Inhibition of human immunodeficiency virus type 1 replication in primary macrophages by using Tat- or CCR5-specific small interfering RNAs expressed from a lentivirus vector - PubMed (original) (raw)

Inhibition of human immunodeficiency virus type 1 replication in primary macrophages by using Tat- or CCR5-specific small interfering RNAs expressed from a lentivirus vector

Ming-Ta M Lee et al. J Virol. 2003 Nov.

Abstract

Although several groups have demonstrated that RNA interference, induced by transfection of small interfering RNA (siRNA) duplexes, can protect cells against a viral challenge in culture, this protection is transient. Here, we describe lentivirus expression vectors that can stably express siRNAs at levels sufficient to block virus replication. We have used these vectors to stably express siRNAs specific for the essential human immunodeficiency virus type 1 (HIV-1) Tat transcription factor or specific for a cellular coreceptor, CCR5, that is required for infection by the majority of primary HIV-1 isolates. These lentivirus vectors are shown to protect cells, including primary macrophages, against HIV-1 infection in culture by inducing selective degradation of their target mRNA species. These data suggest that it should be possible to block the expression of specific viral or cellular genes in vivo by using viral vectors to stably express the appropriate siRNAs.

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Figures

FIG. 1.

Expression of siRNAs by a lentivirus vector. (A) Schematic representation of the NL-SIN-CMV-BLR lentivirus vector. The blasticidin resistance (blr) gene was introduced as a selectable marker. Unique restriction enzyme sites are indicated. An siRNA expression cassette containing the human H1 promoter can be introduced in place of most of the 3′ LTR U3 region. (B) Predicted stem-loop structure of the Tat siRNA precursor encoded by the pNL-H1-siTat lentivirus vector. Arrows indicate the 5′ ends of the mature siRNAs encoded by this vector. (C) Primer extension analysis. pH1, cells transfected with 1 μg of pSuper; pH1-siTat, cells transfected with 1 μg of pH1-siTat; NL-H1, cells transduced with the NL-H1 lentivirus vector; NL-H1-siTat, cells transduced with NL-H1-siTat. A probe that detects the 3′ or antisense strand was used for lanes 2 to 6, and a probe specific for the 5′ or sense strand was used for lanes 7 to 12. Lane 1 contained a labeled 21-nt RNA marker, while lane 7 contained a synthetic 21-nt RNA identical to the first 21 nt of the 5′ arm of the siRNA precursor as a positive (POS) control. Mock, mock-transfected wild-type 293T cells.

FIG. 2.

Inhibition of HIV-1 replication with an siRNA expressed from a lentivirus vector. (A) Normal 293T cells (lane 2) or cells transduced by lentivirus vectors (lanes 3 and 4) were cotransfected with 25 ng each of pBC12/CMV/β-gal and pHIV/Tat. At 48 h posttransfection, protein expression levels were assayed by Western analysis. Mock-transfected 293T cells served as the negative control (NEG). (B) Specific inhibition of HIV-1 replication in 293T cells transduced with the NL-H1-siTat lentivirus vector. 293T cells were infected overnight with VSV-G-pseudotyped HIV-1, and viral replication was measured by p24 Gag antigen production at 48 h after infection. 293T cells infected with wild-type, i.e., nonpseudotyped, HIV-1 served as the negative control (NEG), while transfected, nontransduced 293T cells served as the positive control (POS). The results shown are the average of three experiments with the standard deviation. (C) Total RNA was extracted from 293T cells used in panel B and subjected to Northern analysis with an HIV-1 LTR-specific probe. The approximate sizes of the three classes of HIV-1 transcript are indicated.

FIG. 3.

Inhibition of HIV-1 gene expression by NL-H1-siTat is specific. (A) Sequences of the wild-type (WT) and mutant synthetic (Syn) forms of the Tat siRNA target sequence. (B) 293T cells transduced with NL-H1 (lanes 2 and 4) or NL-H1-siTat (lanes 3 and 5) were transfected with 25 ng each of pBC12/CMV/β-gal and either wild-type pHIV/Tat (lanes 2 and 3) or the mutant pHIV/SynTat expression plasmid (lanes 4 and 5). Western analysis was performed as described in the legend to Fig. 2A. (C) 293T cells transduced with NL-H1 or NL-H1-siTat were transfected with expression plasmids encoding CD4 and CCR5 and, where indicated, 10 ng of either pHIV/Tat or pHIV/SynTat. At 48 h after transfection, the transduced, transfected cells were infected with the NL-Luc-ADA indicator virus and induced luciferase levels were assayed a further 48 h later. NEG, mock-transfected, NL-H1-transduced cells. The average of three experiments and the standard deviation are indicated. NEG, negative control.

FIG. 4.

Stable inhibition of HIV-1 gene expression in NL-H1-siTat-transduced cells. 293T cells transduced with NL-H1-siTat were cultured under selection for either 1 week or 12 weeks. Transduced cells were then transfected with expression plasmids encoding CD4 and CCR5 or CD4 and CXCR4 and infected with the R5-tropic NL-Luc-ADA or X4-tropic NL-Luc-HXB indicator virus, and induced luciferase activities were determined 2 days later. The average of three experiments and the standard deviation are indicated. NEG, negative control.

FIG. 5.

Inhibition of CCR5 expression by a lentivirus-expressed siRNA. (A) Transduced 293T cells were transfected with plasmids expressing CCR5 and GFP. At 48 h after transfection, the level of cell surface CCR5 expression on GFP-positive cells was analyzed by FACS with an anti-CCR5 monoclonal antibody. CCR5 expression on NL-H1-siCCR5-transduced cells is represented by shading, and that on NL-H1-transduced cells is represented by the open area. (B) Transduced 293T cells were transfected with expression plasmids encoding CD4 and CCR5 or CD4 and CXCR4. At 48 h after transfection, CD4+ CCR5+ cells were infected with NL-Luc-ADA while CD4+ CXCR4+ cells were infected with NL-Luc-HXB. Induced luciferase levels were determined 48 h after infection and are indicated. NEG, nontransfected 293T cells. The average of three experiments and the standard deviation are indicated. (C) Transduced or control 293T cells were transfected with CCR5 and CD4 expression plasmids. At 48 h after transfection, total RNA was extracted and subjected to Northern analysis to measure CCR5 mRNA (top) and CD4 mRNA (bottom) expression. Mock-transfected 293T cells served as a negative control (NEG), while wild-type 293T cells transfected with CCR5 and CD4 served as a positive control (POS).

FIG. 6.

Single-cycle assay of HIV-1 replication in primary macrophages. MDM were cultured for 7 days in the presence of macrophage colony-stimulating factor and then transduced twice with a lentivirus vector stock over a 2-day period. The order of vector infection is shown; e.g., siCCR5 denotes MDM transduced twice with NL-H1-siCCR5, while siCCR5/siTat denotes MDM transduced first with NL-H1-siCCR5 and then with NL-H1-siTat. Two days later, the transduced MDM were infected with the R5-tropic reporter virus NL-Luc-ADA (A) or X4-tropic reporter virus NL-Luc-1549 (B) and induced luciferase levels were measured a further 48 h later. Doubly NL-H1-transduced MDM served as the negative control (NEG). The average of three experiments and the standard deviation are indicated.

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Inhibition of human immunodeficiency virus type 1 replication in primary macrophages by using Tat- or CCR5-specific small interfering RNAs expressed from a lentivirus vector - PubMed (original) (raw)