A novel approach for inhibition of HIV-1 by RNA interference: counteracting viral escape with a second generation of siRNAs

Abstract

RNA interference (RNAi) is an evolutionary conserved gene silencing mechanism in which small interfering RNA (siRNA) mediates the sequence specific degradation of mRNA. The recent discovery that exogenously delivered siRNA can trigger RNAi in mammalian cells raises the possibility to use this technology as a therapeutic tool against pathogenic viruses. Indeed, it has been shown that siRNAs can be used effectively to inhibit virus replication. The focus of this review is on RNA interference strategies against HIV-1 and how this new technology may be developed into a new successful therapy. One of the hallmarks of RNAi, its sequence specificity, also presents a way out for the virus, as single nucleotide substitutions in the target region can abolish the suppression. Strategies to prevent the emergence of resistant viruses have been suggested and involve the targeting of conserved sequences and the simultaneous use of multiple siRNAs, similar to current highly active antiretroviral therapy. We present an additional strategy aimed at preventing viral escape by using a second generation of siRNAs that recognize the mutated target sites.

Keywords: HIV-1; RNA interference; combinatorial therapy; gene therapy; lentiviral vector; shRNA; siRNA.

Figure 1.

The HIV-1 life cycle and possible opportunities for RNAi-mediated intervention. (A) After virus infection, the HIV-1 core with the RNA genome will be released into the cytoplasm. The incoming RNA genome represents a desirable target, since destruction of the genome (route 1) prevents reverse transcription and integration of the provirus. After integration of the provirus, viral messenger RNAs are expressed. Viral mRNAs can potentially be degraded in the nucleus (route 2) or in the cytoplasm (route 3), preventing viral protein production and virus particle assembly and release. (B) The proviral DNA genome. Early gene expression results in fully spliced mRNAs encoding for Tat, Rev and Nef. Late gene expression results in partially spliced mRNAs, encoding for Env, Vif, Vpr, and unspliced mRNA for Gag and Gag-Pol proteins or as the genomic RNA.

Figure 2.

Counteracting HIV-1 escape with a second generation of shRNAs. (A) Hairpin structure of the shRNA molecules. Shown is the shNEF-wt and a second generation of shRNA variants that counteract escape mutants of HIV-1. Altered nucleotides are highlighted. (B) Molecular clones with sequence variation in the target were co-transfected with shRNA expressing constructs: either the shLUC control, the shNEF-wt or the matching shRNA variant. The number of basepair complementarity of the predicted siRNA and the target are shown. (C) Co-transfections were performed in 293T cells with 500 ng of pLAI molecular clone, 100 ng of pSUPER plasmid and 2.5 ng pRL as an internal control. Transfection was performed on 1.5 × 10⁵ cells with lipofectamine-2000 according to the manufacturers instructions (Invitrogen). Two days post-transfection, CA-p24 was measured in the cell culture supernatant and Renilla activity measured in cell extract. The ratio between CA-p24 and internal control values yields the relative CA-p24 production, for all pLAI transfections the control shLUC were set at 100%. Black bars represent co-transfections with the control shLUC, transfections with shNEF-wt are shown in grey. The white bars represent co-transfections of the respective pLAI molecular clones with the matching pSUPER-shRNA counterpart.

Figure 3.

Predicting viral escape. The highly conserved tat/rev target is shown with flanking sequences. The 19 nucleotide target sequence encompasses two overlapping reading frames for tat and rev. For each reading frame the codons and amino acid sequence are shown. Numerous silent codon changes are possible in each reading frame (grey box), but a silent mutation in both reading frames is possible only at 2 positons within the target sequence (black box).