead to gene silencing by recruitment of transcriptional repressors including the nucleosome remodeling and histone deacetylation (NuRD) complex (Bird and Wolffe, 1999; Kauder et al., 2009; Wade, 2001; Zhang et al., 1999). In conclusion, more analysis is required to confirm the association between HIV1 latency and CpG methylation. Histone methylation has also been associated with transcriptional regulation. It is considered more labile than DNA methylation (Jones and Baylin, 2007; Kelly et al., 2010; Shi, 2007). Histone methylation has distinct effects on transcriptional controls depending on the specific residues and the pattern of methylation (Spannhoff et al., 2009). For example, methylation of histone H3 lysine-4 and arginine-17 is commonly associated with activation of transcription, but methylation of histone H3 lysine-9 and lysine-27 generally leads to inactivation (Huang et al., 2006; Martin and Zhang, 2005; Spannhoff et al., 2009; Volkel and Angrand, 2007). Because H3K9 methylation is considered responsible for gene silencing, some histone methyltransferases (HMTs) that catalyze H3K9 methylation have been investigated (du Chene et al., 2007; Imai et al., 2010). Methyltransferases other than Su39H1 and G9a, have been identified. These include Suv39H2, GLP (G9a-like protein)/EHMT1, and SETDB1. These H3K9 HMTs all contain a common SET-domain, which acts as the catalytic unit (Dillon et al., 2005; Ogawa et al., 2002; Rea et al., 2000; Schultz et al., 2002; Tachibana et al., 2001; Yang et al., 2003). GLP, a G9a-related mammalian H3K9 specific methyltransferase has been found to be involved in gene repression (Levy et al., 2010; Tachibana et al., 2008). However, the role of GLP in HIV-1 latency remains to be clarified. The mechanism underlying HIV latency is not absolutely clear. We established an in vitro model of HIV-1 latency. On the latency model consisting of four clonal cell lines, we performed a series of experiments, including analyses of HIV-1 integration sites and assessments of CpG and H3K9 methylation in HIV 5′LTR to elucidate the mechanism of HIV-1 latency in our in vitro model and identify potential targets for complete eradication of latent HIV-1.
Results Establishment of an in vitro HIV latency model Plasmid NL43-EGFP was derived from pNL4-3. It is a HIV-1 constructor imported EGFP-encoding gene in a nef open reading frame. It was here mutated into Env and Vpr genes. We used pNL43-EGFP and pVSVG to co-transfect 293 T cells for 72 h and analyzed the relative number of GFP þ cells using FACS analysis. We used viruses at an m.o.i of 0.1 to infect Jurkat T cells. We separated GFP-negative cells using FACS after 72 h infection. Because the GFP-negative population was composed of uninfected cells and cells containing transcriptional silenced proviruses, we treated GFP-negative cells with TSA, PMA, TNF-α, PHA, and lonomycin for 48 h to activate silenced HIV-1 and sorted out GFP-positive cells using FACS (Fig. 1). These GFP-positive cells were cultured as a monoclonal cell line for 4 weeks. Several impure clonal cell lines were discarded, and four clonal cell lines were selected individually for further analyses. Location of HIV-1 integration site from latently infected cells Because each clonal cell line showed nonproductive HIV-1 replication and low, unequal levels of basal transcription, we
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Fig. 1. Sorting of HIV-1 latently infected cells from GFP-negative cells. Because the GFP-negative population were composed of uninfected cells and cells containing transcriptional silenced proviruses, we treated GFP-negative cells with TNF-α at 10 ng/ml, TSA (50 nM, 100 nM, and 200 mM), PMA at 10 ng/ml, PHA at 5 μg/ml, and 1 μM lonomycin for 48 h to activate silenced HIV-1 and sorted out GFP-positive cells by FACS analysis.
cloned and indentified the integration sites of HIV-1 from four clonal cell lines using an inverse PCR strategy (Fig. 2A). Genomic DNA was isolated from latently infected cells, digested with the restriction enzyme PstI, which recognizes a six-base site in gag gene of HIV and ligates into a self-loop under dilute conditions. The circularized DNA was used as a template in inverse PCR and the PCR products were visible on agarose gel (Fig. 2B). Bands were isolated, cloned, and sequenced. A total of four distinct clonal cell lines were used to assay the integration site. Sequences of four unique integration sites were identified and analyzed in a BLAST search of GenBank and the UCSC Bioinformatics Human Genome database. Sequences flanking the 5′LTR of HIV-1 were considered authentic if they showed at least a 99% match to the human genomic sequence. We then examined the distribution of integration sites and found the integration sites of C1, C3, and C11 to be located in introns of gene regions but those in C13 were downstream of a transcriptional unit. As characterized in Table 1, HIV-1 probably preferred to integrate in gene-rich regions. This is consistent with previous in vivo studies (Han et al., 2004).
Hypomethylation of the HIV-1 5′LTR in all four clonal cell lines To determine the possible relationship between latency and HIV promoter methylation, five latently infected Jurkat clonal cell lines C1, C3, C11, and C13, each containing single proviral copy, were used to investigate HIV 5′LTR cytosine methylation. The methylation level was dependent on bisulfite sequencing. After bisulfite conversion, the cytosine of CpG did not change, but the cytosine outside CpG was converted to uracil, which is recognized as thymine in PCR amplification. Sequencing showed that low CpG methylation levels in 5′LTR of these latency models (3.7±2.6% in C1, 6.2±2.7% C3, 1.2±1.2% C11, 2.5±1.6% C13) (mean ±standard error) (Fig. 3). Another analysis was performed to evaluate the methylation of each CpG site in the 5′LTR. No evidence indicated that CpG methylation was associated with HIV-1 promoter silencing. Some mechanisms other than CpG methylation in the 5′LTR may contribute to HIV-1 latency.
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Fig. 2. HIV integration sites are detected in four latently infected cell lines by inverse PCR. (A) Diagram of inverse PCR. Briefly, DNA purified from latency cell lines was digested with PstI, the digested DNA was ligated by T4 ligase and proviral-host DNA junctions were amplified with two couples of primers under a nested reaction. (B) Products of inverse PCR from four clonal cell lines were visible in agarose gels. Each band meant a unique integration event. Control reactions carried out either human genome without HIV infection or no template.
Table 1 Characteristics of HIV-1 integration sites in Jurkat clonal cell lines. Clonal cell line Conjunctional sequencea Chromosome location Host nt. at the junctionb Gene
Integration locus Gene description
C1 C3 C11 C13
Intron Intron Intron Intron
ATGCCTGGAA AAGGCTGGAA GAGATTGGAA AAAGGTGGAA
8q24.3 16p13.3 16p13.3 19q13.33
145153859 366460 2306831 48261159
SHARPIN AXIN1 RNPS1 LOC729626
SHANK-associated RH domain interactor Axin 1 RNA binding protein S1 hypothetical protein
The characteristics of HIV-1 integration sites were analyzed by using Genbank and UCSC database. Sequences flanking to 5′LTR of HIV-1 were considered to be authentic as they showed at least 99% match to human genomic sequence by BLAST search of GenBank. a b
Junction between Human genomic sequence and 5′LTR (first five bases). Nucleotide number at integration site.
