A U1i RNA that Enhances HIV-1 RNA Splicing with an Elongated Recognition Domain Is an Optimal Candidate for Combination HIV-1 Gene Therapy

Figures

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  Figure 1. Structure of the U1 snRNP and Mechanism of Action of U1i RNAs (A) Left, the U1 snRNA with associated proteins U1-70K, U1-A, U1-C, and Sm. Right, a U1i RNA in which the U1 snRNA recognition domain is changed to be complementary to a target RNA sequence. Stem loop (SL)1- and SL2-mutated sequences used for the domain mutation experiment are illustrated. (B) Depiction of the mechanism of action of U1i RNAs targeting 5′ splice donor sites (5′ss) or 3′ terminal exons of targeted HIV-1 mRNA. Left, U1i RNAs targeted to a 5′ss or downstream of a 3′ splice acceptor site (3′ss) enhance splicing at the upstream 3′ss, resulting in an increase in mRNA species containing a particular exon and a decrease in unspliced RNA and mRNA species that do not include that particular exon. Right, binding of U1i RNAs to the 3′ terminal exon of mRNAs results in an inhibition of polyadenylate polymerase (PAP) at the polyadenylation site (PAS).

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  Figure 2. U1i RNAs Targeting the Gag Open Reading Frame of HIV-1 RNA Inhibit HIV-1 Production (A) Outline of the sequences targeted by constructed U1i RNAs as well as an shRNA described in our previous study, shGag. (B) Inhibition of HIV-1 production by shGag, U1-Gag1, U1-Gag2, U1-Gag3, and U1-Gag4 when co-transfected with HIV-1 pNL4-3 (100 ng) at 5, 25, 50, and 100 ng. The dotted line in the graph represents the level at which 50% of HIV-1 production is inhibited. Results are expressed as a percentage of HIV-1 reverse transcriptase (RT) activity in culture supernatants relative to supernatants from cells co-transfected with pNL4-3 and the U1-WT or empty shRNA expression plasmid. All data are represented as the mean ± SEM of at least three independent transfections with two to three replicates (n = 6–9). A two-way ANOVA with a Bonferroni post-test was used to compare replicate means to the means of U1-WT or empty shRNA expression-plasmid-transfected cells. The level of significance is indicated above each mean value that was significantly different from its control (*p < 0.05, **p < 0.01, ***p < 0.001).

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  Figure 3. U1i RNAs that Enhance Splicing and U1-Gag2 Are More Potent Inhibitors of HIV-1 Production When Compared to U1i RNAs that Inhibit Polyadenylation (A) Illustration of the U1i RNA target sites relative to the HIV-1 genome and the major splice donor and acceptor sites. (B) Inhibition of HIV-1 production by U1i RNAs at 0.5, 1, 5, 25, 50, and 100 ng. The dotted line in the graph represents the level at which 50% of HIV-1 production is inhibited. Results are expressed as a percentage of HIV-1 reverse transcriptase (RT) activity in culture supernatants relative to supernatants from cells co-transfected with pNL4-3 and the U1-WT plasmid. All data are represented as the mean ± SEM of at least three independent transfections with two to three replicates (n = 6–9). A two-way ANOVA with a Bonferroni post-test was used to compare replicate means to the means of U1-WT-plasmid-transfected cells. The level of significance is indicated above each mean value that was significantly different from its control (*p < 0.05, **p < 0.01, ***p < 0.001). (C) Conservation estimates of sequences targeted by the various U1i RNAs at each nucleotide in the target site relative to the consensus nucleotide.

