Combinatorial RNAi against HIV-1 using extended short hairpin RNAs

Abstract

RNA interference (RNAi) is a widely used gene suppression tool that holds great promise as a novel antiviral approach. However, for error-prone viruses including human immunodeficiency virus type 1(HIV-1), a combinatorial approach against multiple conserved sequences is required to prevent the emergence of RNAi-resistant escape viruses. Previously, we constructed extended short hairpin RNAs (e-shRNAs) that encode two potent small interfering RNAs (siRNAs) (e2-shRNAs). We showed that a minimal hairpin stem length of 43 base pairs (bp) is needed to obtain two functional siRNAs. In this study, we elaborated on the e2-shRNA design to make e-shRNAs encoding three or four antiviral siRNAs. We demonstrate that siRNA production and the antiviral effect is optimal for e3-shRNA of 66 bp. Further extension of the hairpin stem results in a loss of RNAi activity. The same was observed for long hairpin RNAs (lhRNAs) that target consecutive HIV-1 sequences. Importantly, we show that HIV-1 replication is durably inhibited in T cells stably transduced with a lentiviral vector containing the e3-shRNA expression cassette. These results show that e-shRNAs can be used as a combinatorial RNAi approach to target error-prone viruses.

Figure 1

Design of e-shRNAs encoding 3 and 4 siRNAs. (a) The HIV-1 genome and the position of target sequences for the original 21 bp shRNAs (shNef, shPol, shR/T, and shGag). (b) Structure of the e2-shRNA and the e-shRNAs encoding 3 or 4 siRNAs of 63, 66, 88, or 92 bp. The e3 and e4 constructs were made with mutations in the passenger strand of the hairpins (indicated in red), which results in destabilizing G-U bp in the hairpin stem. The guide strand sequences are marked in colors. bp, base pair; HIV-1, human immunodeficiency virus type 1; shRNA, short hairpin RNA; siRNA, small interfering RNA.

Figure 2

RNAi activity of the different siRNAs produced by e-shRNAs. Luciferase reporter constructs encoding 50-nt HIV-1 sequences (a) Luc-nef, (b) Luc-pol, (c) Luc-r/t, and (d) Luc-gag, the e-shRNA variants and the renilla luciferase expression plasmid (pRL) were co-transfected into HEK 293T cells. Two days post-transfection, firefly and renilla luciferase expression levels were determined and the ratio was plotted as relative luciferase activity. Luciferase expression in the presence of the pBS control was set at 100%. A scrambled hairpin (SCR) was used as a negative control and the original shRNAs (shNef, shPol, shR/T, and shGag) were included as positive controls. Bars represent the average values from five independent transfections and error bars show the SD. HEK, human embryonic kidney; HIV, human immunodeficiency virus; nt, nucleotide; RNAi, RNA interference; shRNA, short hairpin RNA; siRNA, small interfering RNA.

Figure 3

RNAi activity of lhRNAs of different hairpin stem length. (a) Design of two sets of lhRNAs targeting a consecutive sequence of the HIV-1 nef and r/t gene of 66, 88, and 92 bp in length (lhNef and lhR/T). The shRNA targeting nef and r/t is placed at the base of the hairpin and is marked in green or purple, respectively. Mutations were introduced in the passenger strand of the hairpin (indicated in red) to create G-U bp in the hairpin stem (b) HEK 293T cells were co-transfected with the lhRNA constructs, the corresponding Luc-nef or Luc-r/t reporter and pRL. Two days post-transfection luciferase activities were measured and used to calculate the relative luciferase expression (firefly/renilla ratio). Luciferase expression in the presence of pBS was set at 100%. A scrambled hairpin (SCR) was used as negative control and the original shRNA, shNef, or shR/T, as positive control. Bars represent the average values from five independent transfections and error bars show the SD. bp, base pair; HEK, human embryonic kidney; lhRNA, long hairpin RNA; RNAi, RNA interference; shRNA, short hairpin RNA.

