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User name	Arndt Benecke
Email	arndt@ihes.fr
Phone / Fax number	+33160926665 / +33160926609
Location	Institut des Hautes Etudes Scientifiques (Integrative Biology) - 35, route de Chartres - 91440 Bures sur Yvette, France

Scientific description

The TAR structure of the nascent HIV-1 transcript is critically involved in the recruitment of inactive P-TEFb to the promoter proximal paused RNA Polymerase II. The viral transactivator Tat is responsible for subsequent P-TEFb activation in order to start efficient viral transcription elongation. In the absence of Tat, e.g. during latency or in early stages of HIV transcription, TAR mediates an interaction of P-TEFb with its inhibitor HEXIM1, keeping P-TEFb in its inactive form. In this study, we address the function of HIV-1 TAR in the absence of Tat by analyzing consequences of a HIV-1 TAR overexpression on host cellular gene expression. Therefore, we designed an RNA chimera of Epstein Barr viral EBER2 RNA and HIV-1 TAR, assuring a robust overexpression of TAR. In HEK293 cells such an overexpression results in the differential expression of more than 800 human genes of which a significant proportion is involved in the suppression of cellular immune responses, among these a significant set of 7SK-responsive P-TEFb target genes. Our findings identify a novel role for HIV-1 TAR in the absence of Tat consisting in the interference with host cellular immune responses by targeting 7SK RNA-mediated gene expression and P-TEFb inactivation.

Technical description

For microarray analyses, RNA amplification, labeling, hybridization and detection were performed following the protocols supplied by Applied Biosystems using the corresponding kits (Applied Biosystems, ProdNo: 4339628 and 4336875). The data obtained were analyzed as described previously [18, 31]. Briefly, the raw data were quality controlled [43], NeoNORM normalized using k=0.2 [44], and then analyzed as outlined in [45, 46, 47]. For each condition, three biological replicates were merged and compared to the merged signals of three biological replicates of the corresponding control condition. Canonical pathway enrichment studies were performed using Ingenuity Pathway Analysis® software (Ingenuity® Systems, Qiagen) were performed as recommended by the manufacturer.

References:

[18] Eilebrecht, S., Brysbaert, G., Wegert, T., Urlaub, H., Benecke, B.J. & Benecke, A. (2011) 7SK small nuclear RNA directly affects HMGA1 function in transcription regulation. Nucleic Acids Res 39: 2057-2072.
[31] Howe, J.G. & Shu, M.D. (1989) Epstein-Barr virus small RNA (EBER) genes: unique transcription units that combine RNA polymerase II and III promoter elements. Cell 57: 825-834.
[43] Brysbaert, G., Pellay, F.X., Noth, S. & Benecke, A. (2010) Quality assessment of transcriptome data using intrinsic statistical properties. Genomics Proteomics Bioinformatics 8: 57-71.
[44] Noth, S., Brysbaert, G. & Benecke, A. (2006) Normalization using weighted negative second order exponential error functions (NeONORM) provides robustness against asymmetries in comparative transcriptome profiles and avoids false calls. Genomics Proteomics Bioinformatics 4: 90-109.
[45] Noth, S. & Benecke, A. (2005) Avoiding inconsistencies over time and tracking difficulties in Applied Biosystems AB1700/Panther probe-to-gene annotations. BMC Bioinformatics 6: 307.
[46] Noth, S., Brysbaert, G., Pellay, F.X. & Benecke, A. (2006) High-sensitivity transcriptome data structure and implications for analysis and biologic interpretation. Genomics Proteomics Bioinformatics 4: 212-229.
[47] Benecke, A. (2003) Genomic plasticity and information processing by transcription coregulators. ComPlexUs 1: 65-76.

Reference

Sebastian Eilebrecht*, Bernd-Joachim Benecke, Arndt G. Benecke* (2017) Latent HIV-1 TAR targets 7SK-RNA-mediated immune responses in the absence of Tat.