Inhibition of human immunodeficiency virus type 1 by RNA interference using long-hairpin RNA - PubMed (original) (raw)

Inhibition of human immunodeficiency virus type 1 by RNA interference using long-hairpin RNA

P Konstantinova et al. Gene Ther. 2006 Oct.

Abstract

Inhibition of virus replication by means of RNA interference has been reported for several important human pathogens, including human immunodeficiency virus type 1 (HIV-1). RNA interference against these pathogens has been accomplished by introduction of virus-specific synthetic small interfering RNAs (siRNAs) or DNA constructs encoding short-hairpin RNAs (shRNAs). Their use as therapeutic antiviral against HIV-1 is limited, because of the emergence of viral escape mutants. In order to solve this durability problem, we tested DNA constructs encoding virus-specific long-hairpin RNAs (lhRNAs) for their ability to inhibit HIV-1 production. Expression of lhRNAs in mammalian cells may result in the synthesis of many siRNAs targeting different viral sequences, thus providing more potent inhibition and reducing the chance of viral escape. The lhRNA constructs were compared with in vitro diced double-stranded RNA and a DNA construct encoding an effective nef-specific shRNA for their ability to inhibit HIV-1 production in cells. Our results show that DNA constructs encoding virus-specific lhRNAs are capable of inhibiting HIV-1 production in a sequence-specific manner, without inducing the class I interferon genes.

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Figures

Figure 1

Figure 1

Scheme of the human immunodeficiency virus type 1 (HIV-1) pLAI proviral genome and target sequences used for the design of long-hairpin RNAs (lhRNAs). The target sequences are indicated as bars below the HIV-1 coding regions. lhRNA (300 basepairs (bp)) tat fuses tat exon 1 (gray bar, 5422–5626) and tat exon 2 (black bar, 7972–8017) sequences, rev fuses rev exon 1 (gray bar, 5562–5626) and rev exon 2 (black bar, 7972–8206) and nef1 contains _nef_-LTR sequences (8519–8818). Double-stranded RNA nef2 is a duplex of two separate, complementary sense and antisense nef sequences (8416–8695). The positive control sh-nef is a 21-bp hairpin consisting of nef sequences (8552–8571).

Figure 2

Figure 2

Inhibition of human immunodeficiency virus type 1 (HIV-1) by in vitro transcribed nef2 double-stranded RNA (dsRNA), in vitro diced si-nef2 and sh-nef RNA. (a) 2% MetaPhor agarose gel stained with ethidium bromide, showing the 300 basepairs (bp) nef2 dsRNA and the diced 19 bp si-nef2 products; M, 20 bp marker. (b) Cotransfection of human embryonic kidney (HEK) 293T cells with 10 ng of the indicated RNA, 500 ng pLAI and 2.5 ng pRL as an internal control. Transfections were performed with Lipofectamine 2000 and 1.5 × 105 cells. Virus production was measured in the culture supernatant 2 days after transfection. CA-p24 values are given as percentage of the pLAI production without inhibitory RNA. Standard error bars represent the means of four independent experiments. (c) Cotransfection of HEK 293T cells with 10 ng of the indicated dsRNA, 100 ng pGL3-Nef and 2.5 ng pRL as an internal control. Transfections were performed as described above. After 72 h cells were lysed and firefly and renilla luciferase expression was measured.

Figure 8

Figure 8

Long-hairpin RNA (lhRNA) constructs do not induce the interferon (IFN) response. Human embryonic kidney (HEK) 293T cells (1.5 × 105) were cotransfected with pLAI and the indicated lhRNA/double-stranded RNA (dsRNA)-expression constructs using Lipofectamine 2000. Construct pT7-nef2 was cotransfected with pT7-pol. Renilla luciferase (pRL) was used as an internal control. The pUC19 plasmid was used as a negative control. In vitro transcribed ds-nef2 RNA and poly (I:C) act as positive controls for IFN-β induction. Two separate transfections were performed, which were processed for either IFN-β mRNA expression or renilla measurement. No significant differences in Renilla expression were measured, except for the toxic treatment with ds-nef2 RNA and poly(I:C) (results not shown). In addition, we measured CA-p24 in the supernatant, which showed the inhibition characteristics described earlier. Total RNA was isolated from the cells 24 h after transfection. The IFN-β expression level was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). _β_-Actin mRNA expression was analyzed as an internal control. PUC19 RT- and poly (I:C) RT- are control reactions without RT step.

Figure 3

Figure 3

Expression vectors for long-hairpin RNA (lhRNA), double-stranded RNA (dsRNA) and short-hairpin RNA (shRNA). Long-hairpin RNAs were created by cloning the 300-nucleotide (nt) inverted repeats from tat, rev and nef1 (see Figure 1) downstream of the EF1_α_, 7tetO or long-terminal repeat (LTR) promoters. The 1 kb EF1_α_ intron is positioned downstream of the EF1_α_ promoter. A schematic representation of the final hairpin structures is shown on the right. In pEF1_α_ constructs, the two complementary RNA strands are separated by a 46 nt loop. In the p7tetO and pLTR constructs, the complementary sequences are separated by a 1 kb spacer that contains splice donor and acceptor sites. Vector pLTRΨ-nef1 is a derivative of pLTR-nef1 in which the human immunodeficiency virus type 1 (HIV-1) leader sequence (Ψ, 76–630, marked as a gray box) was inserted. The predicted transcript will have the Ψ domain upstream of the RNA hairpin. All transcripts contain a polyadenylation signal (pA) downstream of the hairpin sequences. Vector pT7-nef2 has 300 basepairs (bp) long double-stranded nef sequences flanked by T7 promoters (T7) and terminators (φ) at both 5′ and 3′ ends. Two separate complementary RNA chains, potentially capable of forming dsRNA, are transcribed from the convergent promoters by T7 RNA polymerase (encoded by expression plasmid pT7-pol). Vectors pT7sh-nef and pH1sh-nef express sh-nef from the T7 and H1 promoters, respectively.

