Arch Virol DOI 10.1007/s00705-016-2755-5

ORIGINAL ARTICLE

Expression of HIV-encoded microRNA-TAR and its inhibitory effect on viral replication in human primary macrophages

1,2 1 3 3 1 1 Li Li • Haimin Feng • Qin Da • Honglin Jiang • Lang Chen • Linlin Xie • 1,2 1,2 1,2 2 4 Qiuling Huang • Hairong Xiong • Fan Luo • Lei Kang • Yan Zeng • 5 1,2 1,2 Haitao Hu • Wei Hou • Yong Feng

Received: 24 August 2015 / Accepted: 7 January 2016 Ó Springer-Verlag Wien 2016

Abstract A number of -encoded microRNAs have Introduction been shown to play important roles in virus replication and virus-host interactions, although the expression and func- MicroRNAs (miRNAs) are a class of small, single-stranded tion of miR-TAR-3p derived from the human immunode- that inhibit the expression of specific messenger ficiency virus type 1 (HIV-1) TAR element remain RNAs (mRNAs) by binding to complementary target controversial. In this study, miR-TAR-3p was detected in sequences within the mRNAs [3]. It is increasingly clear human peripheral blood monocyte-derived macrophages that cellular miRNAs play pivotal roles in the inhibition of (MDMs) infected by HIV-1. Overexpression of miR-TAR- viral gene expression and replication [7, 9, 15, 21, 27, 33]. 3p impaired viral replication, while inhibition of miR- Certain also encode their own miRNAs [10]. The TAR-3p enhanced it. Additionally, miR-TAR-3p repressed first viral miRNAs were discovered in human B cells viral and replication by targeting the TAR latently infected with Epstein Barr virus (EBV). Many element in the HIV-1 5’-LTR in a sequence-specific other viral miRNAs were subsequently identified in various manner. These results confirm the presence of miR-TAR- viruses, including virus (HSV) type 1 and 3p in HIV-1-infected MDMs and suggest that its function type 2, human cytomegalovirus (HCMV), human her- might be used as a mechanism to modulate HIV-1 repli- pesvirus 6 (HHV-6), Kaposi’s sarcoma-associated her- cation through the expression of a negative regulatory pesvirus (KSHV), adenovirus serotype 5 (ADV-5), JC factor. polyomavirus, BK polyomavirus, and Merkel cell poly- omavirus [2, 5, 6, 18, 25, 29, 35]. Virus-derived small & Wei Hou RNAs have also been detected in cells infected by dengue [email protected] virus, vesicular stomatitis virus (VSV), poliovirus, hepatitis & Yong Feng C virus (HCV), and West Nile virus [14]. Bovine leukemia [email protected] virus (BLV), a with an RNA genome, encodes a conserved cluster of miRNAs that are transcribed by RNA 1 School of Basic Medical Sciences, Wuhan University, 185 polymerase III [20]. Donghu Road, Wuchang, Wuhan 430070, Hubei, People’s HIV-1 has so far been shown to encode for four viral Republic of China 2 miRNAs: miR-TAR, miR-N367, miR-H1, and miR-H3 State Key Laboratory of Virology, Wuhan University, [19, 22–24, 28, 34]. The HIV-1 TAR-element-derived Wuhan 430072, Hubei, People’s Republic of China 3 small RNA called miR-TAR has been estimated to be Hubei Center for Disease Control and Prevention, present at 3 9 103 copies per HIV-1 NL4-3-infected MT4 Wuhan 430079, Hubei, People’s Republic of China 4 cell, suggesting that it might contribute to HIV-1 infection Department of Zoology, College of Life Sciences, Nanjing and HIV-host interaction [31]. However, due to lack of Agriculture University, Nanjing 210095, Jiangsu, People’s Republic of China evidence of miR-TAR expression in HIV-1-infected cells, the question of whether miR-TAR is produced during HIV- 5 Department of Microbiology and Immunology, Sealy Center for Vaccine Development, University of Texas Medical 1 infection remains controversial. The goal of this study, Branch, Galveston, TX 77555, USA therefore, was to examine the presence of miR-TAR in 123 L. Li et al. naturally HIV-1-infected MDMs and its possible effects on transfected with infectious HIV-1 clone pNL4-3 using HIV-1 viral replication. Lipofectamine 2000 according to the manufacturer’s pro- tocol (Invitrogen). Viral supernatant was collected 2 days after transfection and used to infect MT-2 cells. Materials and methods Real-time reverse transcription PCR (RT-qPCR) Plasmids Total RNA was isolated from MDMs infected with the The HIV-1 proviral plasmid pNL4-3 from Dr. Malcolm HIV-1 Bal strain, using TRIzol Reagent according to the Martin [1] and the luciferase reporter construct pNL4- manufacturer’s protocol. Real-time PCR was performed 3.Luc.R-E- from Dr. Nathaniel Landau [8, 16] were using a MyiQ single-color real-time PCR detection system obtained through the NIH AIDS Reagent Program, Divi- (Bio-Rad Laboratories, Hercules, CA, USA). The products sion of AIDS, NIAID, NIH, as described previously [11]. were detected using SYBR Green I (Bio-Rad Laborato- The plasmids pGL3-LTR-AD, pGL3-LTR-AC, and pGL3- ries). Data were normalized to U6 or GAPDH as appro- TAR-BD were constructed by inserting the HIV-1 LTR priate and expressed as the fold change in induced cells (with or without the TAR element) into the pGL3-Basic relative to control. Thermal cycling conditions were as plasmid vector upstream of a luciferase gene. The pCMV- follows: initial denaturation at 95 °C for 3 minutes, fol-

