Results and discussion To investigate whether RNAi effectors regulate HIV-1 replication, we analyzed virus replication in cells where expression of RNAi effectors was reduced using specific siRNA. HeLa cells were transfected with siRNA specific to RCK/p54, GW182, LSm-1 or XRN1. As controls, HeLa cells were transfected with scrambled siRNA (Scr) or CDK9 specific siRNA and subsequently infected with HIV-1 (Figure 1a). Knockdown of RCK/p54, GW182, LSm-1 and XRN1 enhanced virus replication by up to 10 fold (Figure 1b). As we have previously shown, knockdown of Drosha [ 21] and DGCR8 (Figure 1b), the two subunits of the microprocessor complex, increased virus production while knockdown of the CDK9 subunit of the PTEFb complex that is required for viral gene expression, reduced HIV-1 production (Figure 1b). Interestingly, analysis of HIV-1 cytoplasmic mRNA distribution on glycerol gradient showed that knockdown of RCK/p54 shifted HIV-1 mRNA from the non-polysomal fraction to polysomes as compared to control siRNA transfected cells (Figure 2, upper panel). As control, we analyzed the distribution of endogenous mRNA expressed from a gene encoding Hdm2. Knockdown of RCK/p54 did not affect Hdm2 mRNA distribution (Figure 2, lower panel). These experiments show that GW182, RCK/p54, LSm-1 and XRN1, factors required for RNAi, are repressors of HIV-1 gene expression that act by preventing HIV-1 mRNA translation. ![Figure 1 Figure 1](picrender.fcgi?artid=2657893&blobname=1742-4690-6-26-1.gif) | Figure 1miRNA effectors are repressors of HIV-1 replication. HeLa cells were transfected with siRNA as indicated. 48 hours post transfection, cells were analyzed for RCK/p54, LSm-1, GW182, XRN1, DGCR8, DROSHA and CDK9 expression by Western blotting (a), or infected (more ...) |
![Figure 2 Figure 2](picrender.fcgi?artid=2657893&blobname=1742-4690-6-26-2.gif) | Figure 2RCK/p54 restricts HIV-1 mRNA association with polysomes. Cytoplasmic extracts from HeLa cells that were transfected with the indicated siRNA and infected with HIV-1-VSVG-luc were run on glycerol gradient (7% to 47%). Fractions were collected and their (more ...) |
We next investigated the physical interaction between RNAi effectors and HIV-1 mRNA. 293 cells were mock transfected or transfected with combinations of pNL4-3, Myc-Ago2, a central component of the RISC complex, or its RNA-binding mutant Myc-Ago2PAZ9 constructs as indicated in figures 3 and 4. First, we verified that Myc-Ago2 and Myc-Ago2PAZ9 were equally expressed (Figure 3a). Second, cytoplasmic extracts were prepared, and a fraction was used for total RNA extraction while the rest was subjected to immunoprecipitation using anti-Myc antibody to purify Myc-Ago2 associated mRNP. Both total RNA (Figure 3b, left panels) and Myc-Ago2 associated RNA (Figure 3b, right panels) were reverse transcribed and subjected to PCR amplification using oligonucleotides specific for HIV-1 TAR RNA (a structured motif associated with all HIV-1 mRNAs) or HIV-1 unspliced mRNA, Hdm2 mRNA or GAPDH mRNA. PCR analysis of total RNA showed that equal amounts of HIV-1, Hdm2 and GAPDH mRNAs were present in all samples (Figure 3b, left panels). HIV-1 mRNAs (both TAR and unspliced) were associated with Myc-Ago2, but not with Myc-Ago2PAZ9 mutant (Figure 3b, right panels). In agreement with the