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Suppression of chemokine receptor expression by RNA interference allows for inhibition of HIV-1 replication

Martínez, Miguel Aa; Gutiérrez, Arantxaa; Armand-Ugón, Mercedesa; Blanco, Juliàa; Parera, Marionaa; Gómez, Jordib; Clotet, Bonaventuraa; Esté, José Aa

Basic Science

Objectives: Duplexes of 21 base pair RNA, known as short-interfering RNA (siRNA), have been shown to inhibit gene expression by a sequence-specific RNA degradation mechanism termed RNA interference (RNAi). The objective of our study was to evaluate the effect of chemokine receptor gene suppression by RNAi on the entry and replication of HIV-1.

Methods: A flow cytometry and microscopy evaluation of HIV co-receptor expression of cells transfected with siRNA. An evaluation of the effect of siRNA on HIV entry and replication by intracellular p24 antigen detection, and virus production by infected cells, respectively.

Results: siRNA that target CXCR4 and CCR5 could effectively impede cell surface protein expression and their consequent function as HIV co-receptors. The inhibitory effect of RNAi directed to CXCR4 was detected 48 h after transfection of CXCR4+ U87-CD4+ cells. The expression of CXCR4 and CCR5 was blocked in 63 and 48% of positive cells by the corresponding siRNA. However, siRNA directed to CXCR4 or CCR5 did not have an effect on CD4 cells or green fluorescence protein expression. siRNA directed to CXCR4 did not suppress CCR5 expression or vice versa. The suppression of HIV-1 co-receptor expression effectively blocked the acute infection of CXCR4+ or CCR5+ U87-CD4+ cells by X4 (NL4-3) or R5 (BaL) HIV-1 strains. Inhibition of virus replication occurred regardless of the multiplicity of infection employed.

Conclusion: Our results demonstrate that RNAi may be used to block HIV entry and replication through the blockade of cellular gene expression. Gene silencing by siRNA may become a valid alternative for HIV intervention.

From the aRetrovirology Laboratory IrsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916 Badalona, Spain; and bLiver Unit, Department of Internal Medicine, Hospital General Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, 08035 Barcelona, Spain.

Correspondence to: Dr. José A. Esté, Fundació irsiCaixa, Retrovirology Laboratory, Hospital Universitari Germans Trias i Pujol, 08916 Badalona, Spain. Tel: +34 93 4656374; fax: +34 93 4653968; e-mail: jaeste@ns.hugtip.scs.es

Received: 26 June 2002; revised: 1 August 2002; accepted: 16 September 2002.

© 2002 Lippincott Williams & Wilkins, Inc.