Flag-2B-Tat plasmid, which expresses the HIV-1NL4-3 Tat lowed by 40 cycles of 95 °C for 30 seconds and 60 °C for protein, was graciously provided by Professor Jianguo Wu 30 seconds. (State Key Lab of Virology and College of Life Sciences, For measurement of miRNA expression, specific Bulge- Wuhan University). Loop miR primer sets for miR-TAR-3p and U6 were purchased from Guangzhou RiboBio Co., Ltd. The primers Cells and virus infection for gag (forward, 5’-ATAATCCACCTATCCCAGTAGG AGAAA-3’; reverse, 5’-TTTGGTCCTTGTCTTATGTCC Human blood MDMs were used in this study as model AGAATGC-3’) and GAPDH (forward, 5’-GGTGGTCTC HIV-1 reservoir systems during latent infection after they CTCTGACTTCAACA-3’; reverse, 5’-GTTGCTGTAGC- were infected by the HIV-1 Bal strain. Peripheral blood CAAATTCGTTGT-3’) were purchased from Wuhan samples were obtained from Zhongnan Hospital of Wuhan GeneCreate Co., Ltd. University. Donors gave informed consent for their blood to be used in this research. Healthy samples were screened miRNA and plasmid transfection for common blood-borne pathogens and certified to be pathogen-free. Monocytes were isolated from peripheral The sequence of miR-TAR-3p used in this study was blood mononuclear cells. To achieve differentiation of reported previously by Ouellet et al. [24]. The sequences of monocytes into macrophages, monocytes were cultured miR-TAR-3p and its mutants are shown in Table 1. The with 20 lg of human granulocyte-macrophage colony- miR-TAR-3p mimic, miRNA control mimic, miR-TAR-3p stimulating factor (GM-CSF) (Pepro Tech) per ml for 5 to inhibitor, miRNA inhibitor control, and miRNA mutation 7 days, with half of the culture medium being replaced mimics were purchased from Guangzhou RiboBio Co., with fresh media every 2 days. Ltd. Macrophages were plated in 12-well plates (0.5 9 106 The macrophage-tropic R5 strain Bal was obtained from cells/well) in 1 ml of medium supplemented with 10 % the AIDS Research and Reference Program, NIH (Bethesda, FBS or in 96-well plates (105 cells/well) in 200 llof MD, USA). Macrophages maintained in culture for 5 to medium. Transfection of miRNA mimics was performed 7 days (seeded at a density of 105 cells/well in 96-well with InterferIN (Polyplus-Transfection, New York, NY, plates) were infected with cell-free HIV-1 for 2 hours at USA) according to the manufacturer’s instructions. Mac- 37 °C. The amount of HIV-1 was standardized according to rophages were then incubated at 37 °C for 48 hours. The the concentration of the p24 antigen. Cells were then washed three times with Dulbecco’s modified Eagle medium (DMEM) to remove unabsorbed virus, and fresh medium Table 1 Sequences of miR-TAR-3p and its mutants was added. The cell cultures were replaced with fresh med- miRNA mimic Sequence ium once every 4 days after infection with HIV-1. miR-TAR-3p 30-ACCCAAGGGAUCAAUCGGUCUCU-50 HEK293T cells were cultured in DMEM supplemented miR Mut1 30-ACCCAAGGGAUCAAUCGGUGACU-50 with 10 % fetal bovine serum (FBS), 2 mM L-glutamine, miR Mut2 30-ACCCAAGGGAUCAUACGGUCUCU-50 100 lg of streptomycin per ml, and 100 U of penicillin per miR Mut3 30-ACCCAAGGGAUCAUACGGUGACU-50 ml. Sixty-percent confluent HEK293T cells were 123 HIV-encoded microRNA-TAR medium was replaced with fresh medium before HIV-1 (Promega). Luciferase activity was typically measured for infection. 10 seconds using a luminometer (Turner Designs TD-20/ 20). Assays were performed in triplicate, and the results are P24 ELISA assays and western blotting expressed as the mean ± standard deviation (SD) of luci- ferase activity. For HIV-1 p24 assay, MDM culture supernatants were collected at the designated time points after HIV-1 infec- Statistical analysis tion and analyzed by ELISA as described by the manu- facturer (Zeptometrix Corp. Buffalo. NY. USA). Where appropriate, data are expressed as mean ± SD of For western blot assays, macrophages were washed with triplicate cultures. For comparison of the means of two PBS and lysed in SDS lysis buffer for 5 minutes. The total groups, statistical significance was assessed using Student’s cell lysate was boiled for 10 minutes. Equal amounts of t-test. If there were more than two groups, one-way repe- protein were separated by 10 % or 12.5 % SDS-PAGE and ated-measures ANOVA was used. Statistical analysis was then transferred to a nitrocellulose membrane. The mem- performed with GraphPad InStat statistical software brane was probed with a mouse monoclonal HIV-1 p24 (GraphPad Software, La Jolla, CA, USA). Statistical sig- antibody (Fitzgerald, Acton, MA, USA) and HRP goat nificance was defined as P \ 0.05. anti-mouse secondary antibody. Proteins were visualized using electrochemiluminescence (ECL) western blotting substrate. Results

