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Background:  The recent discovery of RNA-based gene silencing (RNAi) has fuelled functional genomics and target validation in mammalian cells. The major challenge remains the triggering of the desired cellular response in the absence of unspecific response. In the present study, we compare the transient knock-down (siRNA) of the recently described integrase co-factor, LEDGF/p75, with that of stable gene silencing by small hairpin RNA (shRNA), encoded by a lentiviral vector.

Methods:  We compared the effect of siRNA and shRNA of a potential co-factor of HIV-1 integrase, LEDGF/p75, to establish a strategy to validate the role of HIV-1 co-factors during viral replication. Unrelated or double mismatch siRNA/shRNA were used as controls. Hereto we transfected HeLaP4 cells with specific siRNA or generated polyclonal HeLaP4 cells stably expressing a specific shRNA. Cells were infected with HIV-1, strain NL4.3, in various dilutions and the virus replication was analyzed at different time points using p24 ELISA and β-galactosidase measurement. Cytotoxicity in the knock-downs was investigated by FACS.

Results:  Transient siRNA-based silencing of LEDGF/p75 resulted in protein levels below the detection limit in Western blot and HIV-1 replication was inhibited 8- to 10-fold compared to wild type cells. Reproducible target validation was dependent on the standardization of cell culture conditions. Stable silencing of the co-factor in polyclonal cell lines resulted in a partial knock-down of gene expression and a 2-fold inhibition of HIV-1 replication. Cells expressing shRNA were less susceptible to cell death than cells transfected with siRNA.

Conclusion:  RNAi is a powerful tool to validate potential cellular co-factors of HIV-1 replication. Both siRNA and shRNA proved valuable in the validation process of LEDGF/p75. Inhibition of HIV-1 replication correlated with the level of gene silencing. Although a more profound gene silencing was obtained with siRNA, a significantly lower degree of cellular toxicity was observed with shRNA. This may be related to the lower extent of gene silencing, to the absence of unspecific RNA-induced cellular response, or to adaptation during selection.

Keywords: co-factors; RNAi; integrase