Fig. 3. CpG methylation profiles of 5′LTR in HIV-1 from four Jurkat clonal cell lines. (A) Distributions of CpG dinucleotides in 5′LTR of latent HIV-1 LTR-driven retroviral vector pNL4-3 are shown as open circles. Genomic DNA from latency cell lines was converted by bisulfite treatment and subsequently was used to amply. (B) Different level of methylation in HIV-1 5′LTR in Jurkat clonal cell lines C1, C3, C11, and C13. Analyses of nine molecules are shown by QUMA online software. Only sequences with at least 98% conversion of cytosines outside CpG dinucleotides were calculated. Levels of CpG methylation are indicated as a mean±standard error for each clonal cell line.
MTA treatment induces HIV-1 transcription in Jurkat clonal cell line C11 MTA is competitive with the SAM cofactor, which can provide methyl groups for histone methlytransferase. In this way, it plays an important role in inhibiting methlytransferase activity. The presence of hypomethylation in CpG islands in vitro latency cell lines might indicate that some other mechanisms, such as histone methylation, are involved in HIV-1 latency. Four clonal cell lines were seeded in 96-well plates at 4×105 cells/ml and treated with MTA under a concentration gradient (0, 1 μM, 10 μM, 100 μM, 1000 μM) incubated at 37 °C with 5% CO2 for 48 h. As indicated by FACS analysis, after MTA treatment lasting 48 h, the number of GFP-positive cells increased obviously in C11 but C1, C3, and C13 showed no significant differences from untreated controls (Fig. 4).
In this way, MTA was shown to act as a histone methyltransferase inhibitor. It can apparently induce HIV-1 transcription to reactivate latent HIV in cell line C11. Dimethylation of H3K9 leads to HIV latency in C11 MTA is a broad-spectrum histone methyltransferase inhibitor (Diehl et al., 2007). It can down-regulate gene expression by inhibiting some histone methyltransferase such as MLL and set1 due to methylation of H3K4 (Huang et al., 2006; Diehl et al., 2007). Methylation at different residues has distinct effects on gene expression. For example, methylation at H3K4, H3K36, and H3K79 is associated with gene activation, and methylation at H3K9, H3K27, and H4K20 is associated with gene silencing (Spannhoff et al., 2009). Here, we found MTA treatment increased
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Fig. 4. MTA treatment induces HIV-1 transcription in latent cell line C11. (A) After latent cell lines were treated with or without 1mM MTA for 48 h, GFP-positive cells were analyzed by FACS (FACS Calibur, BD Science) in four clonal cell lines. GFP-positive cells were increased 5.5-fold after 1mM MTA for 48 h in C11. Data was shown as mean ±standard error by three independent experiments. (B) Percentage of GFP-positive cells after 1mM MTA treatment in C11 by FACS analysis. Latent HIV-1 harbored in C11 was reactivated at the ratio of 13.1% after 1mM MTA treatment for 48 h.
the number of GFP-positive cells in latent cell line C11. A reaction between latent HIV-1 and MTA indicated the presence of a pathway involving histone methylation due to the HIV-1 latency. To demonstrate whether histone methylation was associated with HIV-1 latency in C11, a ChIP assay was performed to measure the level of H3K9 me2 and me3 in the LTR promoter of HIV-1. First, C11 were incubated with 1mM MTA for 24 h, immunoprecipitated DNA fragments were obtained using antibodies against H3K9 me2 and H3K9 me3. These fragments were subjected to real-time quantitative PCR analysis. The level of H3K9 me2 was shown to be down-regulated by approximately 2.27-fold after MTA treatment in C11 cells. The level of H3K9 me3 showed no obvious change (Fig. 5A).
GLP knockdown up-regulates the transcriptional activation of HIV-1 LTR Because GLP was reported to be involved in the dimethylation of H3K9, an assay on endogenous GLP knock-down was performed (Levy et al., 2010). HEK 293 cells were planted and co-transfected with HIV-1 LTR-luc and either GLP-specific siRNA or scramble siRNA. After 48 h incubation, cells were harvested and subjected to luciferase activity according to the manufacture's protocol. Luciferase assay showed that GLP knockdown increased the transcription of HIV-1 LTR over that of cells exposed to scramble siRNA (2.1-fold change). TNF-α treatment was found to promote HIV-1 LTR expression (2.3-fold change compared to control siRNA) (Fig. 6). This experiment indicates that GLP is involved in silencing the expression of HIV-1 LTR.
Discussion Although HAART can reduce viral load in plasma and improve patient quality of life, persistence of latent reservoir becomes an obstacle to prohibit antiretroviral drugs from eliminating HIV-1 completely. For this reason, understanding the mechanism of HIV1 latency is considered crucial in our present study. To establish the HIV-1 latency model, we used an HIV-1derived vector to infect Jurkat T cells. This produced latently infected cells containing a single copy of HIV-1. Studies on in vitro models of HIV latency have been reported previously. These models include U1 and ACH2, which exhibit low levels of basal transcription, despite mutations in Tat (U1) and TAR (ACH2) (Folks et al., 1986, 1987). HIV latency has also been reported in Jurkat T cells harboring HIV provirus with deletion of NF-κB (Antoni et al., 1994). Although these cell lines have served as classical models in studies on HIV-1 latency, the mutations that they carry may raise concerns about whether they can hold up a mirror to HIV-1 latency in vivo (Yang, 2011). Recently, an in vitro HIV latency model was developed in Jurkat T cells infected with Tat-driven vector (Jordan et al., 2001, 2003). In this present study, we used pNL4-3-EGFP, which was reconstructed from pNL4-3 by introducing in gene encoding enhanced green fluorescent protein and mutating in Vpr and Env, to infect Jurkat T cells. We finally obtained four latently infected cell lines. In these latent cells, HIV-1 DNA integrates into the host genome and exhibits low basal transcription. Integration causes a stable, latent state that allows the virus to evade the immune system and antiretroviral drugs. Because integration sites have been shown to play a significant role in HIV-1 latency, several studies have suggested that integration favors in active transcriptional gene
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Fig. 5. The level of H3K9 me2 on 5′LTR of HIV-1 is decreased after MTA treatment. (A) As described, ChIP assay was performed after treating C11 either with or without 1mM MTA for 24 h. Recruitments of H3K9 me2 and H3K9 me3 on 5′LTR of HIV-1 were measured by real-time quantitative PCR. Normal mouse IgG was used as a negative control. Data are determined by three independent experiments. (B) 20 μl Products of real-time quantitative PCR were visible in agarose gels.