Figure 4

RNAi activity of hairpins with increasing hairpin stem length. The relative luciferase activity of the base shRNA of the e-shRNA constructs and the two lhRNA series were plotted against the hairpin stem length. Luciferase expression in the absence of inhibitor was set at 100%. lhRNA, long hairpin RNA; RNAi, RNA interference; shRNA, short hairpin RNA.

Figure 5

Northern blot analysis of the e-shRNAs derived siRNAs. Total RNA was purified from HEK 293T cells transfected with the indicated e-shRNAs and lhRNAs and 10 µg was used to detect siRNAs against nef, pol, r/t, and gag with 19-nt complementary LNA oligonucleotide probes. As negative controls, the pSUPER vector (ctr) and unrelated shRNAs were used. The original shRNAs were used as positive controls, showing the processed ~22 nt siRNA. Precursor hairpin RNAs are indicated by asterisks. Bands that correspond to siRNAs are indicated by arrows. Ethidium bromide staining of the 5S rRNA band is shown below each panel as a control for equal sample loading. M represents the RNA size marker (in nt) and the probe name is indicated next to the blot. lhRNA, long hairpin RNA; LNA, locked nucleic acid; nt, nucleotide; shRNA, short hairpin RNA; siRNA, small interfering RNA.

Figure 6

The e-shRNA and lhRNA transcripts do not induce the interferon response. Total RNA was purified from HEK 293T cells transfected with (a) e-shRNAs and (b) lhRNAs at an efficacious dose. The IFN-β, OAS, RIG-I, ISG56, STAT-1, and MxA expression levels were determined by RT-PCR. β-actin mRNA expression serves as an internal control. We included poly I:C transfected cells as positive control. As negative controls, cells were Mock transfected or with the pSUPER vector (ctr). We also performed a control PCR on RNA extracts derived from poly I:C transfected cells that were not subjected to reverse transcription. Either the 200 or 400 bp lane of the SmartLadder (M) (Eurogentec) is shown as size reference. HEK, human embryonic kidney; IFN-β, interferon-β; lhRNA, long hairpin RNA; MxA, myxovirus resistant gene A; OAS, 2′-5′ oligoadenylate synthase; RIG-I, retinoic acid-inducible gene-I; shRNA, short hairpin RNA; STAT-1, signal transduction and activator of transcription-1.

Figure 7

Inhibition of wild-type and mutant HIV-1 variants by e-shRNAs. (a) HEK 293T cells were co-transfected with the HIV-1 molecular clone pLAI, pRL, and increasing amounts of e3-shRNA constructs. Two days post-transfection, HIV-1 production was measured by CA-p24 quantification in the culture supernatant. CA-p24 levels were normalized for renilla expression; virus production with the pSUPER vector (ctr) was set at 100%. The original shRNAs were used as positive controls. The SCR served as negative control. (b) Inhibition of HIV-1 production by e-shRNAs at low concentrations. Transfections were performed as described for a, but now including the e2 design and we used reduced shRNA concentrations to carefully examine their potency. The SCR and pBS served as negative control. (c) Inhibition of RNAi-escape viruses by e-shRNAs. Sequence of the L2, L3, L6, and L7 HIV-1 variants that contain mutations in the R/T siRNA target region. Inhibition of CA-p24 expression was determined as described above. The mean values and standard deviations are based on three independent experiments. HEK, human embryonic kidney; HIV, human immunodeficiency virus; RNAi, RNA interference; SCR, scrambled hairpin; shRNA, short hairpin RNA.

Figure 8

Suppression of HIV-1 replication in e3-66 expressing T cells. SupT1 cells were transduced with the lentiviral vector encoding e3-66 shNef, shPol, or shR/T. The empty JS1 vector was used as a negative control. (a) Transduced GFP-positive cells were selected by sorting and infected with low (left panel) and high (right panel) virus input. Virus replication was monitored for 49 days by measuring CA-p24 in the culture supernatant. (b) Clonal sequencing reveals the genetic alterations in the three target regions in different viral clones. GFP, green fluorescent protein; HIV, human immunodeficiency virus.