A novel HIV-1-encoded microRNA enhances its viral replication by targeting the TATA box region
© Zhang et al.; licensee BioMed Central Ltd. 2014
Received: 1 November 2013
Accepted: 18 February 2014
Published: 12 March 2014
A lot of microRNAs (miRNAs) derived from viral genomes have been identified. Many of them play various important roles in virus replication and virus-host interaction. Cellular miRNAs have been shown to participate in the regulation of HIV-1 viral replication, while the role of viral-encoded miRNAs in this process is largely unknown.
In this report, through a strategy combining computational prediction and deep sequencing, we identified a novel HIV-1-encoded miRNA, miR-H3. MiR-H3 locates in the mRNA region encoding the active center of reverse transcriptase (RT) and exhibits high sequence conservation among different subtypes of HIV-1 viruses. Overexpression of miR-H3 increases viral production and the mutations in miR-H3 sequence significantly impair the viral replication of wildtype HIV-1 viruses, suggesting that it is a replication-enhancing miRNA. MiR-H3 upregulates HIV-1 RNA transcription and protein expression. A serial deletion assay suggests that miR-H3 targets HIV-1 5′ LTR and upregulates the promoter activity. It interacts with the TATA box in HIV-1 5′ LTR and sequence-specifically activates the viral transcription. In addition, chemically-synthesized small RNAs targeting HIV-1 TATA box activate HIV-1 production from resting CD4+ T cells isolated from HIV-1-infected patients on suppressive highly active antiretroviral therapy (HAART).
We have identified a novel HIV-1-encoded miRNA which specifically enhances viral production and provide a specific method to activate HIV-1 latency.
MiRNA represents a class of small RNA ranged from 21-24 nts, which plays important regulatory roles in animal, plant, and fungi [1, 2]. Virus-encoded miRNAs were initially identified from Epstein-Barr viruses . Since then, increasing virus-encoded miRNAs have been identified [4, 5]. Most of these miRNAs were reported in DNA viruses such as Herpes and Polyoma viruses, but rarely in RNA viruses . Because of the rapid development of deep sequencing technology which is much more sensitive and quantitative than the conventional cDNA clone sequencing method, more RNA virus-derived miRNAs have been discovered especially from HIV-1, WNV and BLV [6–9].
Most miRNAs repress gene expression through targeting the 3′ UTR of mRNA in cytoplasmic RISC for translation repression or mRNA degradation [1, 10–12]. It has been revealed that 5′ UTR and exons could also be the targets of miRNAs for translation repression [13, 14]. In addition, miRNAs could also enter the nucleus and modulate gene expression at transcriptional level [15–17]. These findings reveal multiple action modes are exploited by miRNAs for gene expression regulation.
MiRNAs play important roles in the interaction between parasites and their hosts. Cellular miRNAs could affect the viral replication, latency and mediate antiviral defense. For example, miR-122 that is enriched in the liver plays a key role in the accumulation of viral RNAs of hepatitis C viruses . A cellular miRNA effectively restricts the accumulation of the retrovirus primate foamy virus type 1 (PFV-1) in human cells . Our group reported that several miRNAs from resting human CD4+ T cells repress the translation of viral proteins and contribute to the latency of HIV-1 . Conversely, viral miRNAs could facilitate viral infection through reducing the viral antigens or impairing the host antiviral immune response. For instances, SV40 miR-S1 down-modulates the production of the viral T antigen (TAg), an early protein which is not required during late infection . An EBV-encoded miRNA miR-BART5 targets the proapoptotic factor PUMA to promote host cell survival . Furthermore, an hCMV miRNA, miR-UL112-1, was reported to inhibit the expression of the stress-induced ligand MICB and enable hCMV to escape from the immune surveillance by NK cells .
It has been reported that human immunodeficiency virus type 1 (HIV-1) also encode several miRNAs and other small RNAs. Bennasser et al. first performed a computational prediction on HIV-1 encoded miRNAs and found five pre-miRNAs candidates . Subsequently, several groups identified HIV-1 encoded miRNAs from the nef or the TAR element [25–28]. Through the new generation sequencing method, a number of HIV-1-encoded small RNAs were discovered, some of which exhibit the features of miRNA or small interfering RNA (siRNA) [7, 29].