Fig. 6. GLP knockdown up-regulates the transcriptional activation of HIV-1 LTR. 293 cells were co-transfected with 160ng HIV-1 LTR-luc and 100 nM siRNAs (GLP siRNA or control siRNA) using Lipofectamine 2000 (Invitrogen). After 24 h transfection, Cells were either treated or untreated with 10 ng/ml TNF-α for additional 24 h. Cells were finally harvested after 48 h transfection and subjected to luciferase activity assay. Results were shown as fold change (mean value±standard error).
regions in CD4 þ T cells from patients on HAART or in CD4 þ T cells infected with retroviral vector (Han et al., 2004; Brady et al., 2009). However, analyses of integration sites in a Jurkat T cell model show that integration distributes in or near the alphoid repeats found in heterochromatin in most latent cell lines (Jordan et al., 2001). In
contrast, analysis of our small sample set indicated that three out of four integration sites were located in active gene regions (Table 1), which is consistent with other in vitro studies (Lewinski et al., 2006; Schröder et al., 2002). Our conclusion is probably limited by the small sample size. According to previous reports, LEDGF/p75 protein is associated with HIV-1 integration into active transcriptional genes and this protein can vary in expression depending on different cell types (Brady et al., 2009; Shun et al., 2007), this may explain its preference for the integration site. After the integration of HIV-1 DNA into the host chromatin, proviral expression is largely governed by host cellular factors. LTR promoter is the driver of HIV-1 gene expression, including a number of binding sites essential to the regulation of transcriptional activity (Calman et al., 1988; Parrott et al., 1991; Sune and Garcia-Blanco, 1995). In this way, epigenetic modifications of the HIV-1 5′LTR region have been involved in studies into the mechanism by which HIV-1 latency is established. DNA methylation has been shown to be associated with inactivation of HIV LTR (Bednarik et al., 1987, 1990). In our latent cell lines, we analyzed CpG methylation of HIV-1 5′LTR and observed hypomethylation in CpG of HIV 5′LTR in four Jurkat clonal cell lines. These CpG methylation levels varied from 1.2% to 6.2%. The phenomenon observed in our study is consistent with the findings of studies performed in other Jurkat clonal cells and on primary resting memory T lymphocytes infected with retroviral vectors (Blazkova et al., 2009; Pion et al., 2003). Hypermethylation of CpG of HIV-1 5′LTR is observable in latently infected cells selected after two-step reactivation. This has also been reported in long-term-HIV-1infected aviremic individuals but not in viremic patients (Blazkova et al., 2009). These findings suggest that methylation of CpG of HIV-1 5′LTR is probably accompanied by continuous development of HIV-1 latency. The observation made in ACH-2 cells demonstrates the existence of 5′LTR-selective methylation (Ishida et al., 2006). Overall, our work suggests that CpG methylation levels of 5′LTR in HIV-1 are usually low. No 5′LTR-selective methylation was observed in our in vitro model of HIV-1 latency. Because hypomethylation was observed in HIV-1 5′LTR, we speculated that the mechanism of DNA methylation might not be correlated with initial establishment of HIV-1 latency in our latent cell lines. Previous studies showed that histone H3K9 methylation has a deep influence on regulation of transcription and establishment of HIV-1 latency (du Chene et al., 2007; Imai et al., 2010; Kubicek et al., 2007; Kelly et al., 2010; Seligson et al., 2005). We used MTA, a broad-spectrum histone methyltransferase inhibitor,
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to treat these clonal cell lines and assess them for histone methylation. With 1mM MTA treatment, the ratios of GFPpositive cells were analyzed by FACS after 48 h. Latent HIV-1 in cell line C11 was reactivated at the ratio of 13.1% by MTA compared to 2.5% without MTA, but no obvious reactivation was observed in other clonal cell lines. This suggested that histone methylation plays a considerable role in HIV-1 latency. To demonstrate this, we performed a ChIP assay with anti-H3K9 me2 and anti-H3K9 me3 antibodies. The precipitated DNA segments were subjected to realtime quantification PCR. The results showed that level of dimethylation of H3K9 was reduced (2.27-fold change) after MTA treatment. The ChIP assay also confirmed that dimethylation of H3K9 was associated with HIV-1 latency in clonal cell line C11. Although HIV-1 latency has been shown to be dependent on H3K9 me2 in C11, no similar results were observed under MTA treatment in other latent cell lines. Histone acetylation was also found to be involved in HIV-1 latency, though some studies have also shown it to be less effective than histone methylation (Colin and Van Lint, 2009; du Chene et al., 2007). We used a HDAC inhibitor (trichostatin A, TSA) to reactivate our latent cell lines at 100 nM. Finally, partial latent HIV-1 was reactivated in these clonal cell lines in the presence of obvious cytotoxicity (data not shown). The distinct roles of histone methylation in different latent cell lines could be explained by a study of computational prediction, This would show whether epigenetic modifications are associated with diverse HIV-1 integration sites (Wang et al., 2007). Some HMTs have been found to participate in the catalysis of H3K9 methylation. Studies show that SuvH1 and G9a can silence genes by increasing levels of H3K9 me3,and H3K9 me2 (du Chene et al., 2007; Imai et al., 2010). Another HMT GLP is also an important epigenetic marker of gene repression. Previous studies have reported that GLP knockdown can dramatically reduce the activity of H3K9 me2 and activate silenced genes (Link et al., 2009; Tachibana et al., 2005). Here, our work shows that HIV-1 LTR expression was significantly induced after GLP knockdown. The observations indicate that HMT GLP has been involved in the maintenance of HIV-1 latency.
Conclusion Curing AIDS has been a major goal for many years, ever since the disease was discovered. HAART can slow the progression of the disease, but it has limitations, many of which revolve around the existence of the HIV-1 reservoir. Studies show that epigenetic modifications, including histone acetylation and methylation, are closely associated with HIV-1 latency. Although HMT SuvH1 and G9a have been found to be recruited in latent HIV-1, other HMTs involved in HIV-1 latency are still poorly understood. In summary, analyses carried on our in vitro model of HIV-1 latency suggest that H3K9 dimethylation, which is catalyzed by HMT GLP, may play a more significant role in the maintenance of HIV-1 latency than CpG methylation does. These findings present novel evidence that HMT GLP takes part in HIV-1 latency and may be a suitable target for therapy in HIV-1infected individuals. Considering that the process of HIV latency is subjected to multiple forms of regulation, clarification of the molecular mechanism of HIV-1 latency requires further investigation in the hope of providing an effective platform for eradication of latent HIV-1.