results shown in figure 2, Hdm2 mRNA was not detected in Myc-Ago2 mRNPs, suggesting that under these conditions Hdm2 is not regulated by RNAi. A similar experiment was performed to analyze the association of HIV-1 multispliced mRNA with Myc-Ago2 mRNPs. The RT-PCR reactions were performed in the presence of 32P-α ATP and were analyzed by autoradiography (Figure 3c). HIV-1 multispliced mRNAs associated with Myc-Ago2 (compare lane 3 to 2) and weakly with Myc-Ago2PAZ9 (compare lane 4 to lanes 3 and 2). Co-localization of HIV-1 mRNA and effectors of RNAi such as Ago2 and RCK/p54 within the P-bodies was also observed by immunofluorescence using HIV-1 containing MS2 binding sites and MS2-GFP constructs (Figure 4). Indeed, HIV-1 mRNAs visualized through their binding to MS2-GFP colocalized with endogenous RCK/p54 and ectopically expressed Myc-Ago2 (Figure 4). Our results show that HIV-1 mRNAs physically associate with Ago2, a central component of RISC, and co-localize with cellular proteins required for miRNA-mediated silencing such as RCK/p54 and Ago2 in P-bodies. We observed that all HIV-1 mRNA species associated with RISC. Accordingly, Huang et al. had identified 5 cellular miRNAs able to target the 3'UTR sequence present in all HIV-1 mRNAs [ 22]. Additionally, other cellular miRNAs able to target regions out side the 3'UTR may also participate [ 23]. ![Figure 3 Figure 3](picrender.fcgi?artid=2657893&blobname=1742-4690-6-26-3.gif) | Figure 3HIV-1 mRNAs associate with Argonaute 2. 293 cells were transfected with HIV-1 molecular clone pNL4-3, Myc-Ago2 or Myc-AgoPAZ9 as indicated. 48 hours later cells were harvested and cytoplasmic extracts were prepared. Total RNA was purified from a fraction (more ...) |
![Figure 4 Figure 4](picrender.fcgi?artid=2657893&blobname=1742-4690-6-26-4.gif) | Figure 4HIV-1 mRNA co-localizes with RCK/p54 and Ago2. HeLa cells were transfected with Myc-Ago2 expression vector either alone (top panels) or co-transfected with HIV-1 vector containing 24 repeats of MS2 binding sites and MS2-GFP expression vectors [64,65] (more ...) |
Emerging evidence suggests the physical and functional interactions between P-bodies and the viral life cycles [ 24]. Viral mRNA trafficking through P-bodies may represent a pool of translationally repressed viral transcripts otherwise used for efficient packaging or formation of viral-replication complexes. Indeed, yeast retrotransposons Ty1 and Ty3 mRNA associate with P-bodies, and this association is required for efficient retrotransposition [ 25- 27]. In the case of BMV (Brome Mosaic Virus), formation of the virus replication complex occurs in P-bodies [ 28]. In addition, P-bodies may also function in host defences against viruses and transposable elements. Indeed, the cellular factors APOBEC 3G (A3G) and 3F (A3F), which are viral restriction factors, are found to accumulate in P-bodies [ 29, 30]. It has been suggested that A3G and A3F mediated HIV-1 restriction may involve viral mRNA targeting to P-bodies leading to their translational inhibition [ 30]. We, therefore, asked whether P-bodies are positive or negative regulators of HIV-1 replication. Thus, we analyzed HIV-1 replication in cells where P-bodies were disrupted by knocking down RCK/p54 or LSm-1 [ 31]. HeLa CD4+ cells were transfected with RCK/p54 or LSm-1 