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


Research Article

J RNAi Gene Silencing (2010), 6(1), 386-400

doi: jrgsxx

Published online: 29 May 2010

Full Text: (html | pdf ~782kb | refs) Erratum (PDF ~ 62kb) published online: 12 July 2010

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

Lawrence Carpio †, Zachary Klase ‡, William Coley ¥, Irene Guendel †, Sarah Choi ¥, Rachel Van Duyne †, Aarthi Narayanan †, Kylene Kehn-Hall †, Laurent Meijer §, Fatah Kashanchi †,*

† National Center for Biodefense and Infectious Disease, 10900 University Blvd MS 1H8, George Mason University, Manassas, VA 20110, USA

‡ Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, MD 20892, USA

¥ The Department of Microbiology, Immunology and Tropical Medicine, The George Washington University School of Medicine, Washington, DC 20037, USA

§ Station Biologique de Roscoff, CNRS, France

*Correspondence toFatah Kashanchi, Email: fkashanc@gmu.edu, Tel: + 703 993 9160, Fax: + 703 993 7022

Received: 20 March 2010, Revised: 21 May 2010, Accepted: 24 May 2010

© Copyright The Authors

—————————————————————————————————————–

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.

KEYWORDS: microRNA, HIV-1, TAR, cdk inhibitor, ATP analogs, Tat transactivation

—————————————————————————————————————–

Leave a Reply

*

captcha *