Identification of functional microRNAs released through asymmetrical processing of HIV-1 TAR element†

The interaction between human immunodeficiency virus type 1 (HIV-1) and RNA silencing pathways is complex and multifaceted. Essential for efficient viral transcription and supporting Tat-mediated transactivation of viral gene expression, the trans-activation responsive (TAR) element is a structured RNA located at the 5′ end of all transcripts derived from HIV-1. Here, we report that this element is a source of microRNAs (miRNAs) in cultured HIV-1-infected cell lines and in HIV-1-infected human CD4+ T lymphocytes. Using primer extension and ribonuclease (RNase) protection assays, we delineated both strands of the TAR miRNA duplex deriving from a model HIV-1 transcript, namely miR-TAR-5p and miR-TAR-3p. In vitro RNase assays indicate that the lack of a free 3′ extremity at the base of TAR may contribute to its low processing reactivity in vivo. Both miR-TAR-5p and miR-TAR-3p down-regulated TAR miRNA sensor activity in a process that required an integral miRNA-guided RNA silencing machinery. miR-TAR-3p exerted superior gene downregulatory effects, probably due to its preferential release from HIV-1 TAR RNA by the RNase III Dicer. Our study suggests that the TAR element of HIV-1 transcripts releases functionally competent miRNAs upon asymmetrical processing by Dicer, thereby providing novel insights into viral miRNA biogenesis.

[1]  F. Kashanchi,et al.  Direct interaction of human TFIID with the HIV-1 transactivator Tat , 1994, Nature.

[2]  Byoung-Tak Zhang,et al.  Molecular Basis for the Recognition of Primary microRNAs by the Drosha-DGCR8 Complex , 2006, Cell.

[3]  J. Burns,et al.  Generation of high-titer pseudotyped retroviral vectors with very broad host range. , 1994, Methods in cell biology.

[4]  A. Reynolds,et al.  The contributions of dsRNA structure to Dicer specificity and efficiency. , 2005, RNA.

[5]  P. Zamore,et al.  A Protein Sensor for siRNA Asymmetry , 2004, Science.

[6]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[7]  B. Samuelsson,et al.  Ribonuclease activity and RNA binding of recombinant human Dicer , 2002, The EMBO journal.

[8]  S. Le,et al.  Evidence that HIV-1 encodes an siRNA and a suppressor of RNA silencing. , 2005, Immunity.

[9]  Xiaodong Wang,et al.  R2D2, a Bridge Between the Initiation and Effector Steps of the Drosophila RNAi Pathway , 2003, Science.

[10]  T. A. Hagan,et al.  Detection , Isolation , and Continuous Production of Cytopathic Retroviruses ( HTLV-III ) from Patients with AIDS and Pre-AIDS , 2022 .

[11]  Kuniaki Saito,et al.  Processing of Pre-microRNAs by the Dicer-1–Loquacious Complex in Drosophila Cells , 2005, PLoS biology.

[12]  B. Haynes,et al.  Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. , 1984, Science.

[13]  R. Berro,et al.  HIV-1 TAR element is processed by Dicer to yield a viral micro-RNA involved in chromatin remodeling of the viral LTR , 2007, BMC Molecular Biology.

[14]  Eric Westhof,et al.  Single Processing Center Models for Human Dicer and Bacterial RNase III , 2004, Cell.

[15]  Mihaela Zavolan,et al.  Effects of Dicer and Argonaute down-regulation on mRNA levels in human HEK293 cells , 2006, Nucleic acids research.

[16]  S. B. Atienza-Samols,et al.  With Contributions by , 1978 .

[17]  M. Zacharias,et al.  The bend in RNA created by the trans-activation response element bulge of human immunodeficiency virus is straightened by arginine and by Tat-derived peptide. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Lin He,et al.  MicroRNAs: small RNAs with a big role in gene regulation , 2004, Nature reviews genetics.

[19]  K. Jeang,et al.  HIV-1 TAR RNA Subverts RNA Interference in Transfected Cells through Sequestration of TAR RNA-binding Protein, TRBP* , 2006, Journal of Biological Chemistry.

[20]  Blossom Damania,et al.  Kaposi's sarcoma-associated herpesvirus expresses an array of viral microRNAs in latently infected cells. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[21]  F. Holstege,et al.  Specific inhibition of gene expression using a stably integrated, inducible small‐interfering‐RNA vector , 2003, EMBO reports.

[22]  N. Hernandez,et al.  Characterization of the inducer of short transcripts, a human immunodeficiency virus type 1 transcriptional element that activates the synthesis of short RNAs , 1993, Molecular and cellular biology.

[23]  M. Carmell,et al.  Posttranscriptional Gene Silencing in Plants , 2006 .

[24]  Anton J. Enright,et al.  Identification of Virus-Encoded MicroRNAs , 2004, Science.

[25]  James M. Pipas,et al.  SV40-encoded microRNAs regulate viral gene expression and reduce susceptibility to cytotoxic T cells , 2005, Nature.