specific siRNA or control siRNA. Forty eight hours later, cells were infected with equal amounts of HIV-1 viral particles (as measured by p24 assay). HIV-1 p24 antigen was measured in cell culture supernatant 48 hours post-infection. As shown in figure 5b, knockdown of RCK/p54 or LSm-1 results in enhanced virus production as compared to infection of control siRNA transfected cells. To assess the infectivity of the produced viruses, an equal volume of supernatant from Scr, RCK/p54 and LSm-1 siRNAtransfected cells was used to infect HeLa CD4+ cells, and p24 release in the culture supernatant was measured 48 hours later (Figure 5c). Virus infectivity correlated with the amount of p24 produced (Figure 5b) showing that virions produced in RCK/p54 and LSm-1 knocked down cells are fully competent for replication and have no defect in steps such as RNA packaging. Since the knockdown of RCK/p54 and LSm-1 was shown to result in the disruption of P-bodies, we concluded from these experiments that accumulation of HIV-1 mRNA in P-bodies limits virus replication. ![Figure 5 Figure 5](picrender.fcgi?artid=2657893&blobname=1742-4690-6-26-5.gif) | Figure 5Disruption of P-bodies through knockdown of RCK/p54 and LSm-1 leads to enhanced production of infectious HIV-1 virions. HeLa CD4+ cells were transfected with siRNA as indicated. 48 hours post transfection cells were analyzed for RCK/p54 and LSm-1 expression (more ...) |
Next, we asked whether A3G-mediated HIV-1 restriction requires effectors of miRNA-mediated mRNA translational inhibition. Thus, we compared A3G-mediated HIV-1 restriction in cells where RCK/p54 or LSm-1 expression was reduced compared to control cells. HeLa cells were transfected with control siRNA or siRNA specific for RCK/p54 or LSm-1 (Figure 6, right panel). Forty-eight hours later, cells were transfected with an HIV-1 molecular clone lacking the vif gene (pNL4-3Δvif) either alone or with wild-type A3G or A3G mutant lacking antiviral activity (A3Gdm). HIV-1 p24 antigen was measured in culture supernatant 48 hours post-transfection. Interestingly, knock down of RCK/p54 or LSm-1 enhanced HIV-1 production regardless of A3G (Figure 6, left upper panel). Similarly, A3G but not A3Gdm reduced virus production regardless of RCK/p54 or LSm-1 expression (Figure 6, left upper panel). These results suggested that RCK/p54 or LSm-1 and A3G -mediated HIV-1 repression involves different mechanisms. We then analyzed the infectivity of HIV-1 produced from siRNA transfected cells. Equal amounts of p24 were used to infected HeLa CD4+ cells, and HIV-1 p24 antigen was measured in culture supernatant 48 hours post-infection. As shown in figure 6 (lower panel), virus produced in Scr siRNA transfected cells in the presence of A3G showed lower infectivity than those produced in its absence or in the presence of A3Gdm. Similar HIV-1-restriction activity of A3G was observed when the virus was produced in RCK/p54 or LSm-1 knocked down cells. This experiment showed that A3G-mediated HIV-1 restriction is independent of RNAi effectors RCK/p54 and LSm-1 and does not require P-bodies. ![Figure 6 Figure 6](picrender.fcgi?artid=2657893&blobname=1742-4690-6-26-6.gif) | Figure 6RNAi effectors and APOBEC 3G-mediated HIV-1 repression involve different pathways. HeLa CD4+ cells were transfected with the indicated siRNA. 48 hours later cells were analyzed for RCK/p54 and LSm-1 expression (right panel) or co-transfected with 1 μg (more ...) |