Materials and methods Cell culture and drug treatment 293 T cells, 293 cells, and TZM-bl cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco) medium supplemented
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with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ ml streptomycin (Invitrogen), and were incubated at 37 °C with 5% CO2. Jurkat T cells were cultivated in the same condition except that DMEM medium was replaced by RPMI 1640 medium (Gibco). 5′-deoxy-5′-methylthioadenosine (MTA), Trichostatin A (TSA), Phorbol 12-Myristate-13-Acetate (PMA), Tumor Necrosis Factor-α (TNF-α), phytohaemagglutinin (PHA), and lonomycin were dissolved in anhydrous dimethyl sulfoxide (DMSO) (Sigma-Aldrich). Cells were treated with these reagents for further analyses in different time phases. Plasmids HIV-1 vector plasmid NL4-3-EGFP was kindly provided by Dr. Jianqing Xu (Institute of Bioscience, Fudan University, Shanghai, China). HIV-1 LTR-luciferase (LTR-luc) plasmid was constructed as described (du Chene et al., 2007). Virus production and cell infection For viral production, 2×105 293T cells were transfected with 15 μg pNL43-EGFP and 7.5 μg pVSVG. Supernatants containing viral particles were harvested after 72 h and centrifugated to remove scraps under 3000 rpm for 5 min. Jurkat T cells were infected with viral suspension at an m.o.i of 0.1 after centrifuging under 1200g for 2 h at room temperature as described previously (Chun et al., 1998; Hauber, 2008). Cells were washed by 1×PBS twice and added 2ml fresh RPMI 1640 medium containing 10% FBS. Cells were cultured at 37 °C with 5% CO2 for 72 h and differentiated GFP negative cells from all by flow cytometry analysis (FACS-Aria machine, BD Science). GFP-negative cells were separated and stimulated with TSA, PMA, TNF-α, PHA, and lonomycin to sort out GFP-positive cells that are the latently infected cells. These latently infected cells were cultured selectively and finally four clonal cell lines were obtained and labeled as Clone 1 (C1), Clone 3 (C3), Clone 11 (C11), and Clone 13 (C13). Detection of HIV integration site Inverse PCR procedure was used to detect HIV integration site in our established in vitro model of HIV latency. In brief, genomic DNA extracted from latent cell lines was digested with PstI, the digested DNA was ligated by T4 ligase and provirus-host DNA junctions were amplified with two couples of primers under a nested reaction as previously described (Han et al., 2004). Bands on agarose gels were purified, cloned and sequenced. The Human sequence flanking to 5′LTR of HIV-1 was identified by BLAST search of GenBank or UCSC Bioinformatics Human Genome database. Cytosine methylation analysis of HIV-1 5′LTR The samples of total genomic DNA from four latent cell lines were extracted and proceeded to analyze according to the procedure provided by EZ DNA Methylation-Gold kit (Zymo Research). As described by Pion et al. (2003), modified DNA was amplified by PCR application using two pairs of primers. PCR was performed by 30 cycles at 95 °C for 60 s, 58 °C for 120 s, and 72 °C for 60 s. PCR products were separated, eluted, cloned in the pMD18-T Vector (Takara), and sequenced finally. Sequences were obtained from at least nine PCR clones after excluding clones with lower than 95% conversion of cytosines outside CpGs. Ultimate results of sequencing were analyzed by accessing to Quantification Tool for Methylation Analysis (QUMA) (Kumaki et al., 2008; Kauder et al., 2009 ).
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Chromatin Immunoprecipitation (ChIP) assay ChIP experiments were performed according to the protocol provided by EZ-ChIP™ Chromatin Immunoprecipitation Kit (Millipore). C11 was incubated either with or without MTA (1 mM) for 24 h before ChIP assays. Cells were cross-linked in 1% formaldehyde for 10 min at room temperature and quenched with 0.125 M Glycine for 5 min. After lysis, chromatin was sheared by sonicator for a total of 12 min (10 s on, 10 s off) on ice to obtain DNA fragments at the length of 200–1000 bp. 10% of total sheared chromatin DNA was used as input DNA. Other sheared chromatin was incubated with antibodies against H3K9me2, H3K9me3 (Abcam), and normal mouse IgG (Millipore) for a negative control. The immunoprecipitated DNA was analyzed by a real-time PCR (ABI Prism 7900 Real-Time PCR System) at 30 cycles with Taq Mastermix (Invitrogen) using primers for 5′LTR of HIV-1 (forward: 5′-TAGCAGTGGCGCCCGAACAGG-3′, reverse: 5′GCCTCTTGCCGTGCGCGCTTC-3′). Transient transfection and RNA interference Small interference RNAs (siRNAs) against GLP and scramble siRNA as negative control were synthesized by Invitrogen. The target sequences are as follows: GLP (5′-GCACAAACAGCGUGGUCAATT-3′) and control (5′-UUCUCCGAACGUGUC ACGUTT-3′). 293 cells were co-transfected with 160 ng HIV-1 LTR-luc and 100 nM siRNAs using Lipofectamine 2000 (Invitrogen). After 24 h transfection, Cells were either treated or untreated with 10 ng/ml TNF-α for additional 24 h. Cells were finally harvested after 48 h transfection and subjected to luciferase activity assay according to Luciferase Assay System™ (Promega).
Acknowledgments This work was supported by the National Grand Program on Key Infectious Disease (2012ZX10001003) and National Natural Science Funding of China (No. 31171247).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2013.02.022.
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Involvement of histone methyltransferase GLP in HIV-1 latency through catalysis of H3K9 dimethylation Donglin Ding, Xiying Qu, Lin Li, Xin Zhou, Sijie Liu, Shiguan Lin, Pengfei Wang, Shaohui Liu, Chuijin Kong, Xiaohui Wang, Lin Liu, Huanzhang Zhu * State key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China
art ic l e i nf o
a b s t r a c t
Article history: Received 6 September 2012 Returned to author for revisions 27 December 2012 Accepted 20 February 2013 Available online 27 March 2013
Understanding the mechanism of HIV-1 latency is crucial to eradication of the viral reservoir in HIV-1infected individuals. However, the role of histone methyltransferase (HMT) G9a-like protein (GLP) in HIV-1 latency is still unclear. In the present work, we established four clonal cell lines containing HIV-1 vector. We found that the integration sites of most clonal cell lines favored active gene regions. However, we also observed hypomethylation of CpG of HIV 5′LTR in all four clonal cell lines. Additionally, 5′-deoxy5′-methylthioadenosine (MTA), a broad-spectrum histone methyltransferase inhibitor, was used to examine the role of histone methylation in HIV-1 latency. MTA was found to decrease the level of H3K9 dimethylation, causing reactivation of latent HIV-1 in C11 cells. GLP knockdown by small interfering RNA clearly induced HIV-1 LTR expression. Results suggest that GLP may play a significant role in the maintenance of HIV-1 latency by catalyzing dimethylation of H3K9. & 2013 Elsevier Inc. All rights reserved.