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  Figure 4. HIV-1 Suppression by All U1i RNAs Is Dependent on the Proper Assembly of the SL1 Domain and Recruitment of U1-70K whereas Only U1i RNAs that Inhibit Polyadenylation Depend on the SL2 Domain and Recruitment of U1-A The effects of U1 snRNA SL1 and SL2 domain mutations on the suppression of HIV-1 production by U1-D2 (A), U1-T6 (B), U1-Env (C), U1-Gag2 (D), U1-Rev (E) and U1-Nef (F). HEK293T cells were co-transfected with HIV-1 pNL4-3 (100 ng) and 1–500 ng of mutated U1i RNAs. Results are expressed as a percentage of RT activity in SL1 or SL2 WT plasmid-transfected cells. Data from Figure 3 for the non-mutated U1i RNAs are shown for comparison. Data were log transformed and a nonlinear regression log(inhibitor) versus response equation with least-squares (ordinary) fit was determined using GraphPad prism. All data points are represented as the mean ± SEM of at least three independent transfections with two to three replicates (n = 6–9).

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  Figure 5. The Effects of U1i RNA Expression on HIV-1 mRNA (A) Total RNA isolated from HEK293T cells transfected with 400 ng of pNL4-3 in the presence or absence of U1i RNAs (1,000 ng) were analyzed by northern blotting. Full-length (FL), incompletely spliced (IS), and completely spliced (CS) RNA species are indicated. (B) The numerous HIV-1 mRNA species produced by alternative splicing at the various splice donor (D1–D5) and splice acceptor (A1–A7) sites. Primers used for RT-PCR analysis are depicted in red for IS and CS. (C) RT-PCR analysis of CS (left) and IS (right) RNA species in the presence of U1i RNAs or U1-WT-expressing plasmid.

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  Figure 6. U1-T6 Demonstrates Superior Inhibition of HIV-1 Replication in Stably Transduced T Lymphocytes when Compared to an shRNA Currently in Clinical Trials (A–C) Sup-T1 cells were transduced with HIV-based lentiviral vectors (HIV-7-EGFP) expressing U1-WT, shRNA-NS, U1-Gag2, U1-D2, U1-T6, U1-Rev, U1-Env, and shRNA-tat/rev or the empty vector (HIV-7). The transduced cells were then infected with NL4-3 at 8750 cpm/mL (A and C) or 1750 cpm/mL (B) and viral replication was monitored for several days after infection by measuring HIV-1 RT activity in culture supernatants. The mean RT activity (cpm) in culture supernatants for infections performed in triplicate (n = 3) (A and B), or across two independent infections performed in triplicate (n = 6) (C), is shown for the various days following infection. All data are represented as the mean ± SEM. A two-way ANOVA with a Bonferroni post-test was used to compare replicate means to the means of U1-WT-transduced cells. The level of significance is indicated next to each mean value that was significantly different from its control (*p < 0.05, **p < 0.01, ***p < 0.001). (D) Competitive growth assay of Sup-T1-transduced cells. Sup-T1 cells transduced…

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  Figure 7. Increasing the Recognition Domain of U1-T6 Is Feasible with Minimal Loss to Its Inhibitory Activity (A) Sequence logo showing the conservation and nucleotide identity of the site targeted by U1-T6 among completely sequenced HIV-1 strains. (A–C) Sites targeted by elongated U1-T6 in molecular clones pNL4-3 (A), pMJ4 (B), and pADA (C) are shown. Nucleotides in red indicate mismatches. To the right, inhibition of HIV-1 production by elongated U1-T6 molecules when co-transfected with 100 ng of pNL4-3 (A), 200 ng of pMJ4 (B), or 200 ng of pADA (C) and 1, 5, or 100 ng of U1-T6 variants are shown. The length variants are named according to the direction of the increased recognition domain (toward the 5′ or 3′ end of the viral genome) and by the number of nucleotides added (5′ + 3, 5′ + 6, 3′ + 3, and 3′ + 6). Results are expressed as a percentage of HIV-1 reverse transcriptase (RT) activity in culture supernatants relative to supernatants from cells co-transfected with pNL4-3, pMJ4, or pADA and the U1-WT plasmid. All data are represented as the mean ± SEM of at least three independent transfections with two to three replicates (n = 6–9). A two-way ANOVA with a Bonferroni post-test was…