These HIV-1 derived small RNAs have been shown to modulate the cellular and/or viral gene expression. A nef-derived miRNA-miR-N367 could block HIV-1 Nef expression in vitro. The expression of the TAR derived miRNA could protect the infected cells from apoptosis by down-regulating cellular genes involved in apoptosis [27, 30]. Since the expression levels of small non-coding RNAs generated from RNA viruses are relatively low, their roles in viral replication remain largely elusive. Here, we initiated our project by searching for new HIV-1-derived miRNA(s) and have identified a surprising new function for a miRNA isolated from the reverse transcriptase sequence.
Computational prediction of HIV-1-encoded miRNAs
Experimental validation of miR-H3
To further reveal whether the generation of miR-H3 is dependent on the miRNA processing pathway, the proteins required for miRNA precursor processing and transport such as Drosha and Exportin-5 were knocked down by siRNAs, and the expression of miR-H3-3p was significantly reduced (Figure 2D). The subcellular distribution analysis of miR-H3-3p suggested that it has equal amount in the nucleus and the cytoplasm, indicating a possible role it plays in the nucleus (Figure 2E).
MiR-H3 enhances viral production and replication
MiR-H3 increases viral RNA accumulation
MiR-H3 targets HIV-1 5′ LTR for promoter activation
MiR-H3 targets HIV-1 TATA box sequence-specifically
The small RNAs targeting HIV-1 TATA box activate viral production from latently infected resting CD4+T cells
In this report, we found that a novel HIV-1-encoded miRNA could upregulate its viral transcription by targeting the TATA box in the 5′ LTR. Several studies have revealed that small non-coding RNAs (e.g. miRNA and siRNA) target to gene promoters are able to induce gene transcription activation or silence. For instance, miR-373 can activate the expression of E-cadherin and cold-shock domain-containing protein C2 (CSDC2) through a target site in their promoters . Another miRNA, miR-423-5p, induces transcriptional silencing by targeting a highly conserved region in the promoter of progesterone receptor (PR) gene . At the same time, the potential of synthetic small RNAs to manipulate gene transcription was also explored. Morris and coworkers initially reported the inhibition of the EF1α promoter with a siRNA targeting approximately 100 bp upstream the EF1α transcription start site (TSS) . Several studies subsequently reported that small RNAs could also induce transcriptional gene activation by targeting gene promoters [38, 39]. These reports indicate the existence of a small RNA guided transcription regulation mechanism in the nucleus of mammalian cells. For the first time, our study revealed that viruses have exploited this host mechanism to regulate viral replication by virus-encoded miRNAs. However, the targeting sites of miRNA or siRNA on gene promoter in previous studies distribute in a wide range (~1000 bp upstream of the TSS). In this study, miR-H3 targets the key position for transcription initiation -TATA box, wherein the polymerase II pre-initiation complexes (PICs) are assembled. This finding raises the possibility that miRNAs directly participate in the transcription initiation regulation in mammalian cells.
The transcription activity of HIV-1 provirus is finely modulated in different host cells, which is accomplished through a lot of cellular transcription factors and viral proteins. Cellular transcription factors such as Sp1, NF-κB, NF-AT, LEF-1/TCF-1α, C/EBP, and CREB are important for the activation of HIV-1 LTR-driven transcription [40–49]; Conversely, cellular factors including LBP-1, TDP-43, YY1 and P53 exhibit inhibitory effects on LTR-driven transcription [50–54]. It is noteworthy that the effects of many cellular factors are cell-type dependent and there are complicated interaction among these factors (more discussion see the review of Rohr et al. ). A key viral regulator of HIV-1 transcription activity is the regulatory protein-Tat, which is produced during early phase of infection and binds to the trans-activation-responsive region (TAR) located at the 5′-end of viral mRNAs . After binding, Tat recruits a diverse series of transcriptional complexes to the viral promoter and activates transcription activity . These complexes include enzymes with histone and factor acetyl transferase (HAT and FAT respectively) activities, which modify chromatin conformation at the proviral integration site , and a protein complex (P-TEFb) that hyper-phosphorylates the carboxy-terminal domain (CTD) of RNA polymerase II, thus promoting the initiation and elongation of viral transcription [59, 60]. Vpr and Nef also have effects on HIV-1 transcription, which through the interaction with Tat or up-regulating the expression of activating factors such as NF-AT, NF-κB, and AP-1 [61–64]. In this study, we showed that miR-H3 is another HIV-1- encoded cis- regulatory element, in addition to Tat, that direct interacts with viral element and regulates transcription activity. It is interesting to investigate whether the effect of miR-H3 is cell-type dependent and identify its cellular co-factors. Our findings further reveal the complexity of transcription regulation for HIV-1.