Taken together, our results show a physically repressive interaction between RNAi effectors and HIV-1 mRNA. Since cellular miRNAs were shown to play a role in HIV-1 latency [ 22], we asked whether RCK/p54, which is required for miRNA-mediated mRNA translational inhibition, contributes to HIV-1 silencing in vivo. Thus, PBMCs isolated from 3 HAART-treated HIV-1-infected patients with undetectable viremia were transfected with control siRNA or with siRNA specific for Drosha, DGCR8 or RCK/p54. Transfected cells were co-cultured with PHA/IL2-activated PBMCs isolated from healthy donors. Virus production was monitored every 3 days by measuring p24 antigen in the culture supernatant (Figure 7). As we have previously shown, knockdown of Drosha resulted in virus reactivation in PBMCs isolated from 3 HAART-treated HIV-1-infected patients [ 21]. Remarkably, viral replication from its natural reservoir resumed also when DGCR8 or RCK/p54 was silenced. No virus was isolated from control siRNA transfected PBMCs suggesting that virus production observed in Drosha, DGCR8 and RCK/p54 knock down was not due to actively infected PBMCs relieved from drug pressure. These results show that endogenous levels of Drosha, DGCR8 and RCK/p54 contribute to HIV-1 latency and/or its maintenance in infected patients. ![Figure 7 Figure 7](picrender.fcgi?artid=2657893&blobname=1742-4690-6-26-7.gif) | Figure 7Implication of RNAi in HIV-1 latency. PBMCs were isolated from three patients undergoing active HAART. Isolated PBMCs were transfected with the indicated siRNA and either analyzed for RCK/p54, DGCR8 and DROSHA expression by Western blotting 48 hours after (more ...) |
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References Brodersen P, Sakvarelidze-Achard L, Bruun-Rasmussen M, Dunoyer P, Yamamoto YY, Sieburth L, Voinnet O. Widespread translational inhibition by plant miRNAs and siRNAs. Science. 2008;320:1185–1190. doi: 10.1126/science.1159151. [PubMed]Filipowicz W, Bhattacharyya SN, Sonenberg N. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet. 2008;9:102–114. doi: 10.1038/nrg2290. [PubMed]Tam OH, Aravin AA, Stein P, Girard A, Murchison EP, Cheloufi S, Hodges E, Anger M, Sachidanandam R, Schultz RM, Hannon GJ. Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes. Nature. 2008;453:534–538. doi: 10.1038/nature06904. [PubMed]Stefani G, Slack FJ. Small non-coding RNAs in animal development. Nat Rev Mol Cell Biol. 2008;9:219–230. doi: 10.1038/nrm2347. [PubMed]Okamura K, Lai EC. Endogenous small interfering RNAs in animals. Nat Rev Mol Cell Biol. 2008;9:673–678. doi: 10.1038/nrm2479. [PubMed]Peters L, Meister G. Argonaute proteins: mediators of RNA silencing. Mol Cell. 2007;26:611–623. doi: 10.1016/j.molcel.2007.05.001. [PubMed]Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song JJ, Hammond SM, Joshua-Tor L, Hannon GJ. Argonaute2 is the catalytic engine of mammalian RNAi. Science. 2004;305:1437–1441. doi: 10.1126/science.1102513. [PubMed]Eulalio A, Huntzinger E, Izaurralde E. GW182 interaction with Argonaute is essential for miRNA-mediated translational repression and mRNA decay. Nat Struct Mol Biol. 2008;15:346–353. doi: 10.1038/nsmb.1405. [PubMed]Landthaler M, Gaidatzis D, Rothballer A, Chen PY, Soll SJ, Dinic L, Ojo T, Hafner M, Zavolan M, Tuschl T. Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs. Rna. 2008;14:2580–96. doi: 10.1261/rna.1351608. [PubMed]Rehwinkel J, Behm-Ansmant I, Gatfield D, Izaurralde E. A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing. Rna. 2005;11:1640–1647. doi: 10.1261/rna.2191905. [PubMed]Behm-Ansmant I, Rehwinkel J, Doerks T, Stark A, Bork P, Izaurralde E. mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. Genes Dev. 2006;20:1885–1898. doi: 10.1101/gad.1424106. [PubMed]Pillai RS, Bhattacharyya SN, Artus CG, Zoller T, Cougot N, Basyuk E, Bertrand E, Filipowicz W. Inhibition of translational initiation by Let-7 MicroRNA in human cells. Science. 2005;309:1573–1576. doi: 10.1126/science.1115079. [PubMed]Bhattacharyya SN, Habermacher R, Martine U, Closs EI, Filipowicz W. Relief of microRNA-mediated translational repression in human cells subjected to stress. Cell. 2006;125:1111–1124. doi: 10.1016/j.cell.2006.04.031. [PubMed]Jakymiw A, Lian S, Eystathioy T, Li S, Satoh M, Hamel JC, Fritzler MJ, Chan EK. Disruption of GW bodies impairs mammalian RNA interference. Nat Cell Biol. 2005;7:1267–1274. doi: 10.1038/ncb1334. [PubMed]Liu J, Rivas FV, Wohlschlegel J, Yates JR, 3rd, Parker R, Hannon GJ. A role for the P-body component GW182 in microRNA function. Nat Cell Biol. 2005;7:1261–1266. [PubMed]Ding SW, Voinnet O. Antiviral immunity directed by small RNAs. Cell. 2007;130:413–426. doi: 10.1016/j.cell.2007.07.039. [PubMed]Gottwein E, Cullen BR. Viral and cellular microRNAs as determinants of viral pathogenesis and immunity. Cell Host Microbe. 2008;3:375–387. doi: 10.1016/j.chom.2008.05.002. [PubMed]Yeung ML, Benkirane M, Jeang KT. Small non-coding RNAs, mammalian cells, and viruses: regulatory interactions? Retrovirology. 2007;4:74. doi: 10.1186/1742-4690-4-74. [PubMed]Han Y, Siliciano RF. Keeping quiet: microRNAs in HIV-1 latency. Nat Med. 2007;13:1138–1140. doi: 10.1038/nm1007-1138. [PubMed]Weinberg MS, Morris KV. Are viral-encoded microRNAs mediating latent HIV-1 infection? DNA Cell Biol. 2006;25:223–231. doi: 10.1089/dna.2006.25.223. [PubMed]Triboulet R, Mari B, Lin YL, Chable-Bessia C, Bennasser Y, Lebrigand K, Cardinaud B, Maurin T, Barbry P, Baillat V, Reynes J, Corbeau P, Jeang KT, Benkirane M. Suppression of microRNA-silencing pathway by HIV-1 during virus replication. Science. 2007;315:1579–1582. doi: 10.1126/science.1136319. [PubMed]Huang J, Wang F, Argyris E, Chen K, Liang Z, Tian H, Huang W, Squires K, Verlinghieri G, Zhang H. Cellular microRNAs contribute to HIV-1 latency in resting primary CD4+ T lymphocytes. Nat Med. 2007;13:1241–1247. doi: 10.1038/nm1639. [PubMed]Ahluwalia JK, Khan SZ, Soni K, Rawat P, Gupta A, Hariharan M, Scaria V, Lalwani M, Pillai B, Mitra D, Brahmachari SK. Human cellular microRNA hsa-miR-29a interferes with viral nef protein expression and HIV-1 replication. Retrovirology. 2008;5:117. doi: 10.1186/1742-4690-5-117. [PubMed]Beckham CJ, Parker R. P bodies, stress granules, and viral life cycles. Cell Host Microbe. 2008;3:206–212. doi: 10.1016/j.chom.2008.03.004. [PubMed]Griffith JL, Coleman LE, Raymond AS, Goodson SG, Pittard WS, Tsui C, Devine SE. Functional genomics reveals relationships between the retrovirus-like Ty1 element and its host Saccharomyces cerevisiae. Genetics. 