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

Abstract

The discovery that RNA interference (RNAi) can induce sequence-specific degradation of target mRNA through short interfering 21-23-mer RNA segments (siRNA) in mammalian cells has opened a new molecular strategy for potential therapeutic intervention against HIV. Our laboratory pioneered the research on siRNA-mediated inhibition of HIV-12 infection in cell lines and primary cells by targeting the viral cellular receptors CCR5 and CD4 and the viral gene Gag. We found that siRNA delivered before as well as after infection effectively reduced HIV replication highlighting the potential of RNAi in both prophylaxis and therapy. However, viral heterogeneity is a major hurdle for potential therapeutic use of RNA interference (RNAi) against HIV-1. To determine the extent of RNAi tolerance to mutations, we tested 3 viral target sites with differing propensity for mutations: a highly variable rev sequence, a gag sequence conserved only among clade B isolates, and a vif sequence highly conserved across clades. Lentiviral expression of all 3 shRNAs inhibited replication of the homologous HIV(IIIB) strain. However, they differed in their ability to protect primary CD4 T cells against multiple isolates within and across HIV clades. The least conserved rev sequence inhibited only 2 of 5 clade B isolates. The gag sequence (conserved within clade B) protected 5 of 5 clade B isolates but not other clade viruses with 2 or 3 mutations in the central region. In contrast, the vif sequence, which was conserved across clades except for single mutations at positions 14 and 17, inhibited viruses from 5 different clades. Moreover, siRNAs with introduced mutations at sites of gag sequence polymorphisms showed reduced antiviral activity, whereas mutations in vif siRNA only modestly decreased silencing. Thus, although 1 or 2 mutations at peripheral sites are tolerated, mutations in the central target cleavage region abolish RNAi activity.