Suppression of HIV-1 replication by microRNA effectors
The rate of HIV-1 gene expression is a key step that determines the kinetics of virus spread and AIDS progression. Viral entry and gene expression were described to be the key determinants for cell permissiveness to HIV. Recent reports highlighted the involvement of miRNA in regulating HIV-1 replication post-transcriptionally. In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression. Here we show that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterize some of the proteins required for miRNA-mediated silencing (miRNA effectors). RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes. Interestingly, knockdown of RCK/p54 or DGCR8 resulted in virus reactivation in PBMCs isolated from HIV infected patients treated with suppressive HAART.
KeywordsBrome Mosaic Virus Cellular miRNAs RNAi Effector mRNA Decapping Latent Viral Reservoir
RNA silencing (RNAi) is a new gene regulatory mechanism conserved from plants to humans. RNAi mediators are small non-coding RNAs (sncRNAs) that function through sequence specific mRNA targeting to either induce their degradation and/or inhibit translation [1, 2]. In mammals, RNAi is mediated by different classes of small non-coding RNAs including piRNAs, microRNAs and siRNAs [3, 4, 5]. MicroRNAs are produced from a primary transcript (pri-miRNA) which is processed in the nucleus by the microprocessor complex containing RNase Drosha and DGCR8. The resulting product or pre-miRNA is exported to the cytoplasm through the exportin-5 pathway. Cytoplasmic pre-miRNA is processed by typeIII RNase Dicer to miRNA/miRNA* duplex of 19 to 25 nucleotides. miRNA/miRNA* is incorporated into the RNA-Induced Silencing Complex (RISC) where miRNA* is degraded while miRNA serves as a guide for mRNA targeting . Key components of miRISC are proteins of the Argonaute family (Ago1 to Ago4) that are required for miRNA-mediated silencing [6, 7]. To ensure mRNA translational inhibition and decay, miRISC, loaded with miRNA and its mRNA targets, associate with proteins involved in mRNA processing . A key factor in this process is the GW182 protein that interacts directly with Argonaute1 (Ago1) , and the human homologs of GW182 that interact with Ago1–4 . GW182 orchestrates both mRNA decapping, through the recruitment of p54/RCK that regulates the activity of the decapping enzymes DCP1/DCP2 , and mRNA deadenylation by recruiting the CCR4-NOT1 complex . mRNA decapping and deadenylation leads to mRNA decay through the action of XRN1, a 5'-3' exonuclease . Interestingly, RNAi effectors, including miRNAs and their target mRNAs, Ago proteins, GW182, RCK/p54, LSm-1 and DCP proteins co-localize in cytoplasmic structures called GW-bodies or P-bodies suggesting that miRNA-mediated silencing occurs at these sites [11, 12, 13, 14, 15]. Emerging evidence suggests that miRNA-mediated gene regulation serves as a defence mechanism against both RNA and DNA viruses in mammals [16, 17, 18, 19, 20]. The present study was designed to explore physical and functional interaction between effectors of miRNA-mediated silencing and HIV-1 replication.
Results and discussion
The outcome of HIV-1 infection results from complex interactions between viral components and host cell factors [32, 33, 34, 35]. In most cases, HIV-1 successfully hijacks cellular pathways and bypasses restriction factors for optimal replication leading to continuous rounds of infection, replication, and cell death. Continuous viral replication causes the loss of CD4+T cells and progression to immunodeficiency in infected individuals. HAART treatment revealed the existence of a pool of resting memory CD4+ T cells harbouring integrated, but silent HIV-1 provirus [36, 37]. This latent reservoir is believed to be the major obstacle for virus eradication by HAART. Therefore, it is critical to understand how HIV-1 latency is established and maintained . Post-integration latency takes place at both transcriptional and post-transcriptional levels . Transcriptional latency involves different mechanisms ranging from integration position effect [40, 41, 42], limitation in transcription factors [43, 44, 45, 46], establishment of chromatin repressive marks and recruitment of chromatin silencers [47, 48, 49, 50, 51]. Post-transcriptional silencing involves defects in mRNA export and translation [52, 53, 54]. All together, these studies show that HIV-1 post-integration latency is a multi-factorial process. In the present study, we show that HIV-1 gene expression is additionally regulated by the miRNA pathway. HIV-1 mRNA associates with components of the RISC complex by a mechanism that does not involve APOBEC3G, but does need sncRNAs. Accordingly, it has been recently shown that the suppressor of RNAi P19 from tomato bushy stunt virus, known to bind and sequester sncRNAs including miRNA, enhances HIV-1 replication . Additionally, the RNAi suppressor function of HIV-1 Tat  could be complemented by VP35 from Ebola virus  and the NS3 protein of rice hoja blanca virus through sequesteration of small non-coding RNAs . HIV-1 mRNAs associated with RISC are sequestered in the non-polysomal fraction, thereby preventing translation. In agreement with two previous reports [19, 21, 22], we show that knockdown of RCK/p54, a protein required for miRNA-mediated silencing, led to virus reactivation from PBMCs isolated from HIV-1 infected patients who were undergoing suppressive HAART.