2003;164:867–879. [PubMed]Devine SE, Boeke JD. Integration of the yeast retrotransposon Ty1 is targeted to regions upstream of genes transcribed by RNA polymerase III. Genes Dev. 1996;10:620–633. doi: 10.1101/gad.10.5.620. [PubMed]Beliakova-Bethell N, Beckham C, Giddings TH, Jr, Winey M, Parker R, Sandmeyer S. Virus-like particles of the Ty3 retrotransposon assemble in association with P-body components. Rna. 2006;12:94–101. doi: 10.1261/rna.2264806. [PubMed]Beckham CJ, Light HR, Nissan TA, Ahlquist P, Parker R, Noueiry A. Interactions between brome mosaic virus RNAs and cytoplasmic processing bodies. J Virol. 2007;81:9759–9768. doi: 10.1128/JVI.00844-07. [PubMed]Gallois-Montbrun S, Kramer B, Swanson CM, Byers H, Lynham S, Ward M, Malim MH. Antiviral protein APOBEC3G localizes to ribonucleoprotein complexes found in P bodies and stress granules. J Virol. 2007;81:2165–2178. doi: 10.1128/JVI.02287-06. [PubMed]Wichroski MJ, Robb GB, Rana TM. Human retroviral host restriction factors APOBEC3G and APOBEC3F localize to mRNA processing bodies. PLoS Pathog. 2006;2:e41. doi: 10.1371/journal.ppat.0020041. [PubMed]Chu CY, Rana TM. Translation repression in human cells by microRNA-induced gene silencing requires RCK/p54. PLoS Biol. 2006;4:e210. doi: 10.1371/journal.pbio.0040210. [PubMed]Goff SP. Host factors exploited by retroviruses. Nat Rev Microbiol. 2007;5:253–263. doi: 10.1038/nrmicro1541. [PubMed]Brass AL, Dykxhoorn DM, Benita Y, Yan N, Engelman A, Xavier RJ, Lieberman J, Elledge SJ. Identification of host proteins required for HIV infection through a functional genomic screen. Science. 2008;319:921–926. doi: 10.1126/science.1152725. [PubMed]König R, Zhou Y, Elleder D, Diamond TL, Bonamy GM, Irelan JT, Chiang CY, Tu BP, De Jesus PD, Lilley CE, Seidel S, Opaluch AM, Caldwell JS, Weitzman MD, Kuhen KL, Bandyopadhyay S, Ideker T, Orth AP, Miraglia LJ, Bushman FD, Young JA, Chanda SK. Global analysis of host-pathogen interactions that regulate early-stage HIV-1 replication. Cell. 2008;135:49–60. doi: 10.1016/j.cell.2008.07.032. [PubMed]Lama J, Planelles V. Host factors influencing susceptibility to HIV infection and AIDS progression. Retrovirology. 2007;4:52. doi: 10.1186/1742-4690-4-52. [PubMed]Chun TW, Engel D, Berrey MM, Shea T, Corey L, Fauci AS. Early establishment of a pool of latently infected, resting CD4(+) T cells during primary HIV-1 infection. Proc Natl Acad Sci USA. 1998;95:8869–8873. doi: 10.1073/pnas.95.15.8869. [PubMed]Finzi D, Hermankova M, Pierson T, Carruth LM, Buck C, Chaisson RE, Quinn TC, Chadwick K, Margolick J, Brookmeyer R, Gallant J, Markowitz M, Ho DD, Richman DD, Siliciano RF. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science. 1997;278:1295–1300. doi: 10.1126/science.278.5341.1295. [PubMed]Marcello A. Latency: the hidden HIV-1 challenge. Retrovirology. 2006;3:7. doi: 10.1186/1742-4690-3-7. [PubMed]Lassen K, Han Y, Zhou Y, Siliciano J, Siliciano RF. The multifactorial nature of HIV-1 latency. Trends Mol Med. 2004;10:525–531. doi: 10.1016/j.molmed.2004.09.006. [PubMed]Jordan A, Bisgrove D, Verdin E. HIV reproducibly establishes a latent infection after acute infection of T cells in vitro. Embo J. 2003;22:1868–1877. doi: 10.1093/emboj/cdg188. [PubMed]Han Y, Lin YB, An W, Xu J, Yang HC, O'Connell K, Dordai D, Boeke JD, Siliciano JD, Siliciano RF. Orientation-dependent regulation of integrated HIV-1 expression by host gene transcriptional readthrough. Cell Host Microbe. 