222 CXCR4 Gene Silencing with Multiple Small Interfering RNAs Inhibits HIV-1 Infection J. Ji*, P. Erb, T. Klimkait, M. Wernli Inst Med Microbiology, Basel, Switzerland
Background: Recently, it was found that small interfering double-stranded RNAs (siRNA) provide a powerful tool to silence a gene of interest. SiRNA duplexes targeting the rev, tat, or gag genes or genomic HIV RNA were reported to inhibit HIV-1 replication, and siRNAs targeting CD4 reduced the ability of HIV-1 to enter the cell. Cellular entry of HIV-1 requires binding to both CD4 and either one of the CCR5 or CXCR4 chemokine receptor. Indeed, the spread of HIV-1 in vitro can be blocked by preventing the interaction between HIV-1 envelope glycoproteins and chemokine receptors. As CXCR4 mediates entry of T-cell line-tropic virus (X4 or R5X4 isolates) and syncytia formation, it is a particularly suited target for drug development. We evaluated the efficacy of specific siRNA duplexes to downregulate CXCR4 and inhibit HIV-1 infection.
Methods: The HeLa CD4-derived reporter cell line SX22-1 was transfected with siRNA duplexes (21-nt dsRNA) targeting CXCR4 mRNA and investigated for down-regulation of CXCR4 expression and inhibition of HIV-mediated cell fusion and infection. Down-regulation of protein and mRNA expression were measured by flowcytometry and TaqMan real-time PCR, respectively. The inhibition of cell fusion and infection was evaluated by the fusion-induced gene stimulation assay (FIGS).
Results: The siRNA duplexes chosen specifically downregulated CXCR4 expression and inhibited CXCR4-mediated cell fusion as well as HIV-1 infection in vitro. Moreover, for the first time we demonstrate that co-transfection of cells with a combination of siRNA duplexes targeting different sites on the same mRNA evoked a cooperative inhibitory effect.
Conclusions: CXCR4, a co-receptor for HIV-1 entry, is widely discussed as a putative therapeutic target for the inhibition of HIV infection. We demonstrate that de-activation of the CXCR4 gene is possible by specific siRNA duplexes leading to the down-regulation of CXCR4 expression and inhibition of HIV infection. Moreover, the application of a combination of specific siRNA duplexes leads to a superior gene silencing effect. Our approach not only demonstrates the successful inhibition of HIV-1 infection, it also extends the use of siRNA for gene-function studies and potential therapy.