Luciferase assay MiR-TAR-3p expression in HIV-1-infected MDMs is associated with viral replication Cells were washed once with phosphate-buffered saline (PBS), and 100 ll of lysis buffer (Promega) was then Although miR-TAR has been identified in cell lines added to each well of a 24-well plate. Fifty microliters of transfected with HIV-1 proviral plasmid [24, 31], evi- sample was mixed with luciferase assay substrate dence of miR-TAR expression in natural HIV-1-infected

Fig. 1 Expression of miR-TAR-3p in an HIV-1 infection model. A- HIV-1 at a p24 protein concentration of 30 pg/ml, which was defined C. Expression of miR-TAR-3p in HIV-1-infected MDMs. MDMs as 1). B. The level of p24 protein was determined using ELISA. were infected with HIV-1 R5 strain Bal at different p24 protein Values are means of triplicate experiments. D-E. Expression of gag concentrations on different days, and levels of the gag gene and miR- and miR-TAR-3p in MT-2 cells infected with HIV-1NL4-3 on different TAR-3p were quantified by RT-qPCR, using GAPDH (A) and U6 days. The data are presented as miR-TAR-3p or gag mRNA levels (C) as internal controls. The data are presented as miR-TAR-3p or relative to the control (at day 5 after infection by HIV-1NL4-3, which is gag mRNA levels relative to the control (at day 2 after infection with defined as 1)