2008;4:134–146. doi: 10.1016/j.chom.2008.06.008. [PubMed]De Marco A, Biancotto C, Knezevich A, Maiuri P, Vardabasso C, Marcello A. Intragenic transcriptional cis-activation of the human immunodeficiency virus 1 does not result in allele-specific inhibition of the endogenous gene. Retrovirology. 2008;5:98. doi: 10.1186/1742-4690-5-98. [PubMed]Weinberger LS, Burnett JC, Toettcher JE, Arkin AP, Schaffer DV. Stochastic gene expression in a lentiviral positive-feedback loop: HIV-1 Tat fluctuations drive phenotypic diversity. Cell. 2005;122:169–182. doi: 10.1016/j.cell.2005.06.006. [PubMed]Williams SA, Chen LF, Kwon H, Ruiz-Jarabo CM, Verdin E, Greene WC. NF-kappaB p50 promotes HIV latency through HDAC recruitment and repression of transcriptional initiation. Embo J. 2006;25:139–149. doi: 10.1038/sj.emboj.7600900. [PubMed]Kim YK, Bourgeois CF, Pearson R, Tyagi M, West MJ, Wong J, Wu SY, Chiang CM, Karn J. Recruitment of TFIIH to the HIV LTR is a rate-limiting step in the emergence of HIV from latency. Embo J. 2006;25:3596–3604. doi: 10.1038/sj.emboj.7601248. [PubMed]Tyagi M, Karn J. CBF-1 promotes transcriptional silencing during the establishment of HIV-1 latency. Embo J. 2007;26:4985–4995. doi: 10.1038/sj.emboj.7601928. [PubMed]Treand C, du Chene I, Bres V, Kiernan R, Benarous R, Benkirane M, Emiliani S. Requirement for SWI/SNF chromatin-remodeling complex in Tat-mediated activation of the HIV-1 promoter. Embo J. 2006;25:1690–1699. doi: 10.1038/sj.emboj.7601074. [PubMed]du Chéné I, Basyuk E, Lin YL, Triboulet R, Knezevich A, Chable-Bessia C, Mettling C, Baillat V, Reynes J, Corbeau P, Bertrand E, Marcello A, Emiliani S, Kiernan R, Benkirane M. Suv39H1 and HP1gamma are responsible for chromatin-mediated HIV-1 transcriptional silencing and post-integration latency. Embo J. 2007;26:424–435. doi: 10.1038/sj.emboj.7601517. [PubMed]Marban C, Suzanne S, Dequiedt F, de Walque S, Redel L, Van Lint C, Aunis D, Rohr O. Recruitment of chromatin-modifying enzymes by CTIP2 promotes HIV-1 transcriptional silencing. Embo J. 2007;26:412–423. doi: 10.1038/sj.emboj.7601516. [PubMed]Pearson R, Kim YK, Hokello J, Lassen K, Friedman J, Tyagi M, Karn J. Epigenetic silencing of human immunodeficiency virus (HIV) transcription by formation of restrictive chromatin structures at the viral long terminal repeat drives the progressive entry of HIV into latency. J Virol. 2008;82:12291–12303. doi: 10.1128/JVI.01383-08. [PubMed]Sadowski I, Lourenco P, Malcolm T. Factors controlling chromatin organization and nucleosome positioning for establishment and maintenance of HIV latency. Curr HIV Res. 2008;6:286–295. doi: 10.2174/157016208785132563. [PubMed]Pomerantz RJ, Seshamma T, Trono D. Efficient replication of human immunodeficiency virus type 1 requires a threshold level of Rev: potential implications for latency. J Virol. 1992;66:1809–1813. [PubMed]Hermankova M, Siliciano JD, Zhou Y, Monie D, Chadwick K, Margolick JB, Quinn TC, Siliciano RF. Analysis of human immunodeficiency virus type 1 gene expression in latently infected resting CD4+ T lymphocytes in vivo. J Virol. 