Keywords: HIV-1 latency Integration site CpG methylation H3K9 methylation GLP
Introduction Highly active antiretroviral therapy (HAART) can suppress HIV1 replication and cripple viral production to the point where viral levels become undetectable in HIV-1-infected individuals (Chun et al., 1999; Gulick et al., 1997; Hammer et al., 1997; Perelson et al., 1997). Despite the great advances that have been made in clinical antiretroviral therapy, interruption of HAART treatment is still closely associated with the recovery of HIV-1 replication and resumption of disease progression. This is because of the viral reservoir that HIV-1 can form in the human body (Chun et al., 2000; Finzi et al., 1997; Wong et al., 1997; Zhang et al., 2000). This reservoir consists of latently infected resting memory CD4 þ T lymphocytes into whose genomes HIV-1 proviruses have integrated (Loetscher et al., 1999; Stevenson, 2003). This latent HIV-1 reservoir can exist for long periods of time and so become a major obstacle to the eradication of HIV in HAART-treated patients. In latently infected cells, HIV-1 gene expression and viral production are inhibited after integration. Integration is a crucial step in the viral infection of CD4 þ T cells and one of the ways in which the virus avoids attack from the host immune system (Colin and Van Lint, 2009; Engelman et al., 1995; Englund et al., 1995; Lassen et al., 2004; Sakai et al., 1993). Because HIV DNA can integrate into the genome of the host cell, it is probable that
* Corresponding author. E-mail address: [email protected] (H. Zhu). 0042-6822/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.virol.2013.02.022
integration can take place in chromatin environments beneficial to latency (Trono et al., 2010). Some investigations of resting CD4 þ T cells purified from infected patients on HAART have suggested that HIV-1 integration acts in a non-random manner in vivo, preferring the intronic regions of active genes (Colin and Van Lint, 2009; Han et al., 2004; Wang et al., 2007). This may be because a transcriptional gene named LEDGF/p75 can induce HIV-1 DNA into the region of actively transcribed genes after interacting directly with viral integrase (Cherepanov et al., 2003; Lewinski et al., 2006; Meehan et al., 2009; Schröder et al., 2002; Vandegraaff et al., 2006). Other studies on the Jurkat cell model have reported that integration sites have many alphoid repeats rich in heterochromatin (Jordan et al., 2001; Jordan et al., 2003; Lenasi et al., 2008). Elucidating the characteristics of the integration may help us more clearly understand the mechanisms of HIV-1 latency. After HIV-1 integration into host genome, chromatin modifications in viral promoter of HIV-1 long terminal repeat (LTR) become involved in transcriptional regulations, including CpG methylation and histone methylation (Bednarik et al., 1987; Bednarik et al., 1990; Coiras et al., 2009; Gutekunst et al., 1993; Marban et al., 2007). One study on Jurkat clonal cell lines transfected by HIV-1 derived vector and primary resting memory T cells infected with HIV-1 showed that CpG methylation levels are universally low. Even after reactivation of latent HIV-1, these CpG islands retain hypomethylation (Pion et al., 2003). However, CpG methylation of HIV-1 5′LTR has been found to be selective and is considered an important epigenetic control in the maintenance of HIV-1 latency (Blazkova et al., 2009; Ishida et al., 2006). Observations made on
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the Jurkat cell model and primary lymphocytes latently infected with HIV-1 indicate that MBD2, a member of the methyl-CpG binding domain family of proteins, can specially bind methylated CpG islands and lead to gene silencing by recruitment of transcriptional repressors including the nucleosome remodeling and histone deacetylation (NuRD) complex (Bird and Wolffe, 1999; Kauder et al., 2009; Wade, 2001; Zhang et al., 1999). In conclusion, more analysis is required to confirm the association between HIV1 latency and CpG methylation. Histone methylation has also been associated with transcriptional regulation. It is considered more labile than DNA methylation (Jones and Baylin, 2007; Kelly et al., 2010; Shi, 2007). Histone methylation has distinct effects on transcriptional controls depending on the specific residues and the pattern of methylation (Spannhoff et al., 2009). For example, methylation of histone H3 lysine-4 and arginine-17 is commonly associated with activation of transcription, but methylation of histone H3 lysine-9 and lysine-27 generally leads to inactivation (Huang et al., 2006; Martin and Zhang, 2005; Spannhoff et al., 2009; Volkel and Angrand, 2007). Because H3K9 methylation is considered responsible for gene silencing, some histone methyltransferases (HMTs) that catalyze H3K9 methylation have been investigated (du Chene et al., 2007; Imai et al., 2010). Methyltransferases other than Su39H1 and G9a, have been identified. These include Suv39H2, GLP (G9a-like protein)/EHMT1, and SETDB1. These H3K9 HMTs all contain a common SET-domain, which acts as the catalytic unit (Dillon et al., 2005; Ogawa et al., 2002; Rea et al., 2000; Schultz et al., 2002; Tachibana et al., 2001; Yang et al., 2003). GLP, a G9a-related mammalian H3K9 specific methyltransferase has been found to be involved in gene repression (Levy et al., 2010; Tachibana et al., 2008). However, the role of GLP in HIV-1 latency remains to be clarified. The mechanism underlying HIV latency is not absolutely clear. We established an in vitro model of HIV-1 latency. On the latency model consisting of four clonal cell lines, we performed a series of experiments, including analyses of HIV-1 integration sites and assessments of CpG and H3K9 methylation in HIV 5′LTR to elucidate the mechanism of HIV-1 latency in our in vitro model and identify potential targets for complete eradication of latent HIV-1.
Results Establishment of an in vitro HIV latency model Plasmid NL43-EGFP was derived from pNL4-3. It is a HIV-1 constructor imported EGFP-encoding gene in a nef open reading frame. It was here mutated into Env and Vpr genes. We used pNL43-EGFP and pVSVG to co-transfect 293 T cells for 72 h and analyzed the relative number of GFP þ cells using FACS analysis. We used viruses at an m.o.i of 0.1 to infect Jurkat T cells. We separated GFP-negative cells using FACS after 72 h infection. Because the GFP-negative population was composed of uninfected cells and cells containing transcriptional silenced proviruses, we treated GFP-negative cells with TSA, PMA, TNF-α, PHA, and lonomycin for 48 h to activate silenced HIV-1 and sorted out GFP-positive cells using FACS (Fig. 1). These GFP-positive cells were cultured as a monoclonal cell line for 4 weeks. Several impure clonal cell lines were discarded, and four clonal cell lines were selected individually for further analyses. Location of HIV-1 integration site from latently infected cells Because each clonal cell line showed nonproductive HIV-1 replication and low, unequal levels of basal transcription, we
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Fig. 1. Sorting of HIV-1 latently infected cells from GFP-negative cells. Because the GFP-negative population were composed of uninfected cells and cells containing transcriptional silenced proviruses, we treated GFP-negative cells with TNF-α at 10 ng/ml, TSA (50 nM, 100 nM, and 200 mM), PMA at 10 ng/ml, PHA at 5 μg/ml, and 1 μM lonomycin for 48 h to activate silenced HIV-1 and sorted out GFP-positive cells by FACS analysis.
cloned and indentified the integration sites of HIV-1 from four clonal cell lines using an inverse PCR strategy (Fig. 2A). Genomic DNA was isolated from latently infected cells, digested with the restriction enzyme PstI, which recognizes a six-base site in gag gene of HIV and ligates into a self-loop under dilute conditions. The circularized DNA was used as a template in inverse PCR and the PCR products were visible on agarose gel (Fig. 2B). Bands were isolated, cloned, and sequenced. A total of four distinct clonal cell lines were used to assay the integration site. Sequences of four unique integration sites were identified and analyzed in a BLAST search of GenBank and the UCSC Bioinformatics Human Genome database. Sequences flanking the 5′LTR of HIV-1 were considered authentic if they showed at least a 99% match to the human genomic sequence. We then examined the distribution of integration sites and found the integration sites of C1, C3, and C11 to be located in introns of gene regions but those in C13 were downstream of a transcriptional unit. As characterized in Table 1, HIV-1 probably preferred to integrate in gene-rich regions. This is consistent with previous in vivo studies (Han et al., 2004).
