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microRNA machinery is an integral component of drug-induced transcription inhibition in HIV-1 infection

Lawrence Carpio1, Zachary Klase2, William Coley3, Irene Guendel1, Sarah Choi3, Rachel Van Duyne1, Aarthi Narayanan1, Kylene Kehn-Hall1, Laurent Meijer4, Fatah Kashanchi1,*
1National Center for Biodefense and Infectious Disease, 10900 University Blvd MS 1H8, George Mason University,
Manassas, VA 20110, USA, 2Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda,
MD 20892, USA, 3The Department of Microbiology, Immunology and Tropical Medicine, The George Washington
University School of Medicine, Washington, DC 20037, USA, 4Station Biologique de Roscoff, CNRS, France
J RNAi Gene Silencing, 2010, 6(1), 386-400
© Copyright The Authors: This is an open access article, published under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/). This license permits noncommercial use, distribution and reproduction of the article, provided the original work is appropriately acknowledged with correct citation details.
The authorship of this article should be read as follows:
Lawrence Carpio1, Zachary Klase2, William Coley3, Irene Guendel1, Sarah Choi3, Rachel Van Duyne1, Aarthi Narayanan1, Kylene Kehn-Hall1, Hervé Galons5, Nassima Oumata5, Benoît Joseph6, Laurent Meijer4, Fatah Kashanchi1,*
5Laboratoire de Chimie Organique 2, CNRS UMR8601, INSERM U 648, Université Paris - Descartes, 4 avenue de l'Observatoire, 75270 Paris cedex 06, France, 6Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Université Claude Bernard - Lyon 1, Bâtiment Curien, 43 Boulevard du 11 Novembre 1918, F-69622 Villeurbanne, France
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Abstract

RNA interference plays a significant role in manipulating cellular and viral mechanisms to maintain latency during HIV-1 infection. HIV-1 produces several microRNAs including one from the TAR element which alter the host’s response to infection. Since cyclin/cdk complexes are important for viral transcription, these studies focus on the possible cdk inhibitors that inhibit viral transcription, without affecting normal cellular mechanisms. Roscovitine and Flavopiridol are well-studied cdk inhibitors that are effective at suppressing their target cdks at a low IC50. These cdk inhibitors and possibly future generations of drugs are affected by microRNA mechanisms. From our studies, we developed a third generation derivative called CR8#13. In cells that lack Dicer there was a higher level of basal viral LTR-reporter transcription. When drugs, specifically Flavopiridol and CR8#13 were added, the transcriptional inhibition of the LTR was less potent in cells that lacked Dicer. Also, after transfection with HIV-1 clone (pNL4.3), CR8 and CR8#13 derivatives were shown to be more effective viral transcription inhibitors in cell lines that contained Dicer (T-cells) as compared to Dicer deficient lines (monocytes). We next asked whether the addition of CR8 or CR8#13 could possibly increase levels of TAR microRNA in HIV-1 LTR containing cells. We demonstrate that the 3’TAR microRNA is produced in higher amounts after drug treatment, resulting in microRNA recruitment to the LTR. MicroRNA recruitment results in chromatin alteration, changes in Pol II phosphorylation and viral transcription inhibition. In conclusion, our results indicate that viral microRNA, specifically the TAR microRNA produced from the HIV-1 LTR is responsible for maintaining latent infections by manipulating host cell mechanisms to limit transcription from the viral LTR promoter. With the microRNA machinery present, cdk inhibitors are able to significantly increase the amount of TAR microRNA, leading to downregulation of viral LTR transcription.

The following text was omitted from acknowledgements during processing:
This research was also funded by the "Conseil Régional de Bretagne", ‘Fonds de Maturation’, « Développement de
molécules inhibitrices de kinases à activité anti-SIDA. Optimisation du mode d’administration par voie orale/intrapéritonéale
et validation sur modèle animal » (LM).
Erratum published online: 12 July 2010