Optimal design and validation of antiviral siRNA for targeting HIV-1

Abstract

We propose rational designing of antiviral short-interfering RNA (siRNA) targeting highly divergent HIV-1. In this study, conserved regions within HIV-1 genomes were identified through an exhaustive computational analysis, and the functionality of siRNAs targeting the highest possible conserved regions was validated. We present several promising antiviral siRNA candidates that effectively inhibited multiple subtypes of HIV-1 by targeting the best conserved regions in pandemic HIV-1 group M strains.

Figure 1

Conservations of siRNA target sequences among HIV-1 group M. (A) A total of 4,417,157 siRNA targets were generated from the 495 HIV-1 sequences, and their conservations within the HIV-1 genomes are represented using a color density plot. The line plot above the color chart represents the highest value in each position. (B) A detailed view of the three conserved regions; 5' LTR, the cPPT/CTS in the integrase gene, and 3' PPT. 'Position' indicates the 5'-most position of each 21-mer. The landmarks of the HIV-1 genome are adjusted to align at the center of the siRNAs by shifting 10 bp to the left. (C) Pie chart indicating the percentage of the 4,417,157 siRNA target sites at each conservation level.

Figure 2

Validation of 41 siRNAs. The antiviral efficacy of each siRNA was tested against four HIV-1 infectious molecular clones: pNL4-2 (subtype B); 95MM-yIDU106 (subtype B'); 93IN101 (subtype C); or 93JP-NH1 (CRF01_AE). The potency of each siRNA was tested using the target mRNA cleavage assay (rightmost panel). The ability of each siRNA to cleave its target was evaluated by the target mRNA cleavage assay.