The advances in next-generation sequencing technology have greatly fueled the discovery of small RNAs, especially the low expressed miRNAs. However, their functions are largely unexploited. One opinion about these low expressed miRNAs is that they may not have function due to the low expression levels. This idea is reasonable according to the well-known paradigm that miRNAs function in cytoplasm through targeting the 3′ UTR of mRNA for translation repression. This model requires a considerable amount of miRNAs for their functions since their targets are relatively highly expressed. But our data suggest miR-H3 and many cellular miRNAs target the core promoter of HIV-1 virus and many important genes (unpublished data), which are on the chromosomal DNA with very limited copy number in the nucleus in contrast to the massive mRNA molecules in the cytoplasm. Thus the requirement of the accumulation level of these TATA box targeting miRNAs is relatively low. Our data and previous studies on promoter targeting miRNAs probably provide a novel function model for the low expressed miRNAs.
Latent infection of HIV-1 is the major barrier for the eradication of the viruses in patients on suppressive HAART. The first step to remove latent viral reservoirs is reactivating the latent proviruses. Several approaches have been developed to activate latent virus transcription including activating T lymphocytes with IL-2 or IL-2 plus anti-CD3/anti-CD28 antibody [65, 66], protein kinase C (PKC) activators (e.g. prostatin ), and activating transcription with small molecule inhibitors of histone deacetylases without inducing host cell activation (such as, valproic acid (VPA), suberoylanilide hydroxamic acid (SAHA))[68–70]. However, the first approach has been shown to cause serious toxic effects, and the latters are speculated about causing global gene expression activation with unpredictable side effects. Thus, a HIV-1 provirus specific activating reagent is ideal for purging the latent reservoir. In this study, we demonstrated a HIV-1 encoded miRNA could activate HIV-1 transcription in a sequence-specific manner, and the synthesized small RNA induced viral production from resting CD4+ T cells from patients receiving suppressive HAART treatment. Together with our previous finding that some cellular miRNAs have contributed to the latency of HIV-1, a combination of the small RNA(s) targeting to HIV-1 TATA box and the inhibitors of these cellular miRNAs will provide a HIV-1 specific approach for eradicating HIV-1 latent reservoir more safely.
In this study, we identified a novel HIV-1-encoded miRNA miR-H3, which potently enhances viral production. Unlike most miRNAs that target the 3′ UTR of mRNA for translation repression, miR-H3 targets the TATA box in HIV-1 5′ LTR to upregulate the promoter activity. It represents another HIV-1-encoded element, in addition to Tat, that activates viral transcription via cis regulation. These findings reveal a new layer of HIV-1 replication regulation and may serve as the basis for an innovative approach to specifically activate latent infected HIV-1 viruses.
This research was approved by the Ethics Review Board of The Eighth People’s Hospital at Guangzhou (Guangzhou Infectious Disease Hospital, Guangzhou, China) and the Ethics Review Board of Sun Yat-Sen University. HIV-1-infected patients were recruited at The Eighth People’s Hospital at Guangzhou and given written informed consent with approval of the Ethics Committees. De-identified human peripheral blood mononuclear cells (PBMCs) from healthy blood donors were obtained from local volunteers. We did not have any interaction with these human subjects or protected information, and therefore no informed consent was required.
