221 RNA Interference of Chemokine Receptor Expression Allows for Inhibition of HIV-1 Replication MA Martinez1, A Gutierrez1, M Armand-Ugon1, J Blanco1, M Parera1, J Gomez2, B Clotet1, J.A. Este*1 1Fundacio IrsiCaixa, Barcelona, Spain and 2Hosp Vall d'Hebron, Barcelona, Spain
Background: Duplexes of 21bp-RNAs, known as short-interfering RNAs (siRNA), have been shown to inhibit gene expression by a sequence-specific RNA degradation mechanism termed RNA interference (RNAi). HIV replication may be blocked by siRNA directed to the HIV genome. The objective of our study was to evaluate the effect of chemokine receptor gene supression by RNAi on the entry and replication of HIV-1.
Methods: Flow cytometry and microscopy evaluation of HIV coreceptor expression of cells transfected with siRNA. 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 coreceptors. The inhibitory effect of RNAi directed to CXCR4 was detected 48h after transfection of CXCR4+ U87-CD4+ cells. 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 or green fluorescence protein (GFP) expression. siRNA directed to CXCR4 did not suppress CCR5 expression or vice versa. 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.
Conclusions: Our results demonstrate that RNAi may be used to block HIV entry and replication through blockade of cellular gene expression. Gene silencing by siRNA may become a valid alternative for HIV intervention.
