Potent and specific inhibition of retrovirus production by coexpression of multiple siRNAs directed against different regions of viral genomes.

siRNA-mediated RNA degradation has been demonstrated to act as an antiviral system in many species. Here we describe inhibition of retrovirus production by multiple siRNAs designed to target various regions of the viral genomes. Using murine leukemia virus (MuLV) as a model, we demonstrate that the virus production can be inhibited by 77% in siLTR2 (a siRNA targeting the U3 region of MuLV) expression vector transfected cells. Coexpression of siLTR2 with siPsi2 (a siRNA targeting the 3' Psi (packaging signal sequence) results in 93% suppression of the virus production, suggesting that an increased inhibition of the virus production can be achieved by coexpression of multiple siRNAs to target different regions of the viral RNA simultaneously. Our results also indicate that not all sequences of the viral RNA are equally accessible to siRNA. We show that U3 region of MuLV is more accessible to siRNA, whereas the packaging signal sequence, especially the region adjacent to 5'LTR, is less accessible to siRNA, partly as a result of the binding of Gag precursors. Furthermore, we demonstrate that coexpression of siLTR2 with siPsi2 in virus producer cells leads to 88% knockdown of viral titer, showing the benefit of coexpression of multiple siRNAs for potent suppression of virus production in the setting of an established infection. Moreover, we demonstrate that infection of MuLV in cells that stably coexpress siLTR2 with siPsi2 diminishes by 77%. Taken together, we establish that siRNA-mediated gene silencing can suppress multiple steps of the retrovirus life cycle, offering a potential for both treating virus-associated diseases and preventing viral infection.