2003;77:7383–7392. doi: 10.1128/JVI.77.13.7383-7392.2003. [PubMed]Ciuffi A, Bleiber G, Muñoz M, Martinez R, Loeuillet C, Rehr M, Fischer M, Günthard HF, Oxenius A, Meylan P, Bonhoeffer S, Trono D, Telenti A. Entry and transcription as key determinants of differences in CD4 T-cell permissiveness to human immunodeficiency virus type 1 infection. J Virol. 2004;78:10747–10754. doi: 10.1128/JVI.78.19.10747-10754.2004. [PubMed]Qian S, Zhong X, Yu L, Ding B, de Haan P, Boris-Lawrie K. HIV-1 Tat RNA silencing suppressor activity is conserved across kingdoms and counteracts translational repression of HIV-1. Proc Natl Acad Sci USA. 2009;106:605–610. doi: 10.1073/pnas.0806822106. [PubMed]Bennasser Y, Le SY, Benkirane M, Jeang KT. Evidence that HIV-1 encodes an siRNA and a suppressor of RNA silencing. Immunity. 2005;22:607–619. doi: 10.1016/j.immuni.2005.03.010. [PubMed]Haasnoot J, de Vries W, Geutjes EJ, Prins M, de Haan P, Berkhout B. The Ebola virus VP35 protein is a suppressor of RNA silencing. PLoS Pathog. 2007;3:e86. doi: 10.1371/journal.ppat.0030086. [PubMed]Schnettler E, de Vries W, Hemmes H, Haasnoot J, Kormelink R, Goldbach R, Berkhout B. The NS3 protein of rice hoja blanca virus complements the RNAi suppressor function of HIV-1 Tat. EMBO Rep. 2009;10:258–63. doi: 10.1038/embor.2009.6. [PubMed]Alexaki A, Liu Y, Wigdahl B. Cellular reservoirs of HIV-1 and their role in viral persistence. Curr HIV Res. 2008;6:388–400. doi: 10.2174/157016208785861195. [PubMed]Palliser D, Chowdhury D, Wang QY, Lee SJ, Bronson RT, Knipe DM, Lieberman J. An siRNA-based microbicide protects mice from lethal herpes simplex virus 2 infection. Nature. 2006;439:89–94. doi: 10.1038/nature04263. [PubMed]Krutzfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M, Stoffel M. Silencing of microRNAs in vivo with 'antagomirs'. Nature. 2005;438:685–689. doi: 10.1038/nature04303. [PubMed]Zimmermann TS, Lee AC, Akinc A, Bramlage B, Bumcrot D, Fedoruk MN, Harborth J, Heyes JA, Jeffs LB, John M, Judge AD, Lam K, McClintock K, Nechev LV, Palmer LR, Racie T, Röhl I, Seiffert S, Shanmugam S, Sood V, Soutschek J, Toudjarska I, Wheat AJ, Yaworski E, Zedalis W, Koteliansky V, Manoharan M, Vornlocher HP, MacLachlan I. RNAi-mediated gene silencing in non-human primates. Nature. 2006;441:111–114. doi: 10.1038/nature04688. [PubMed]Newman EN, Holmes RK, Craig HM, Klein KC, Lingappa JR, Malim MH, Sheehy AM. Antiviral function of APOBEC3G can be dissociated from cytidine deaminase activity. Curr Biol. 2005;15:166–170. doi: 10.1016/j.cub.2004.12.068. [PubMed]Molle D, Maiuri P, Boireau S, Bertrand E, Knezevich A, Marcello A, Basyuk E. A real-time view of the TAR:Tat:P-TEFb complex at HIV-1 transcription sites. Retrovirology. 2007;4:36. doi: 10.1186/1742-4690-4-36. [PubMed]Boireau S, Maiuri P, Basyuk E, de la Mata M, Knezevich A, Pradet-Balade B, Backer V, Kornblihtt A, Marcello A, Bertrand E. The transcriptional cycle of HIV-1 in real-time and live cells. J Cell Biol. 2007;179:291–304. doi: 10.1083/jcb.200706018. [PubMed]Jacquenet S, Mereau A, Bilodeau PS, Damier L, Stoltzfus CM, Branlant C. A second exon splicing silencer within human immunodeficiency virus type 1 tat exon 2 represses splicing of Tat mRNA and binds protein hnRNP H. J Biol Chem. 2001;276:40464–40475. doi: 10.1074/jbc.M104070200. [PubMed]
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