[26]  L Leoni,et al.  Temporal gene regulation during HIV-1 infection of human CD4+ T cells. , 2001, Genome research.

[27]  I. Plante,et al.  Hypothesis: A Role for Fragile X Mental Retardation Protein in Mediating and Relieving MicroRNA-Guided Translational Repression? , 2006, Journal of biomedicine & biotechnology.

[28]  D. Patel,et al.  Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain , 2004, Nature.

[29]  H. Gendelman,et al.  Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone , 1986, Journal of virology.

[30]  W. Filipowicz,et al.  Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP , 2002, The EMBO journal.

[31]  J. Krol,et al.  Ribonuclease dicer cleaves triplet repeat hairpins into shorter repeats that silence specific targets. , 2007, Molecular cell.

[32]  C. Sander,et al.  Identification of microRNAs of the herpesvirus family , 2005, Nature Methods.

[33]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[34]  A. Denli,et al.  Normal microRNA Maturation and Germ-Line Stem Cell Maintenance Requires Loquacious, a Double-Stranded RNA-Binding Domain Protein , 2005, PLoS biology.

[35]  É. Khandjian,et al.  Dicer-Derived MicroRNAs Are Utilized by the Fragile X Mental Retardation Protein for Assembly on Target RNAs , 2006, Journal of biomedicine & biotechnology.

[36]  M. Singh,et al.  HIV‐1 tat protein stimulates transcription by binding to a U‐rich bulge in the stem of the TAR RNA structure. , 1990, The EMBO journal.

[37]  H. Ebhardt,et al.  Extensive 3' modification of plant small RNAs is modulated by helper component-proteinase expression. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[38]  I. MacRae,et al.  Ribonuclease revisited: structural insights into ribonuclease III family enzymes. , 2007, Current opinion in structural biology.

[39]  K. Jeang,et al.  Tat as a transcriptional activator and a potential therapeutic target for HIV-1. , 2000, Advances in pharmacology.

[40]  Sherif Abou Elela,et al.  Evaluation of the RNA Determinants for Bacterial and Yeast RNase III Binding and Cleavage* , 2004, Journal of Biological Chemistry.

[41]  P. Provost,et al.  MicroRNAs in Gene Regulation: When the Smallest Governs It All , 2006, Journal of biomedicine & biotechnology.

[42]  Bryan R. Cullen,et al.  Analysis of the Interaction of Primate Retroviruses with the Human RNA Interference Machinery , 2007, Journal of Virology.

[43]  T. Rana,et al.  Dynamics of nascent mRNA folding and RNA–protein interactions: an alternative TAR RNA structure is involved in the control of HIV-1 mRNA transcription , 2006, Nucleic acids research.

[44]  Eugene Berezikov,et al.  Cloning and expression of new microRNAs from zebrafish , 2006, Nucleic acids research.

[45]  Anne Gatignol,et al.  TRBP, a regulator of cellular PKR and HIV‐1 virus expression, interacts with Dicer and functions in RNA silencing , 2005, EMBO reports.

[46]  Yutaka Tsutsumi,et al.  HIV-1 nef suppression by virally encoded microRNA , 2004, Retrovirology.

[47]  D. Halligan,et al.  Natural Selection Drives Extremely Rapid Evolution in Antiviral RNAi Genes , 2006, Current Biology.

[48]  J. Pipas,et al.  SV 40-encoded microRNAs regulate viral gene expression and reduce susceptibility to cytotoxic T cells , 2005 .

[49]  W. Filipowicz,et al.  Characterization of the interactions between mammalian PAZ PIWI domain proteins and Dicer , 2004, EMBO reports.

[50]  C. Croce,et al.  Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[51]  R. Shiekhattar,et al.  Human RISC Couples MicroRNA Biogenesis and Posttranscriptional Gene Silencing , 2005, Cell.

[52]  D. Frendewey,et al.  Substrate structure requirements of the Pac1 ribonuclease from Schizosaccharmyces pombe. , 1997, RNA.

[53]  M. Tremblay,et al.  Presence of Host ICAM-1 in Laboratory and Clinical Strains of Human Immunodeficiency Virus Type 1 Increases Virus Infectivity and CD4+-T-Cell Depletion in Human Lymphoid Tissue, a Major Site of Replication In Vivo , 2002, Journal of Virology.

[54]  Pascal Barbry,et al.  Suppression of MicroRNA-Silencing Pathway by HIV-1 During Virus Replication , 2007, Science.

[55]  R. Shiekhattar,et al.  TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing , 2005, Nature.

[56]  B. Reinhart,et al.  MicroRNAs in plants. , 2002, Genes & development.

[57]  V. Ambros.  The functions of animal microRNAs , 2004, Nature.

[58]  M. Tremblay,et al.  HIV-l and the microRNA-guided silencing pathway: an intricate and multifaceted encounter. , 2006, Virus research.