A challenge in AIDS treatment is the need to activate latent viral reservoirs in order to eradicate these viruses through HAART. In this respect, targeting the miRNA processing pathway could offer a strategy that could be exploited to activate latent viral reservoirs, for instance, during HAART. Several molecules have been used to reactivate viral reservoirs . However, none of these approaches provides the sequence specific targeting that can be achieved using siRNA. Recent data suggest that siRNA can be used therapeutically in vivo in certain mouse disease models  and more recently in non-human primates [61, 62]. It remains to be explored whether, as suggested here, the in vivo targeting of miRNA-effectors using siRNA can assist in activating latent HIV-1 reservoirs for eradication by HAART.
HIV-1 molecular clone pNL4-3Δvif and expression plasmids for APOBEC3G were gift from Olivier Schwartz (Pasteur, France). APOBEC3G H65R/H257R mutant was previously described . HIV-1 vector containing MS2 binding sites and MS2-GFP expression plasmids [64, 65] were gift from Alessandro Marcello (ICGEB. Trieste, Italy) and Edouard Bertrand (IGMM. Montpellier, France)
PBMCs were transfected with siRNA or miRNA using the Nucleofector II Device with the appropriate Nucleofection solution according to the manufacturer's instructions (Amaxa). siRNA corresponding to DGCR8 (5'-CAUCGGACAAGAGUGUGAU(dTdT)-3'), Drosha (5'-CGAGUAGGCUUCGUGACUU(dTdT)-3'), RCK/p54 (5'-GCAGAAACCCUAUGAGAUUUU(dTdT)-3'), LSm-1 (5'-GUGACAUCCUGCCACCUCACUU(dTdT)-3'), GW182 (5'-UAGCGGACCAGACAUUUCU(dTdT)-3'), XRN1 (5'-AGA UGA ACU UAC CGU AGA A(dTdT)-3') and CDK9 (5'-CCAAAGCUUCCCCCUAUAATT(dTdT)-3') were synthesized (MWG). Expression level of knock down proteins was analyzed by Western blotting 48 hours after transfection. Briefly, cell-extracts were resolved on SDS-PAGE gels. Proteins were transferred to PVDF membrane by semi-dry electroblotting and probed overnight at 4°C with the primary antibody (anti-Drosha, LSm1, GW182 (Abcam), DGCR8 (Proteintech Group), anti-RCK/p54 (Bethyl)or anti-CDK9 (Santa Cruz), washed and incubated with the appropriate secondary antibody (Amersham) for 1 hour. Proteins were visualized by chemiluminescence according to the manufacturer's protocol (Pierce).
PBMC isolation and co-culture assay for virus production
Peripheral blood mononuclear cells of HIV-1 infected patients were isolated by lymphocyte separation medium density centrifugation (Lonza). PBMCs from healthy donors were pre-activated using 5 μg/ml PHA (phytohemagglutinin-P, DIFCO)/10 U/ml IL-2 (interleukin-2, Roche) for 72 hours. They were then washed once with PBS and once with RPMI medium before co-culture assay. siRNA transfected HIV-infected PBMC (106 cells/ml) were co-cultured with pre-activated PBMC (106 cells/ml) from the same healthy donor in the presence of 10 U/ml IL-2. The culture medium was collected every 3 or 4 days. Fresh pre-activated healthy PBMCs were added to the culture every 7 days. Viral production was measured by quantifying the amounts of p24 in the culture medium using an ELISA kit (Ingen).
