Sequence homology required by human immunodeficiency virus type 1 to escape from short interfering RNAs

Abstract

Short interfering RNAs (siRNAs) targeting viral or cellular genes can efficiently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Nevertheless, the emergence of mutations in the gene being targeted could lead to the rapid escape from the siRNA. Here, we simulate viral escape by systematically introducing single-nucleotide substitutions in all 19 HIV-1 residues targeted by an effective siRNA. We found that all mutant viruses that were tested replicated better in the presence of the siRNA than in the presence of the wild-type virus. The antiviral activity of the siRNA was completely abolished by single substitutions in 10 (positions 4 to 11, 14, and 15) out of 16 positions tested (substitution at 3 of the 19 positions explored rendered nonviable viruses). With the exception of the substitution observed at position 12, substitutions at either the 5' end or the 3' end (positions 1 to 3, 16, and 18) were better tolerated by the RNA interference machinery and only in part affected siRNA inhibition. Our results show that optimal HIV-1 gene silencing by siRNA requires a complete homology within most of the target sequence and that substitutions at only a few positions at the 5' and 3' ends are partially tolerated.

FIG. 1.

Inhibition of wild-type HIV-1 by siRNA siRT199 or siNef118. (A) U87-CD4 cells were mock transfected or transfected with different concentrations (pmol) of the appropriate siRNA, siRT199 or siNef118. After 24 h posttransfection, cells were infected with 200 TCID₅₀ of wild-type HIV-1 HXB2. At 96 h postinfection, the supernatant of infected cells was assayed for p24 antigen using an enzyme-linked immunosorbent assay. (B) U87-CD4 cells were mock transfected or transfected with 2 pmol of siRT199 or siNef118. After 24 h posttransfection, cells were infected with 200 TCID₅₀ of wild-type HIV-1 HXB2. At days 1, 2, 3, 4, 5, and 6 after infection, virus replication was monitored by determining the p24 antigen level in the culture supernatant. Values represent the means ± standard deviations (SDs) from at least three independent experiments.

FIG. 2.

Systematic analysis of how single-nucleotide mismatches between siRT199 and its target sequence affect HIV-1 replication silencing. U87-CD4 cells were mock transfected or transfected with siRT199. After 24 h posttransfection, cells were infected with 200 TCID₅₀ of wild-type HXB2 or the different recombinant individual mutated viruses. At 96 h postinfection, the supernatant of infected cells was assayed for p24 antigen using an enzyme-linked immunosorbent assay. p24 levels (upper panel) and percentages of p24 antigen levels relative to that of the respective control (lower panel) are shown. To calculate the inhibitory effect of siRT199, p24 antigen of the control (mock-transfected) samples was normalized to 100%, and levels in test samples were calculated as percentages of the control level. Values represent the means ± SDs from at least three independent experiments. wt, wild type; mut, mutant.

FIG. 3.

Inhibition of the wild type, mutant 8, mutant 9, mutant 10, and mutant 12 by siNef118. U87-CD4 cells were mock transfected or transfected with siNef118. After 24 h posttransfection, cells were infected with 200 TCID₅₀ of wild-type HXB2 or the different recombinant individual mutated viruses. At 96 h postinfection, the supernatant of infected cells was assayed for p24 antigen using an enzyme-linked immunosorbent assay. p24 antigen values (upper panel) and percentages of p24 antigen levels relative to that of the respective control (lower panel) are shown. Values represent the means ± SDs from at least three independent experiments. wt, wild type; m, mutant.

FIG. 4.

Inhibition of wild-type, mutant 9, and mutant 12 HIV-1 by the following siRNA: (A) siRT199, siRT199m9, or siRT199m12 or (B) siNef118, siNef118m9, or siNef118m12. U87-CD4 cells were mock transfected or transfected with the appropriate siRNA, siRT199 or siNef118. After 24 h posttransfection, cells were infected with 200 TCID₅₀ of virus. At 96 h postinfection, the supernatant of infected cells was assayed for p24 antigen using an enzyme-linked immunosorbent assay. p24 antigen values and percentages of p24 antigen levels relative to that of the respective control are shown. Values represent the means ± SDs from at least three independent experiments. wt, wild type; m and mut, mutant.