RNAi-mediated protection of CD4 T cells from multiple clades and primary isolates of HIV-1

Abstract

Viral heterogeneity is a major hurdle for practical therapeutic use of RNAi in HIV infection. To identify suitable RNAi targets for cross clade inhibition of HIV-1, we evaluated 3 viral sequences with differing propensity for mutations. Single or dual specific short hairpin RNAs (shRNAs) targeting viral rev, gag or vif genes were expressed from a lentiviral vector pLL3.7. The endogenous generation of 21 nt siRNAs was confirmed by modified Northern Blots. Primary CD4 T cells transduced with control luciferase or anti HIV shRNAs were challenged with a panel of lab-adapted and primary HIV isolates. With lentiviruses expressing single shRNAs, the least conserved rev target sequence provided protection only against 2 of 5 clade B isolates, the gag sequence, protected against all clade B isolates where the sequence was well conserved but not against common variant strains from other clades that contained 2 or 3 mutations in the centralregion. In contrast, RNAi targeting the vif sequence which is highly conserved in clades A, B, C, D and E, inhibited multiple viruses from all 5 clades, including variant strains with single mutations at positions 14 or 17. Moreover, siRNAs with introduced mutations at sites of gag sequence polymorphisms showed reduced antiviral activity, whereas siRNA mismatches corresponding to natural vif mutations only modestly decreased silencing. Thus, our data demonstrates that RNAi targeting a viral vif sequence that is highly conserved within the central region surrounding the siRNA-mRNA recognition can serve as a potentially useful target for clinical application of RNAi against divergent strains of HIV-1. Studies are currently underway to evaluate the utility of dual shRNA constructs for broader protection and prevention of viral escape.