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Fig. 2 Effects of miR-TAR-3p overexpression on HIV-1 replication. for 24 hours. F, G. MDMs were infected with Bal at a p24 protein A-C. Overexpression of miR-TAR-3p decreases the levels of gag concentration of 3,000 pg/105 cells for 8 days and then transfected mRNA and p24 protein in HEK293T cells. C. DSUs (densitometry with 20 nM miR-TAR-3p mimic or a control mimic for 48 hours. A, scanning units) express the signal intensities of protein bands via F. The data are presented as gag mRNA levels relative to the control scanning. D-G. Overexpression of miR-TAR-3p inhibits HIV-1 (miR Control, which is defined as 1). The results shown are the replication in MDMs. D, E. MDMs isolated from healthy individuals mean ± SD of triplicate cultures, representative of three experiments was transfected with 20 nM miR-TAR-3p mimic or a control mimic (*, P \ 0.05) for 48 hours, and then infected with HIV-1 Bal at 30 pg p24/105 cells cells is rare. To determine whether natural HIV-1 infec- cells) and the expression of miR-TAR-3p was measured tion produces miR-TAR, monocyte-derived macrophages using RT-qPCR. The results showed that expression of (MDM) isolated from healthy donors were infected with miR-TAR-3p increased in a p24-dose-dependent manner, the HIV-1 Bal strain at different p24 protein concentra- suggesting that miR-TAR-3p expression in HIV-1-in- tions (30 pg/105 cells, 300 pg/105 cells, and 3,000 pg/105 fected MDMs is associated with HIV-1 viral replication

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Fig. 3 Effects of miR-TAR-3p inhibitor on HIV-1 replication in MDMs. MDMs isolated from healthy individuals was infected with HIV-1 for 8 days and then transfected with miR-TAR-3p inhibitor, inhibitor control, a mixture of miR-TAR-3p and its inhibitor, or miR-TAR-3p mimic for 48 hours. The cell extracts were used to determine the transcript levels of the gag gene (B, C) and miR-TAR-3p (A), and culture supernatants were collected for the analysis of p24 protein expression (D, E). The data are presented as gag or miR-TAR-3p levels relative to the control (Inhibitor Control, which is defined as 1). The results shown are the mean ± SD of triplicate cultures, representative of three experiments (*, P \ 0.05, **, P \ 0.01)

(Fig. 1A-C). To confirm this observation, miR-TAR-3p MiR-TAR-3p suppresses HIV-1 replication expression in HIV-1 NL4-3-infected MT-2 cells (a T-cell in MDMs line transformed by HTLV-1) was then examined. Again, miR-TAR-3p was detected in MT-2 cells, and its To investigate whether miR-TAR-3p affects HIV-1 repli- abundance was correlated with HIV-1 replication (Fig 1D cation, HEK293T cells were first transfected with a and E). chemically synthesized miR-TAR-3p mimic or a miRNA

