Six Highly Conserved Targets of RNAi Revealed in HIV-1-Infected Patients from Russia Are Also Present in Many HIV-1 Strains Worldwide

Abstract

RNAi has been suggested for use in gene therapy of HIV/AIDS, but the main problem is that HIV-1 is highly variable and could escape attack from the small interfering RNAs (siRNAs) due to even single nucleotide substitutions in the potential targets. To exhaustively check the variability in selected RNA targets of HIV-1, we used ultra-deep sequencing of six regions of HIV-1 from the plasma of two independent cohorts of patients from Russia. Six RNAi targets were found that are invariable in 82%-97% of viruses in both cohorts and are located inside the domains specifying reverse transcriptase (RT), integrase, vpu, gp120, and p17. The analysis of mutation frequencies and their characteristics inside the targets suggests a likely role for APOBEC3G (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G, A3G) in G-to-A mutations and a predominant effect of RT biases in the detected variability of the virus. The lowest frequency of mutations was detected in the central part of all six targets. We also discovered that the identical RNAi targets are present in many HIV-1 strains from many countries and from all continents. The data are important for both the understanding of the patterns of HIV-1 mutability and properties of RT and for the development of gene therapy approaches using RNAi for the treatment of HIV/AIDS.

Keywords: HIV-1; RNAi targets; conserved HIV-1 sequences; gene therapy; ultra-deep sequencing.

Figure 1

Schematic Presentation of RNAi Targets A1–A6 within the HIV-1 Gene Map The positions of 19-bp targets are indicated. The values shown above the targets indicate the 5′ numbering of a target in the reference sequence (GenBank: AF316544).

Figure 2

Alignments of Deep Sequencing Reads from Both Cohorts The top ten alignments are shown. The complete alignments are shown in Figures S1–S12. The reference sequence is shown on the top (GenBank: AF316544). The second line represents the sequences observed in the majority of reads. The number and percentage of reads are indicated. The 19-nt core RNAi sequences are not shaded.

Figure 3

Curve Showing the Frequencies of Nucleotide Substitutions along the 27- to 30-nt Targets for Cohort 1 The horizontal red line corresponds to the threshold of reliable mutation detection (Equation 5 in Materials and Methods). The frequencies were determined against the most invariable RNAi target and were calculated by Equation 1 (Materials and Methods).

Figure 4

Curve Showing the Frequencies of Nucleotide Substitutions along the 27- to 30-nt Targets for Cohort 2 The horizontal red line corresponds to the threshold of reliable mutation detection (Equation 5 in Materials and Methods). The frequencies were determined against the most invariable RNAi target and were calculated by Equation 1 (Materials and Methods).

Figure 5

The Most Conserved Positions inside 19-nt RNAi Core Sequences The regions possessing the mutation frequencies below 10⁻⁴ (see Figures 3 and 4) are shown between the blue lines. The values indicate the number of the nucleotide in a target. Cohort 1 is shown in red. Cohort 2 is shown in black.

Figure 6

Frequencies of All Possible Nucleotide Substitutions along the 27- to 30-nt Targets for Cohort 1 The frequencies were determined against the most invariable RNAi target and were calculated by Equation 1 (Materials and Methods).

Figure 7

Frequencies of All Possible Nucleotide Substitutions along the 27- to 30-nt Targets for Cohort 2 The frequencies were determined against the most invariable RNAi target and were calculated by Equation 1 (Materials and Methods).

Figure 8

Efficiency of RNAi Initiated by the Dicer Substrates Results of co-transfection experiments are shown (see Materials and Methods). Luminescence of the Renilla luc gene (RLU) was normalized to that of the firefly luc gene. *p < 0.005.