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. 2008 Jul 2;3(7):e2602.
doi: 10.1371/journal.pone.0002602.

The efficacy of generating three independent anti-HIV-1 siRNAs from a single U6 RNA Pol III-expressed long hairpin RNA

Sheena Saayman  1 , Samantha Barichievy, Alexio Capovilla, Kevin V Morris, Patrick Arbuthnot, Marc S Weinberg
Affiliations

The efficacy of generating three independent anti-HIV-1 siRNAs from a single U6 RNA Pol III-expressed long hairpin RNA

Sheena Saayman et al. PLoS One. .

Abstract

RNA Interference (RNAi) effectors have been used to inhibit rogue RNAs in mammalian cells. However, rapidly evolving sequences such as the human immunodeficiency virus type 1 (HIV-1) require multiple targeting approaches to prevent the emergence of escape variants. Expressed long hairpin RNAs (lhRNAs) have recently been used as a strategy to produce multiple short interfering RNAs (siRNAs) targeted to highly variant sequences. We aimed to characterize the ability of expressed lhRNAs to generate independent siRNAs that silence three non-contiguous HIV-1 sites by designing lhRNAs comprising different combinations of siRNA-encoding sequences. All lhRNAs were capable of silencing individual target sequences. However, silencing efficiency together with concentrations of individual lhRNA-derived siRNAs diminished from the stem base (first position) towards the loop side of the hairpin. Silencing efficacy against HIV-1 was primarily mediated by siRNA sequences located at the base of the stem. Improvements could be made to first and second position siRNAs by adjusting spacing arrangements at their junction, but silencing of third position siRNAs remained largely ineffective. Although lhRNAs offer advantages for combinatorial RNAi, we show that good silencing efficacy across the span of the lhRNA duplex is difficult to achieve with sequences that encode more than two adjacent independent siRNAs.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. HIV-1 C subtype genome with sites targeted by lhRNA and shRNAs.
A. Organization of HIV-1 subtype C genome indicating open reading frames (ORFs) together with the 5′ and 3′ long terminal repeats (LTRs). Arrows show the sites targeted by each of shRNA vif, shRNA tat and shRNA rev, as well as the lhRNAs. B. Schematic illustration of lhRNAs comprising 69 bp in the stem. G∶U pairings are indicated as corrugated sense strand. A sequence of 2 U residues that are derived from the transcription termination signal is shown. The intended mechanism of transcription and processing of the lhRNAs to form 3 anti HIV-1 siRNAs is illustrated. C. Sequences and predicted structure of lhRNAs and shRNAs. The order of the siRNA-encoding sequences within the lhRNAs is indicated along the extent of the duplex. G∶U and U∶G pairings are indicated with an arrowhead.
Figure 2
Figure 2. Knockdown of target-reporter fusion gene expression.
A. psiCheck-derived vectors that include indicated HIV-1 target sequences inserted downstream of the Renilla luciferase ORF. The control Firefly luciferase cassette, present on the same plasmid, is also shown. Both cassettes are under control of constitutively active transcription regulatory elements: Herpes simplex virus thymidine kinase (HSV TK) and Simian virus 40 (SV40) promoters. B. Average normalized ratios of the Renilla∶Firefly luciferase activity when cells were transfected with psiCheck tat-rev-vif dual luciferase reporter plasmid together with lhRNA- or shRNA-encoding plasmid vectors. C. Average normalized ratios of the Renilla∶Firefly luciferase activity when cells were transfected with psiCheck tat, psiCheck rev or psiCheck vif dual luciferase reporter plasmids together with lhRNA- or shRNA-encoding plasmid vectors. The average values from three independent transfection experiments, with standard deviations, are given (*, p<0.05, t-test, relative to mock transfected control).
Figure 3
Figure 3. Northern blot analysis of RNA extracted from HEK293 cells that had been transfected with the indicated lhRNA and shRNA-expressing plasmids.
A. A single blot was probed with an oligonucleotide that was complementary to putative tat, rev and vif guide sequences. B. The blot was probed with two 14-mer LNA oligonucleotides (LNA nucleotides underlined) which were complementary to the rev siRNA guide sequence and adjacent nucleotides as indicated in the illustration. Both blots (from A and B) were stripped and reprobed with an oligonucleotide complementary to U6 snRNA to control for equal RNA loading.
Figure 4
Figure 4. Effect of nucleotide spacing between tat, rev and vif siRNA-encoding sequences on silencing efficacy.
A. Schematic illustration of hairpin sequences with boundaries between tat, rev and vif duplexes indicated. The sequences and numbers of bases inserted or deleted at the junctions of the RNAi effecter-encoding sequences are indicated for each of lhRNA rev-vif-tat a to lhRNA rev-vif-tat g. B. Average normalized Renilla∶Firefly luciferase activity determined 48 hours after transfecting HEK293 cells with the psiCheck tat-rev-vif target together with each of lhRNA rev-vif-tat a to lhRNA rev-vif-tat g. C. Average normalized Renilla∶Firefly luciferase activity determined 48 hours after transfecting HEK293 cells with the psiCheck tat, psiCheck rev or psiCheck vif target together with each of lhRNA rev-vif-tat a to lhRNA rev-vif-tat g. Results are given as the average values with standard deviations from three independent transfection experiments. (*, p<0.05, t-test, relative to mock transfected control). Mock transfected cells received the empty backbone U6+1 plasmid D. Northern blot analysis of RNA extracted from HEK293 cells that had been transfected with the indicated lhRNA and shRNA-expressing plasmids. The blot was probed with an oligonucleotide that was complementary to putative rev guide sequence. The blot was stripped and reprobed with an oligonucleotide complementary to U6 snRNA to control for equal RNA loading.
Figure 5
Figure 5. HIV-1 challenge assay.
U87.CD4.CCR5 cells were transfected with plasmids expressing the indicated hairpins and then subjected to infection with an equivalent of TCID50 1000 particles of the HIV-1 FV5 viral isolate. Concentrations of HIV-1 p24 in the culture supernatant and viral particle equivalents (A) were determined 6 days after infection. Results are expressed as the means with standard deviations of three independent experiments (*, p<0.05, t-test, relative to mock transfected control). B. Replication kinetics of a representative experiment included in (A). C. Sequence of lhRNA- and shRNA-derived tat, rev and vif guide sequences and complementary regions targeted within FV5 and HXB2 HIV-1 isolates. Mismatches between the putative guide and target sequences are shown. Sequence numbering is based on isolate HXB2, accession K03455. D. Knockdown of FV5 vif and HXB2 vif target-reporter fusion gene expression by shRNA vif and representative lhRNAs in HEK293 cells. Results are expressed as the means with standard deviations of three independent experiments (*; p<0.05, t-test, between annotated samples).
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