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Journal of Virology, May 2007, p. 5121-5131, Vol. 81, No. 10
0022-538X/07/$08.00+0     doi:10.1128/JVI.01511-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Small Interfering RNAs against the TAR RNA Binding Protein, TRBP, a Dicer Cofactor, Inhibit Human Immunodeficiency Virus Type 1 Long Terminal Repeat Expression and Viral Production

Helen S. Christensen,¹ Aïcha Daher,² Kaitlin J. Soye,^2,3 Lisa B. Frankel,^2,3, Marina R. Alexander,¹ Sébastien Lainé,^2,3 Sylvie Bannwarth,^2,3, Chi L. Ong,¹ Sean W. L. Chung,¹ Shahan M. Campbell,¹ Damian F. J. Purcell,^1,* and Anne Gatignol^2,3,4,*

Department of Microbiology and Immunology, University of Melbourne, Parkville, Australia,¹ Virus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research,² Departments of Microbiology and Immunology,³ Experimental Medicine, McGill University, Montréal, Québec, Canada⁴

Received 14 July 2006/ Accepted 25 February 2007

RNA interference (RNAi) is now widely used for gene silencing in mammalian cells. The mechanism uses the RNA-induced silencing complex, in which Dicer, Ago2, and the human immunodeficiency virus type 1 (HIV-1) TAR RNA binding protein (TRBP) are the main components. TRBP is a protein that increases HIV-1 expression and replication by inhibition of the interferon-induced protein kinase PKR and by increasing translation of viral mRNA. After HIV infection, TRBP could restrict the viral RNA through its activity in RNAi or could contribute more to the enhancement of viral replication. To determine which function will be predominant in the virological context, we analyzed whether the inhibition of its expression could enhance or decrease HIV replication. We have generated small interfering RNAs (siRNAs) against TRBP and found that they decrease HIV-1 long terminal repeat (LTR) basal expression 2-fold, and the LTR Tat transactivated level up to 10-fold. In the context of HIV replication, siRNAs against TRBP decrease the expression of viral genes and inhibit viral production up to fivefold. The moderate increase in PKR expression and activation indicates that it contributes partially to viral gene inhibition. The moderate decrease in micro-RNA (miRNA) biogenesis by TRBP siRNAs suggests that in the context of HIV replication, TRBP functions other than RNAi are predominant. In addition, siRNAs against Dicer decrease viral production twofold and impede miRNA biogenesis. These results suggest that, in the context of HIV replication, TRBP contributes mainly to the enhancement of virus production and that Dicer does not mediate HIV restriction by RNAi.

Published ahead of print on 14 March 2007.

Present address: Biotech Research and Innovation Center, University of Copenhagen, Copenhagen, Denmark.

Present address: Laboratoire de Génétique Moléculaire, Hôpital de l'Archet 2, Nice, France.

The laboratories of A.G. and D.F.J.P. contributed equally to this work.