Pseudotyped virion production and single-round infections
The plasmid pNL4-3-env-Luc+ harboring a luciferase gene (obtained from the NIAID AIDS Reagent Program) was co-transfected with the envelope plasmid pMD.2G encoding the G protein of vesicular stomatitis virus (VSV.G) into human embryonic kidney cells-293T. The virions, named HIV-1VSV-Luc, were collected and filtered using 0.45 μm filters 48 hours post-transfection. HeLa or HeLa CD4+ cells were infected over-night at 37°C, washed and resuspended in DMEM containing 10% FCS. Virus production was monitored in culture supernatant by measuring p24 antigen (Ingen) and by following luciferase activity according to the manufacturer's instructions (Promega).
Cytoplasmic extracts analysis on sucrose gradients
To isolate cytoplasmic extracts, cells were lysed for 10 minutes in buffer B (5 mM Tris-HCl pH 7.4, 1.5 mM KCl, 2.5 mM MgCl2, 0.5% NP40 and protease inhibitor). Nuclei were pelleted by centrifugation for 10 minutes at 10,000 rpm. 2 mg of cytoplasmic extracts were loaded on a 7–47% sucrose gradient. Briefly, 5 layers of 7 to 47% sucrose were prepared in sucrose buffer (20 mM Tris-HCl pH7.4, 80 mM NaCl, 5 mM MgCl2, 1 mM DTT and protease inhibitors) and diffused at 4°C for 16 hours to obtain a linear sucrose gradient. 2 mg of cytoplasmic extracts were loaded on the top of the column, and centrifuged for 3 hours at 36,000 rpm in a SW41Ti rotor. After ultracentrifugation, 28 fractions were collected and OD at 254 nm was measured in each fraction using a Nanodrop apparatus (Labtech).
293 cells were grown in 60 mm dishes and transfected with the indicated plasmids using calcium-phosphate. Cells were harvested 48 hours after transfection, lysed for 15 minutes in RIP buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 2.5 mM MgCl2 × 6H2O, 250 mM sucrose, 0.05% NP40, 0.5% Triton X-100) containing RNASIN (Promega) and 1 mM DTT, and centrifuged to pellet debris. Supernatants were incubated overnight with mouse anti-Myc mAb 9E10 (Amersham) at 4°C followed by 2 hours incubation with protein G-Sepharose. Immunoprecipitates were washed with RIP buffer, and nucleic acids were extracted with phenol/chloroform/isoamyl alcohol, isopropanol-precipitated, ethanol-washed and resuspended in RNase-free water. Total RNA was DNase I treated and reverse-transcribed using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). RT products were PCR-amplified using either GAPDH (GAPDH forward: GTA TTG GGC GCC TGG TCA CC; reverse: CGC TCC TGG AAG ATG GTG ATG G), HIV-1 (HIV-1 forward: TAG TGT GTG CCC GTC TGT T; reverse: CTC TGG TTT CCC TTT CGC TTT C or Gag-reverse: GAT GGT TGT AGC TGT CCC AG for unspliced HIV RNA), or HDM2 specific oligonucleotides (HDM2 forward: GTA CCT GAG TCC GAT GAT TCC; reverse: ACC TAC TGA TGG TGC TGT AAC). PCR products were resolved on 1.5% agarose/TAE gels containing ethidium bromide. In vivo splicing assay and oligonucleotides BSS and SJ4.7A have been described 
We are grateful to Kiernan R, Jeang KT, Emiliani S, and Voinnet O for helpful discussions and for critically reading the manuscript. Work in MB's laboratory was supported by ANRS, SIDACTION, ANR and FRM. OM was supported by fellowship from "infectiopôle grand sud". DL was supported by ANRS scholarship. AZ was supported by SIDACTION fellowship.
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