710. Comparison of the Activity of siRNAs and shRNAs Against Furin: A Cellular Target for HIV-1 Inhibition

RNA interference (RNAi) is a sequence-specific post-transcriptional gene regulation mechanism triggered by double-stranded RNA. HIV-1 replication can be inhibited by RNAi using synthetic siRNA or shRNA gene constructs directed against viral sequences. However, this approach has the disadvantage of viral escape through mutation of the target sequence. Targeting of cellular genes that encode viral co-factors could therefore present a solution against viral escape from RNAi. Furin is a membrane-associated cellular endoprotease that belongs to the subtilisin-like proprotein convertase family. Furin cleaves the HIV-1 precursor envelope glycoprotein gp160 into the biologically active gp120-gp41 trimer. Silencing of furin by RNAi may present a potential cellular target for inhibition of HIV-1. To study this, we designed and tested the activity of seven siRNAs and matching shRNA-expression plasmids targeting furin sequences. We initially determined the si/shRNA inhibitory potential on luciferase-furine reporter constructs. Four si/shRNA pairs showed a correlation between the activity of a siRNA and the shRNA. In contrast, for the other three si/shRNA pairs, either the siRNA or the shRNA was active. siRNAs and shRNAs that inhibited reporter-furin expression were also highly effective against furin. The impact on HIV-1 gp160 cleavage was tested by Western blot analysis. Inhibition of furin resulted in decreased gp160 processing. Lentiviral vectors were constructed expressing the effective shRNAs. SupT1 T cell lines, which are susceptible for HIV-replication, were stably transduced. HIV-1 virus replication was inhibited in these transduced cell lines. Experiments are in progress to determine the impact of furin silencing on HIV-1 replication in primary T cells.