MiRNA in silicoprediction
The genomic sequence of pNL4-3 was downloaded from NCBI and submitted to web server of mireval (http://mimirna.centenary.org.au/mireval/). A total of 9 miRNA candidates including two associated with TAR regions were suggested. These candidates were further filtered by comparing their secondary structure with that of HIV-1 genomic RNA  and coincident ones were chosen for experimental validation. The miRNA precursor structure prediction was performed with the Mfold webserver . Representative sequences of major HIV-1 subtypes were downloaded from the HIV sequence database (http://www.hiv.lanl.gov/content/sequence/HIV/mainpage.html). Alignment of these sequences was performed with Clustal X program. The miRNA binding sites on HIV-1 were predicted with RNA-hybrid web server (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid).
Sup-T1 and HEK293T cells were obtained from ATCC (American Type Culture Collection, Manassas, VA) and cultured according to ATCC recommendations. TZM-bl cells were obtained from the AIDS Research and Reference Reagent Program, NIAID, US NIH. Human PBMCs were isolated from the whole blood of healthy donors by Ficoll-Hypaque Solution (HAO YANG, Tianjin, China). The resting primary CD4+ T lymphocytes were then isolated from PBMCs with CD4+ T Cell Isolation Kit II (BD). Human primary PBMCs and CD4+ T cells were grown in the RPMI 1640 conditioned media supplemented with 10% fetal bovine serum (FBS), 50 U/ml penicillin and 50 μg/ml streptomycin.
Plasmids, siRNAs and antibodies
The infectious HIV-1 clone (pNL4-3) and Env-defective HIV-1 clone (pNL4-3-deltaE-EGFP) were obtained through the AIDS Research and Reference Reagent Program, NIAID, US NIH. The precursor of miR-H3 and hsa-miR-150 were amplified by PCR and directionally cloned into the downstream of the EGFP gene in the pEGFP-C1 vector (BD Biosciences). The pNL4-3-miR-H3MT plasmid was constructed by introducing mutations in the region for miR-H3-3p mature miRNA without changing amino acid code. Similar mutations were also introduced into non-infectious HIV-1 clone, pNL4-3-deltaE-EGFP. HIV-pro-Luc plasmid was constructed by replacing the promoter of Luciferase gene in the pMIR-REPORT Luciferase vector (Invitrogen) with HIV-1 5′ LTR sequence. MMLV-Luc-HIV_3LTR plasmid was constructed by replacing the promoter of Luciferase gene in the pMIR-REPORT vector with the MMLV promoter and inserting the HIV-1 3′ LTR downstream the luciferase gene. Several mutations were introduced into miR-H3 binding site in the 5′ LTR sequence. HIV-pro-Luc-mtTAR plasmid was constructed with deleting 5′ half region of TAR motif (470 bp to 492 bp region in HIV-1 5′ LTR) to abolish its functional secondary structure. RSV-pro-Luc plasmid was constructed by replacing the promoter of Luciferase gene in the pMIR-REPORT vector with the RSV promoter. All the constructs were verified by sequencing. The siRNAs against Drosha and Exportin-5 genes were purchased from Dharmacon. Anti-β-actin antibody (D6A8) was purchased from CST (Danvers, MA). Anti-human CD3 and anti-human CD28 antibodies were from BD (Palo Alto, CA). The rabbit anti-P24 antibody was prepared by our lab.
Infection and transfection
Infectious HIV-1 clone pNL4-3 was transfected into 60% confluent HEK293T cells (100 mm plate) using Lipofectamine 2000 according to the manufacturer’s protocol (Invitrogen). Viral supernatant was collected 2 days after transfection and viral production was determined by P24 ELISA kit. Five ng P24 f infectious HIV-1 viruses were used to infect 2X 106 activated human CD4+ T lymphocytes for 3 hrs at 37°C. The cells were then washed three times with cold PBS and add fresh conditioned medium containing IL-2 (10 ng/ml). Supernatants of cell culture were collected in 2-3 days interval and subject to P24 ELISA detection. Transfection of HEK293T and TZM-bl cells was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Transfection of primary CD4+ cells with small RNA was performed with RNAiMAX (Invitrogen) according to the manufacturer’s protocol.