Hypomethylation of the HIV-1 5′LTR in all four clonal cell lines To determine the possible relationship between latency and HIV promoter methylation, five latently infected Jurkat clonal cell lines C1, C3, C11, and C13, each containing single proviral copy, were used to investigate HIV 5′LTR cytosine methylation. The methylation level was dependent on bisulfite sequencing. After bisulfite conversion, the cytosine of CpG did not change, but the cytosine outside CpG was converted to uracil, which is recognized as thymine in PCR amplification. Sequencing showed that low CpG methylation levels in 5′LTR of these latency models (3.7±2.6% in C1, 6.2±2.7% C3, 1.2±1.2% C11, 2.5±1.6% C13) (mean ±standard error) (Fig. 3). Another analysis was performed to evaluate the methylation of each CpG site in the 5′LTR. No evidence indicated that CpG methylation was associated with HIV-1 promoter silencing. Some mechanisms other than CpG methylation in the 5′LTR may contribute to HIV-1 latency.
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Fig. 2. HIV integration sites are detected in four latently infected cell lines by inverse PCR. (A) Diagram of inverse PCR. Briefly, DNA purified from latency cell lines was digested with PstI, the digested DNA was ligated by T4 ligase and proviral-host DNA junctions were amplified with two couples of primers under a nested reaction. (B) Products of inverse PCR from four clonal cell lines were visible in agarose gels. Each band meant a unique integration event. Control reactions carried out either human genome without HIV infection or no template.
Table 1 Characteristics of HIV-1 integration sites in Jurkat clonal cell lines. Clonal cell line Conjunctional sequencea Chromosome location Host nt. at the junctionb Gene
Integration locus Gene description
C1 C3 C11 C13
Intron Intron Intron Intron
ATGCCTGGAA AAGGCTGGAA GAGATTGGAA AAAGGTGGAA
8q24.3 16p13.3 16p13.3 19q13.33
145153859 366460 2306831 48261159
SHARPIN AXIN1 RNPS1 LOC729626
SHANK-associated RH domain interactor Axin 1 RNA binding protein S1 hypothetical protein
The characteristics of HIV-1 integration sites were analyzed by using Genbank and UCSC database. Sequences flanking to 5′LTR of HIV-1 were considered to be authentic as they showed at least 99% match to human genomic sequence by BLAST search of GenBank. a b
Junction between Human genomic sequence and 5′LTR (first five bases). Nucleotide number at integration site.
Fig. 3. CpG methylation profiles of 5′LTR in HIV-1 from four Jurkat clonal cell lines. (A) Distributions of CpG dinucleotides in 5′LTR of latent HIV-1 LTR-driven retroviral vector pNL4-3 are shown as open circles. Genomic DNA from latency cell lines was converted by bisulfite treatment and subsequently was used to amply. (B) Different level of methylation in HIV-1 5′LTR in Jurkat clonal cell lines C1, C3, C11, and C13. Analyses of nine molecules are shown by QUMA online software. Only sequences with at least 98% conversion of cytosines outside CpG dinucleotides were calculated. Levels of CpG methylation are indicated as a mean±standard error for each clonal cell line.
MTA treatment induces HIV-1 transcription in Jurkat clonal cell line C11 MTA is competitive with the SAM cofactor, which can provide methyl groups for histone methlytransferase. In this way, it plays an important role in inhibiting methlytransferase activity. The presence of hypomethylation in CpG islands in vitro latency cell lines might indicate that some other mechanisms, such as histone methylation, are involved in HIV-1 latency. Four clonal cell lines were seeded in 96-well plates at 4×105 cells/ml and treated with MTA under a concentration gradient (0, 1 μM, 10 μM, 100 μM, 1000 μM) incubated at 37 °C with 5% CO2 for 48 h. As indicated by FACS analysis, after MTA treatment lasting 48 h, the number of GFP-positive cells increased obviously in C11 but C1, C3, and C13 showed no significant differences from untreated controls (Fig. 4).
In this way, MTA was shown to act as a histone methyltransferase inhibitor. It can apparently induce HIV-1 transcription to reactivate latent HIV in cell line C11. Dimethylation of H3K9 leads to HIV latency in C11 MTA is a broad-spectrum histone methyltransferase inhibitor (Diehl et al., 2007). It can down-regulate gene expression by inhibiting some histone methyltransferase such as MLL and set1 due to methylation of H3K4 (Huang et al., 2006; Diehl et al., 2007). Methylation at different residues has distinct effects on gene expression. For example, methylation at H3K4, H3K36, and H3K79 is associated with gene activation, and methylation at H3K9, H3K27, and H4K20 is associated with gene silencing (Spannhoff et al., 2009). Here, we found MTA treatment increased
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Fig. 4. MTA treatment induces HIV-1 transcription in latent cell line C11. (A) After latent cell lines were treated with or without 1mM MTA for 48 h, GFP-positive cells were analyzed by FACS (FACS Calibur, BD Science) in four clonal cell lines. GFP-positive cells were increased 5.5-fold after 1mM MTA for 48 h in C11. Data was shown as mean ±standard error by three independent experiments. (B) Percentage of GFP-positive cells after 1mM MTA treatment in C11 by FACS analysis. Latent HIV-1 harbored in C11 was reactivated at the ratio of 13.1% after 1mM MTA treatment for 48 h.
the number of GFP-positive cells in latent cell line C11. A reaction between latent HIV-1 and MTA indicated the presence of a pathway involving histone methylation due to the HIV-1 latency. To demonstrate whether histone methylation was associated with HIV-1 latency in C11, a ChIP assay was performed to measure the level of H3K9 me2 and me3 in the LTR promoter of HIV-1. First, C11 were incubated with 1mM MTA for 24 h, immunoprecipitated DNA fragments were obtained using antibodies against H3K9 me2 and H3K9 me3. These fragments were subjected to real-time quantitative PCR analysis. The level of H3K9 me2 was shown to be down-regulated by approximately 2.27-fold after MTA treatment in C11 cells. The level of H3K9 me3 showed no obvious change (Fig. 5A).
GLP knockdown up-regulates the transcriptional activation of HIV-1 LTR Because GLP was reported to be involved in the dimethylation of H3K9, an assay on endogenous GLP knock-down was performed (Levy et al., 2010). HEK 293 cells were planted and co-transfected with HIV-1 LTR-luc and either GLP-specific siRNA or scramble siRNA. After 48 h incubation, cells were harvested and subjected to luciferase activity according to the manufacture's protocol. Luciferase assay showed that GLP knockdown increased the transcription of HIV-1 LTR over that of cells exposed to scramble siRNA (2.1-fold change). TNF-α treatment was found to promote HIV-1 LTR expression (2.3-fold change compared to control siRNA) (Fig. 6). This experiment indicates that GLP is involved in silencing the expression of HIV-1 LTR.