123 L. Li et al. control mimic (a randomized RNA oligonucleotide) and Fig. 4 Effects of miR-TAR-3p mimic on HIV-1 transcription. c pNL4-3 plasmid. The transcript level of the gag gene was A. HEK293T cells were co-transfected with the luciferase reporter plasmid pNL4-3R-E- and miR-TAR-3p mimic or control mimic. determined using real-time PCR. p24 protein production in Luciferase activity was measured. B. the effect of miR-TAR-3p on the supernatants and in the cells was determined by ELISA expression of the HIV-1 TAR element. HEK293T cells were co- and Western blotting, respectively. The results showed that transfected with the luciferase reporter plasmid pNL4-3R-E- and miR- the miR-TAR-3p mimic significantly downregulated the TAR-3p mimic or control mimic. Luciferase activity was measured. The transcript levels of Gag and TAR were determined by RT-qPCR expression of the HIV-1 gag gene (Fig. 2A) and the p24 (miRNA Control, which is defined as 1). C. Schematic diagrams of protein (Fig. 2B and C) when compared with the control. pGL3-LTR reporters. D. Luciferase reporter expression after treat- Next, the question of whether miR-TAR-3p regulates ment of HEK293T cells transfected with pCMV-Flag2B-Tat and HIV-1 replication in MDMs was assessed. As shown in pGL3-LTR or mutants with miR-TAR-3p. E. Predicted binding site of miR-TAR-3p in the TAR region. Nucleotides of interest are indicated Fig. 2D-G, the miR-TAR-3p mimic decreased the expres- in red and modified in miR-TAR Mut 1-3. F. The effects of wild-type sion of gag mRNA and p24 protein under two infection and mutant miR-TAR-3p on HIV-1 transcription were determined conditions: 30 pg of p24 per 105 cells or 3000 pg of p24 per using a luciferase assay. The results shown are the mean ± SD of 105 cells. In addition, as miR-125b has been reported to triplicate cultures, representative of three experiments (*, P \ 0.05; **, P \ 0.01) inhibit HIV-1 replication [17], it was used as a positive control (Fig. 2F and G). These results indicate that miR- TAR-3p can suppress HIV-1 replication in this MDM infection model. a dose-dependent manner (Fig. 4A). As the luciferase gene These results together suggest that miR-TAR-3p sup- expression reflects HIV-1 transcription, this finding sug- presses HIV-1 replication in MDMs. gests that miR-TAR-3p could suppress HIV-1 genome transcription. Inhibition of miR-TAR-3p increases HIV-1 We next asked whether miR-TAR-3p affects the HIV-1 replication in MDMs promoter LTR. Real-time PCR was performed with primers designed to amplify the TAR RNA [22] in HEK293T cells Because it was found that miR-TAR-3p could inhibit HIV- transfected with the miR-TAR-3p mimic and the luciferase - - 1 replication in MDMs, we next investigated the question reporter plasmid pNL4-3.Luc.R E . We observed that miR- of whether inhibition of miR-TAR-3p could increase HIV- TAR-3p reduced TAR RNA levels in cells consistently with 1 replication. We first infected MDMs with the HIV-1 R5 reduced luciferase expression and Gag mRNA (Fig. 4B). strain Bal at a p24 protein concentration of 3000 pg/105 Next, we investigated whether the HIV-1 TAR element cells for 8 days, followed by transfection for 48 hours with is targeted by miR-TAR-3p. Three HIV-1 LTR promoters one of four experimental options: miR-TAR-3p inhibitor (a that drive luciferase expression were constructed. LTR-AD synthetic oligonucleotide with sequences complementary contained all of the upstream regulatory elements as well as to miR-TAR-3p); inhibitor control (a randomized RNA the TAR element adjacent to the transcription initiation oligonucleotide); a mixture of miR-TAR-3p plus miR- site. LTR-AC lacked the TAR element, and LTR-BD TAR-3p inhibitor; or miR-TAR-3p mimic. The miR-TAR- lacked the upstream regulatory elements (Fig. 4C). These 3p inhibition results showed that miR-TAR-3p inhibitor promoters were each tested in the presence of the HIV-1 successfully decreased HIV-induced miR-TAR-3p expres- Tat protein, whose binding to TAR is a key step in HIV-1 sion in MDMs (Fig. 3A). Measurement of HIV-1 replica- transcription. It was found that miR-TAR-3p downregu- tion revealed that miR-TAR-3p inhibitor significantly lated luciferase expression only when the TAR element upregulated the expression of both gag mRNA and p24 was present in the promoters (Fig. 4D). Thus, miR-TAR-3p protein in a dose-dependent manner (Fig. 3B-E). These might downregulate HIV-1 LTR transcription by targeting results suggested that inhibition of miR-TAR-3p could the TAR element, leading to reduced viral replication. increase HIV-1 replication in MDMs. Computational analysis predicted a miR-TAR-3p bind- ing site in the TAR region. To test the interactions between MiR-TAR-3p suppresses HIV-1 replication miR-TAR-3p and the TAR region, three miR-TAR-3p by targeting the TAR element in the HIV-1 50 LTR mutants (Mut 1-3) were constructed as shown in Figure 4E and inhibiting genome transcription (sequences shown in Table 1). The TAR sequence was not mutated in this study to avoid altering the Tat-TAR inter- To investigate how miR-TAR-3p inhibits HIV-1 replica- action. MiR-TAR-3p Mut1 inhibited HIV-1 replication as tion, HEK293T cells were transfected with the miR-TAR- well as the wild-type miR-TAR-3p, while Mut 2 and Mut 3 3p mimic and the luciferase reporter plasmid pNL4- had no effect (Fig. 4F). These results together suggest that 3.Luc.R-E-. It was found that luciferase expression was targeting the TAR element is essential for miR-TAR-3p- significantly downregulated by the miR-TAR-3p mimic in induced suppression of HIV-1 transcription. 123 HIV-encoded microRNA-TAR