Quantitative real-time RT–PCR analysis
Total RNA from HEK293T or CD4+ T cells was isolated with Trizol reagent (Invitrogen) and then subjected to cDNA synthesis using PrimeScript RT reagent Kit (Takara). All primers were annealed at 37°C and RT was processed at 42°C. Quantitative PCR was performed with SYBR Premix ExTaq II Kit (Takara) by following the manufacturer’s instructions. The expressions of HIV-1 total RNAs were determined with the primer pair HIVTotRNA-5 F/R. The HIV-1 RT activity assay was performed by following the method described in Vermeire et al. . An in vitro-synthesized HIV-1 RNA, after quantification , was used as the external control for measuring virion-associated viral RNA. Quantification was normalized to the housekeeping gene U6 or β-actin. All primers for HIV-1 gene detection were listed in the additional files. The relative expression levels were calculated using the following equation: A = 2[Ct(ref) − Ct(ref ‒ control)] − [Ct(sample) − Ct(sample ‒ control)].
Dual-luciferase reporter assay
HEK293T cells were seeded in 48-well plates (Corning) at a density of 20,000 cells per well one day before transfection. One to 5 ng of HIV-1 wildtype or mutated promoter driven-firefly luciferase (FL) reporter and 2 ng renilla luciferase (RL) constructs were co-transfected with miRNA precursor/mock control into HEK293T cells using Lipofectamine 2000 (Invitrogen) by following the manufacturer’s protocol. After 24-48 hrs, FL and RL activities were measured with the Dual-Glo luciferase assay system according to the manufacturer’s instructions (Promega).
Infectious or defective viral particle production in cell cultures was determined with P24 ELISA kit by following the manufacturer’s protocol. Western blotting was carried out as described previously with some minor modifications . The anti-P24 or anti-β-actin antibodies were used to detect HIV-1 P55, P41 and P24 or β-actin protein respectively.
RNase protection assay (RPA)
The activated CD4+ T-cells were infected with viruses produced from HIV-1 clone pNL4-3 or mock infected. At 48 hrs post infection, the fresh CD4+ T-cells from the same donor were co-cultured with the infected CD4+ T-cells with a ratio of 3:1 for another 72 hrs. Then the total RNAs were isolated with Trizol Reagent (Invitrogen) and the small RNAs (<200 nt) were enriched by using the mirVana miRNA isolation kit (Ambion). The procedure of RPA described by Gilman was followed with some minor modifications . The RNA probe was a sequence complementary to nt 34/71 of miR-H3 precursor and was synthesized from DNA templates by in vitro transcription using the T7 RNA polymerase (NEB) and radiolabeled by random incorporation of α-32 P UTP (Perkin Elmer). The radiolabeled probe was hybridized with 10 ug of small RNA overnight at 45°C. After the hybridized RNAs were then treated with RNases A/T1, the protected RNAs were separated by denaturing PAGE (12%) and visualized by autoradiography.
Chromatin immunoprecipitation (ChIP) assay
ChIP assay was performed with Magna ChIP™ A/G Kit (Millipore) by following the manufacture’s instruction. Briefly, HEK293T cells were co-transfected with pNL4-3-deltaE-EGFP and pre-miR-H3 or the empty vector. At 48 hrs post transfection, the cells were collected to carry out ChIP assay with anti-Pol II (8WG16, Covance), TBP (Abcam), or normal IgG antibodies. The HIV-1 promoter sequence corresponding to -50 - (+)50 bp relative to the TSS was detected with qPCR.
This work was funded in part by the National Special Research Program for Important Infectious Diseases (No.2013ZX10001004), National Basic Research Program of China (973 Program) (No.2010CB912202), Guangdong Innovative Research Team Program (No.2009010058), National Natural Science Foundation of China (No.30972620). Natural Science Foundation of Guangdong (No.9251008901000022), Research Fund for the Doctoral Program of Higher Education of China (No.20090171110083) to H.Z. National Natural Science Foundation of China (No. 81301431), China Postdoctoral Science Foundation (No. 2012 M511866 and No. 2013 T60824) to Y.J.Z.
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