Discussion Although HAART can reduce viral load in plasma and improve patient quality of life, persistence of latent reservoir becomes an obstacle to prohibit antiretroviral drugs from eliminating HIV-1 completely. For this reason, understanding the mechanism of HIV1 latency is considered crucial in our present study. To establish the HIV-1 latency model, we used an HIV-1derived vector to infect Jurkat T cells. This produced latently infected cells containing a single copy of HIV-1. Studies on in vitro models of HIV latency have been reported previously. These models include U1 and ACH2, which exhibit low levels of basal transcription, despite mutations in Tat (U1) and TAR (ACH2) (Folks et al., 1986, 1987). HIV latency has also been reported in Jurkat T cells harboring HIV provirus with deletion of NF-κB (Antoni et al., 1994). Although these cell lines have served as classical models in studies on HIV-1 latency, the mutations that they carry may raise concerns about whether they can hold up a mirror to HIV-1 latency in vivo (Yang, 2011). Recently, an in vitro HIV latency model was developed in Jurkat T cells infected with Tat-driven vector (Jordan et al., 2001, 2003). In this present study, we used pNL4-3-EGFP, which was reconstructed from pNL4-3 by introducing in gene encoding enhanced green fluorescent protein and mutating in Vpr and Env, to infect Jurkat T cells. We finally obtained four latently infected cell lines. In these latent cells, HIV-1 DNA integrates into the host genome and exhibits low basal transcription. Integration causes a stable, latent state that allows the virus to evade the immune system and antiretroviral drugs. Because integration sites have been shown to play a significant role in HIV-1 latency, several studies have suggested that integration favors in active transcriptional gene
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Fig. 5. The level of H3K9 me2 on 5′LTR of HIV-1 is decreased after MTA treatment. (A) As described, ChIP assay was performed after treating C11 either with or without 1mM MTA for 24 h. Recruitments of H3K9 me2 and H3K9 me3 on 5′LTR of HIV-1 were measured by real-time quantitative PCR. Normal mouse IgG was used as a negative control. Data are determined by three independent experiments. (B) 20 μl Products of real-time quantitative PCR were visible in agarose gels.
Fig. 6. GLP knockdown up-regulates the transcriptional activation of HIV-1 LTR. 293 cells were co-transfected with 160ng HIV-1 LTR-luc and 100 nM siRNAs (GLP siRNA or control siRNA) using Lipofectamine 2000 (Invitrogen). After 24 h transfection, Cells were either treated or untreated with 10 ng/ml TNF-α for additional 24 h. Cells were finally harvested after 48 h transfection and subjected to luciferase activity assay. Results were shown as fold change (mean value±standard error).
regions in CD4 þ T cells from patients on HAART or in CD4 þ T cells infected with retroviral vector (Han et al., 2004; Brady et al., 2009). However, analyses of integration sites in a Jurkat T cell model show that integration distributes in or near the alphoid repeats found in heterochromatin in most latent cell lines (Jordan et al., 2001). In
contrast, analysis of our small sample set indicated that three out of four integration sites were located in active gene regions (Table 1), which is consistent with other in vitro studies (Lewinski et al., 2006; Schröder et al., 2002). Our conclusion is probably limited by the small sample size. According to previous reports, LEDGF/p75 protein is associated with HIV-1 integration into active transcriptional genes and this protein can vary in expression depending on different cell types (Brady et al., 2009; Shun et al., 2007), this may explain its preference for the integration site. After the integration of HIV-1 DNA into the host chromatin, proviral expression is largely governed by host cellular factors. LTR promoter is the driver of HIV-1 gene expression, including a number of binding sites essential to the regulation of transcriptional activity (Calman et al., 1988; Parrott et al., 1991; Sune and Garcia-Blanco, 1995). In this way, epigenetic modifications of the HIV-1 5′LTR region have been involved in studies into the mechanism by which HIV-1 latency is established. DNA methylation has been shown to be associated with inactivation of HIV LTR (Bednarik et al., 1987, 1990). In our latent cell lines, we analyzed CpG methylation of HIV-1 5′LTR and observed hypomethylation in CpG of HIV 5′LTR in four Jurkat clonal cell lines. These CpG methylation levels varied from 1.2% to 6.2%. The phenomenon observed in our study is consistent with the findings of studies performed in other Jurkat clonal cells and on primary resting memory T lymphocytes infected with retroviral vectors (Blazkova et al., 2009; Pion et al., 2003). Hypermethylation of CpG of HIV-1 5′LTR is observable in latently infected cells selected after two-step reactivation. This has also been reported in long-term-HIV-1infected aviremic individuals but not in viremic patients (Blazkova et al., 2009). These findings suggest that methylation of CpG of HIV-1 5′LTR is probably accompanied by continuous development of HIV-1 latency. The observation made in ACH-2 cells demonstrates the existence of 5′LTR-selective methylation (Ishida et al., 2006). Overall, our work suggests that CpG methylation levels of 5′LTR in HIV-1 are usually low. No 5′LTR-selective methylation was observed in our in vitro model of HIV-1 latency. Because hypomethylation was observed in HIV-1 5′LTR, we speculated that the mechanism of DNA methylation might not be correlated with initial establishment of HIV-1 latency in our latent cell lines. Previous studies showed that histone H3K9 methylation has a deep influence on regulation of transcription and establishment of HIV-1 latency (du Chene et al., 2007; Imai et al., 2010; Kubicek et al., 2007; Kelly et al., 2010; Seligson et al., 2005). We used MTA, a broad-spectrum histone methyltransferase inhibitor,
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to treat these clonal cell lines and assess them for histone methylation. With 1mM MTA treatment, the ratios of GFPpositive cells were analyzed by FACS after 48 h. Latent HIV-1 in cell line C11 was reactivated at the ratio of 13.1% by MTA compared to 2.5% without MTA, but no obvious reactivation was observed in other clonal cell lines. This suggested that histone methylation plays a considerable role in HIV-1 latency. To demonstrate this, we performed a ChIP assay with anti-H3K9 me2 and anti-H3K9 me3 antibodies. The precipitated DNA segments were subjected to realtime quantification PCR. The results showed that level of dimethylation of H3K9 was reduced (2.27-fold change) after MTA treatment. The ChIP assay also confirmed that dimethylation of H3K9 was associated with HIV-1 latency in clonal cell line C11. Although HIV-1 latency has been shown to be dependent on H3K9 me2 in C11, no similar results were observed under MTA treatment in other latent cell lines. Histone acetylation was also found to be involved in HIV-1 latency, though some studies have also shown it to be less effective than histone methylation (Colin and Van Lint, 2009; du Chene et al., 2007). We used a HDAC inhibitor (trichostatin A, TSA) to reactivate our latent cell lines at 100 nM. Finally, partial latent HIV-1 was reactivated in these clonal cell lines in the presence of obvious cytotoxicity (data not shown). The distinct roles of histone methylation in different latent cell lines could be explained by a study of computational prediction, This would show whether epigenetic modifications are associated with diverse HIV-1 integration sites (Wang et al., 2007). Some HMTs have been found to participate in the catalysis of H3K9 methylation. Studies show that SuvH1 and G9a can silence genes by increasing levels of H3K9 me3,and H3K9 me2 (du Chene et al., 2007; Imai et al., 2010). Another HMT GLP is also an important epigenetic marker of gene repression. Previous studies have reported that GLP knockdown can dramatically reduce the activity of H3K9 me2 and activate silenced genes (Link et al., 2009; Tachibana et al., 2005). Here, our work shows that HIV-1 LTR expression was significantly induced after GLP knockdown. The observations indicate that HMT GLP has been involved in the maintenance of HIV-1 latency.