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Discussion affects miRNA expression greatly [12, 13]. One possible cause of low miR-TAR expression is the suppression of Several groups have reported the identification of miR- RNA silencing (SRS) effect generated by HIV-1 Tat pro- TAR using bioinformatic prediction, Northern blotting, tein. Tat blocks Dicer processing of the TAR hairpin into RNase protection assays, deep sequencing of RNA isolated mature miRNAs [4], which might help HIV-1 to bypass in vitro, or HIV-1 (pNL4-3)-transfected cell lines [24, 31]. miR-TAR-3p interference and replicate successfully in miR-TAR was also detectable in CD4? T cells infected host cells. Consequently, in this study, chemically syn- with HIV-1NL4-3 viruses [34] and exosomes derived from thesized miR-TAR-3p mimic was used instead of the TAR HIV-1-infected cells [22]. In this study, we first examined hairpin (to produce miR-TAR-3p) to investigate the func- whether miR-TAR could be produced in natural HIV-in- tion and mechanism of action of miR-TAR-3p. fected MDMs. MDMs from healthy subjects infected with It is a subject of debate whether HIV-1 encodes such a an R5-tropic HIV-1 Bal strain were then used to detect TAR-derived miRNA that acts as a negative factor against miR-TAR-3p expression. Abundant expression of miR- its own replication. In our opinion, 1) the TAR element TAR-3p was observed in MDMs exhibiting a high viral plays pivotal roles during virus infection and replication, load (Fig. 1A, B, and C). Collectively, this study provides while miR-TAR arises as a by-product when TAR RNA evidence that HIV-1 infection generates miR-TAR-3p in hairpin is processed into miR-TAR via a particular mech- primary MDMs. anism; 2) many micro-organisms including viruses have Previous studies on HIV-encoded miRNAs used the developed mechanisms to slow down their replication to strategy of overexpressing proviral clones in cell lines. In avoid been recognized and cleared by the host immune this study, human PBMC-derived primary macrophages system, or they reduce their growing population to protect infected with the HIV-1 Bal strain were used to investigate the host cells from apoptosis or death; 3) and as mentioned the effect of miR-TAR-3p on viral replication. It was found above, HIV-1 has developed a possible mechanism to that miR-TAR-3p inhibited HIV-1 replication in the MDM control over-processing of miR-TAR by Tat protein to infection model (Fig. 2). The miR-TAR-3p inhibitor may avoid viral replication failure. have enhanced the HIV-1 replication level by negating the effect of endogenous miR-TAR-3p in infected MDMs (Fig. 3). Our data revealed that miR-TAR-3p might serve Conclusion as a negative regulator of HIV-1 replication. Most miRNAs repress gene expression by targeting the This study provides evidence for miR-TAR-3p expression 3’-UTR of mRNAs in the cytoplasmic RNA-induced in HIV-1-infected primary human monocyte-derived mac- silencing complex (RISC) for translation repression or rophages (MDMs). miR-TAR-3p suppressed HIV-1 repli- mRNA degradation. However, miRNAs could also enter cation in MDMs, possibly by targeting the TAR element in the nucleus and regulate gene expression at the transcrip- the 5’LTR, thus inhibiting HIV-1 genome transcription. tional level [26, 32]. Interestingly, a novel HIV-1-encoded These findings reveal a novel class of mechanisms for miR-H3 was recently reported to enhance HIV-1 replica- regulation of HIV-1 replication. tion by targeting the TATA box region [34]. In our study, when cells were co-transfected with pNL4-3.Luc.R-E- and Acknowledgments This work was supported by research grants from the National Natural Science Foundation of China (Nos. the miR-TAR-3p mimic, a significant decrease in luciferase 81271818 and 81471940), the Young Scholarship from the Health gene expression was observed, which suggests that the Department of Hubei to Y. Feng (QJX2012-10), the National Natural miR-TAR-3p mimic may interfere with viral genome Science Foundation of China (No. 30972754), the Specialized transcription. It was also shown that miR-TAR-3p sup- Research Fund for the Doctoral Program of Higher Education, Min- istry of Education of China (20120141110075), and the Science and presses HIV-1 LTR transcription by targeting the TAR Technology Ministry of China as part of a major project of infectious element in a sequence-specific manner (Fig. 4). disease control and prevention carried out by W. Hou The mechanism of biogenesis of miR-TAR remains (2014ZX10001003). unclear, although the Microprocessor complex (Drosha/ Dgcr8 complex) has been shown to bind and cleave the HIV-1 TAR stem-loop RNA [30]. Interestingly, Narayanan References et al. showed evidence that exosomes originating from 1. 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