Figure 4

Figure 4

Marginal inhibition of human immunodeficiency virus type 1 (HIV-1) production by pEF1_α_- and p7tetO-driven long-hairpin RNA (lhRNA) constructs. (a) Cells (C33A, human embryonic kidney (HEK) 239T and Vero) were lipofectamine-transfected with 500 ng pLAI, 500 ng inhibitory construct, 3 ng pCMV-rtTA and 2.5 ng pRL. Vector pEF1_α_-green fluorescent protein (GFP) was used as a control expressing an irrelevant lhRNA against GFP. Vectors pH1sh-nef and the empty vector were used as negative and positive controls, respectively. Virus production was determined as described in the legend to Figure 2. Standard error bars represent the means of three independent experiments. The sh-nef control construct was not tested (n.t.) in C33A cells. (b) HEK 239T cells were cotransfected with 100 ng pLAI, 100 ng p7tetO-tat, p7tetO-nef1 or pH1sh-nef, 3 ng pCMV-rtTA and 2.5 ng pRL as an internal control. Culture medium was refreshed after 16 h and 1 _μ_g/ml doxycycline was added. Virus production was determined as described in the legend to Figure 2. Standard error bars represent the means of four independent experiments.

Figure 5

Figure 5

Antiviral long-hairpin RNA (lhRNA) production from the human immunodeficiency virus (HIV)-inducible long-terminal repeat (LTR) promoter. (a) Inhibition of HIV-1 production by lhRNA expressed from the Tat-inducible HIV-1 LTR. Human embryonic kidney (HEK) 239T cells were cotransfected with 100 ng of pLAI and 100 ng pLTR-tat, pLTR-nef1, pLTRΨ-nef1 or pLTRasΨ-nef1. Equal amounts of a pH1sh-nef expression vector and the empty vector were added as positive and negative controls, respectively. (b) Sequence-specific inhibition of HIV-1 production by pLTRΨ-nef1. HEK 239T cells were cotransfected with 100 ng of pLAI and 10 ng pLTRΨ, pLTRΨ-nef1 or pH1sh-nef. On the _y_-axis, the CA-p24 values (ng/ml) are presented. (c) Effect of the trans-activator protein Tat on the inhibitory effect of pLTRΨ-nef1. pLAI (100 ng) was cotransfected with 0–10–30–100 ng pLTRΨ-nef1 with or without 20 ng pcDNA3-Tat. Virus production was determined as described in the legend to Figure 2. Standard error bars represent the means of five independent experiments.

Figure 6

Figure 6

Inhibition of human immunodeficiency virus type 1 (HIV-1) by cytoplasmically expressed long double-stranded RNA (dsRNA). (a) pT7-luc (100 ng) construct was cotransfected in human embryonic kidney (HEK) 293T cells with or without 30 ng pT7-pol. At 2 days after transfection, cells were lysed and the expression of firefly luciferase was measured. (b) pT7-nef2 (100 ng) and pT7sh-nef vectors were linearized 3′ of the T7 termination signal with _Xba_I and _Bam_HI, respectively, and cotransfected with 100 ng pLAI, 30 ng pT7-pol and 2.5 ng pRL in HEK 293T cells. Two separate complementary RNA chains, potentially forming dsRNA, are transcribed in the cell from convergent T7 promoters. Equal amounts of a pH1sh-nef expression vector and the empty vector were added as positive and negative controls, respectively. Cotransfections were also performed without pT7-pol to check for non-specific effects of the T7 plasmids. Virus production was determined as described in the legend to Figure 2. Standard error bars represent the means of four independent experiments. Cells were lysed 2 days after transfection to measure Renilla luciferase. (c) Sequence-specific inhibition of the pGL3-Nef reporter, containing the 250-nucleotide (nt) nef2 target sequence downstream of the luciferase coding domain, by pT7-nef2. HEK 239T cells were cotransfected with 100 ng of pGL3-Nef, 100 ng pT7-nef2, 30 ng pT7-pol and 2.5 ng pRL. pH1sh-nef (10 ng) expression vector and the empty vector were added as positive and negative controls, respectively. (d) Titration of T7 polymerase. pLAI (100 ng) was cotransfected with increasing amounts (0–3–10–30–100 ng) of pT7-pol and 100 ng pT7-nef2 or pT7sh-nef.

Figure 7

Figure 7

In vivo RNA expression from long-hairpin RNA (lhRNA) constructs. HEK 293T cells were transfected with 500 ng of the indicated lhRNA/double-stranded RNA (dsRNA)-expression constructs using Lipofectamine 2000. Constructs pLTR-nef1 and pLTRΨ-nef1 were cotransfected with 30 ng pTat. Construct pT7-nef2 was cotransfected with 100 ng pT7-pol. The pUC19 plasmid and poly (I:C) were used as negative controls and pT7-nef2 plasmid DNA was used as a positive control. Total RNA was isolated from the cells 48 h after transfection. The nef expression level was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) with primers Nef-B/X and antiU3-att, which create a 110 basepairs (bp) amplification product. _β_-Actin mRNA expression was analyzed as an internal control. pT7-nef2 RT− is a control reaction without RT step.

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