Conclusion Curing AIDS has been a major goal for many years, ever since the disease was discovered. HAART can slow the progression of the disease, but it has limitations, many of which revolve around the existence of the HIV-1 reservoir. Studies show that epigenetic modifications, including histone acetylation and methylation, are closely associated with HIV-1 latency. Although HMT SuvH1 and G9a have been found to be recruited in latent HIV-1, other HMTs involved in HIV-1 latency are still poorly understood. In summary, analyses carried on our in vitro model of HIV-1 latency suggest that H3K9 dimethylation, which is catalyzed by HMT GLP, may play a more significant role in the maintenance of HIV-1 latency than CpG methylation does. These findings present novel evidence that HMT GLP takes part in HIV-1 latency and may be a suitable target for therapy in HIV-1infected individuals. Considering that the process of HIV latency is subjected to multiple forms of regulation, clarification of the molecular mechanism of HIV-1 latency requires further investigation in the hope of providing an effective platform for eradication of latent HIV-1.
Materials and methods Cell culture and drug treatment 293 T cells, 293 cells, and TZM-bl cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco) medium supplemented
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with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ ml streptomycin (Invitrogen), and were incubated at 37 °C with 5% CO2. Jurkat T cells were cultivated in the same condition except that DMEM medium was replaced by RPMI 1640 medium (Gibco). 5′-deoxy-5′-methylthioadenosine (MTA), Trichostatin A (TSA), Phorbol 12-Myristate-13-Acetate (PMA), Tumor Necrosis Factor-α (TNF-α), phytohaemagglutinin (PHA), and lonomycin were dissolved in anhydrous dimethyl sulfoxide (DMSO) (Sigma-Aldrich). Cells were treated with these reagents for further analyses in different time phases. Plasmids HIV-1 vector plasmid NL4-3-EGFP was kindly provided by Dr. Jianqing Xu (Institute of Bioscience, Fudan University, Shanghai, China). HIV-1 LTR-luciferase (LTR-luc) plasmid was constructed as described (du Chene et al., 2007). Virus production and cell infection For viral production, 2×105 293T cells were transfected with 15 μg pNL43-EGFP and 7.5 μg pVSVG. Supernatants containing viral particles were harvested after 72 h and centrifugated to remove scraps under 3000 rpm for 5 min. Jurkat T cells were infected with viral suspension at an m.o.i of 0.1 after centrifuging under 1200g for 2 h at room temperature as described previously (Chun et al., 1998; Hauber, 2008). Cells were washed by 1×PBS twice and added 2ml fresh RPMI 1640 medium containing 10% FBS. Cells were cultured at 37 °C with 5% CO2 for 72 h and differentiated GFP negative cells from all by flow cytometry analysis (FACS-Aria machine, BD Science). GFP-negative cells were separated and stimulated with TSA, PMA, TNF-α, PHA, and lonomycin to sort out GFP-positive cells that are the latently infected cells. These latently infected cells were cultured selectively and finally four clonal cell lines were obtained and labeled as Clone 1 (C1), Clone 3 (C3), Clone 11 (C11), and Clone 13 (C13). Detection of HIV integration site Inverse PCR procedure was used to detect HIV integration site in our established in vitro model of HIV latency. In brief, genomic DNA extracted from latent cell lines was digested with PstI, the digested DNA was ligated by T4 ligase and provirus-host DNA junctions were amplified with two couples of primers under a nested reaction as previously described (Han et al., 2004). Bands on agarose gels were purified, cloned and sequenced. The Human sequence flanking to 5′LTR of HIV-1 was identified by BLAST search of GenBank or UCSC Bioinformatics Human Genome database. Cytosine methylation analysis of HIV-1 5′LTR The samples of total genomic DNA from four latent cell lines were extracted and proceeded to analyze according to the procedure provided by EZ DNA Methylation-Gold kit (Zymo Research). As described by Pion et al. (2003), modified DNA was amplified by PCR application using two pairs of primers. PCR was performed by 30 cycles at 95 °C for 60 s, 58 °C for 120 s, and 72 °C for 60 s. PCR products were separated, eluted, cloned in the pMD18-T Vector (Takara), and sequenced finally. Sequences were obtained from at least nine PCR clones after excluding clones with lower than 95% conversion of cytosines outside CpGs. Ultimate results of sequencing were analyzed by accessing to Quantification Tool for Methylation Analysis (QUMA) (Kumaki et al., 2008; Kauder et al., 2009 ).
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Chromatin Immunoprecipitation (ChIP) assay ChIP experiments were performed according to the protocol provided by EZ-ChIP™ Chromatin Immunoprecipitation Kit (Millipore). C11 was incubated either with or without MTA (1 mM) for 24 h before ChIP assays. Cells were cross-linked in 1% formaldehyde for 10 min at room temperature and quenched with 0.125 M Glycine for 5 min. After lysis, chromatin was sheared by sonicator for a total of 12 min (10 s on, 10 s off) on ice to obtain DNA fragments at the length of 200–1000 bp. 10% of total sheared chromatin DNA was used as input DNA. Other sheared chromatin was incubated with antibodies against H3K9me2, H3K9me3 (Abcam), and normal mouse IgG (Millipore) for a negative control. The immunoprecipitated DNA was analyzed by a real-time PCR (ABI Prism 7900 Real-Time PCR System) at 30 cycles with Taq Mastermix (Invitrogen) using primers for 5′LTR of HIV-1 (forward: 5′-TAGCAGTGGCGCCCGAACAGG-3′, reverse: 5′GCCTCTTGCCGTGCGCGCTTC-3′). Transient transfection and RNA interference Small interference RNAs (siRNAs) against GLP and scramble siRNA as negative control were synthesized by Invitrogen. The target sequences are as follows: GLP (5′-GCACAAACAGCGUGGUCAATT-3′) and control (5′-UUCUCCGAACGUGUC ACGUTT-3′). 293 cells were co-transfected with 160 ng HIV-1 LTR-luc and 100 nM siRNAs using Lipofectamine 2000 (Invitrogen). After 24 h transfection, Cells were either treated or untreated with 10 ng/ml TNF-α for additional 24 h. Cells were finally harvested after 48 h transfection and subjected to luciferase activity assay according to Luciferase Assay System™ (Promega).
Acknowledgments This work was supported by the National Grand Program on Key Infectious Disease (2012ZX10001003) and National Natural Science Funding of China (No. 31171247).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2013.02.022.
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