Suppression of chemokine receptor expression by RNA interference allows for inhibition of HIV-1 replication
Martínez, Miguel Aa; Gutiérrez, Arantxaa; Armand-Ugón, Mercedesa; Blanco, Juliàa; Parera, Marionaa; Gómez, Jordib; Clotet, Bonaventuraa; Esté, José Aa
From the aRetrovirology Laboratory IrsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916 Badalona, Spain; and bLiver Unit, Department of Internal Medicine, Hospital General Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, 08035 Barcelona, Spain.
Correspondence to: Dr. José A. Esté, Fundació irsiCaixa, Retrovirology Laboratory, Hospital Universitari Germans Trias i Pujol, 08916 Badalona, Spain. Tel: +34 93 4656374; fax: +34 93 4653968; e-mail: email@example.com
Received: 26 June 2002; revised: 1 August 2002; accepted: 16 September 2002.
Objectives: Duplexes of 21 base pair RNA, known as short-interfering RNA (siRNA), have been shown to inhibit gene expression by a sequence-specific RNA degradation mechanism termed RNA interference (RNAi). The objective of our study was to evaluate the effect of chemokine receptor gene suppression by RNAi on the entry and replication of HIV-1.
Methods: A flow cytometry and microscopy evaluation of HIV co-receptor expression of cells transfected with siRNA. An evaluation of the effect of siRNA on HIV entry and replication by intracellular p24 antigen detection, and virus production by infected cells, respectively.
Results: siRNA that target CXCR4 and CCR5 could effectively impede cell surface protein expression and their consequent function as HIV co-receptors. The inhibitory effect of RNAi directed to CXCR4 was detected 48 h after transfection of CXCR4+ U87-CD4+ cells. The expression of CXCR4 and CCR5 was blocked in 63 and 48% of positive cells by the corresponding siRNA. However, siRNA directed to CXCR4 or CCR5 did not have an effect on CD4 cells or green fluorescence protein expression. siRNA directed to CXCR4 did not suppress CCR5 expression or vice versa. The suppression of HIV-1 co-receptor expression effectively blocked the acute infection of CXCR4+ or CCR5+ U87-CD4+ cells by X4 (NL4-3) or R5 (BaL) HIV-1 strains. Inhibition of virus replication occurred regardless of the multiplicity of infection employed.
Conclusion: Our results demonstrate that RNAi may be used to block HIV entry and replication through the blockade of cellular gene expression. Gene silencing by siRNA may become a valid alternative for HIV intervention.
In many eukaryotes, double-stranded RNA inhibits gene expression in a sequence-specific manner by triggering the degradation of messenger RNA . This effect, referred to as RNA interference (RNAi), has been studied most extensively in Caenorhabditis elegans and Drosophila melanogaster  by introducing specifically designed, gene-targeted dsRNA that interacts with homologous single-stranded RNA. After recognition, dsRNA is degradated by the DICER RNase into short fragments of 21 nt in length, called short- interfering RNA (siRNA). This mechanism of RNA suppression has been termed ‘post-transcriptional gene silencing'.
RNAi is now believed to work in a variety of organisms, including mammals [3–5]. Unlike long (> 500 base pair) dsRNA, short (21 bp) siRNA duplexes avoid the generation of antiviral defence mechanisms, such as the activation of protein kinase R, a kinase that induces a non-specific translational shutdown, the activation of non-specific ribonuclease RNase L, and the production of IFN-α . RNAi provides a new tool for studying gene function in mammalian cells and their eventual use as gene-specific therapeutic agents. Before this is achieved, proof of the concept that specific cellular genes can be blocked to prevent an unwanted physiological effect needs to be demonstrated. Here, we show that specific siRNA blocked the expression of CXCR4 and CCR5 that are known to function as HIV entry co-receptors . We also show that the suppression of co-receptor expression through RNAi inhibited the infection by X4 or R5 HIV-1 strains of otherwise permissive cells. The functionality of HIV co-receptor is thus blocked by RNA interference.
Materials and methods
Short-interfering RNA preparation
ssRNA, 21-nucleotide RNA were chemically synthesized by Cruachem (Glasgow, Scotland). Oligonucleotides were purified using high-performance liquid chromatography and resuspended in water. siRNA sequences targeting CXCR4 and CCR5 corresponded to the coding regions +330 to +348 of CXCR4 (GCAGUCCAUGUCAUCUACA-dTdT, RNAx42i) and +554 to +572 (GUCAGUAUCAAUUCUGGAA- dTdT, RNAR53i) of CCR5 relative to the start codon, respectively. For the annealing of siRNA duplexes, 20 mM single strands were incubated in annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate) for 1 min at 90°C followed by 1 h at 37°C. The integrity of siRNA was confirmed by polyacrylamide gel electrophoresis.
Short-interfering RNA transfection
The monocitoid U87-CD4, CCR5, CXCR4 cells  or HeLa cells (105 cells) that stably express green fluorescent protein (GFP) were transfected with 200 pmol of the appropriate siRNA using the LipofectAMINE-2000 reagent (Life Technologies, London, UK). Alternatively, CXCR4-negative cells were transfected with pc.Fusin (CXCR4) plasmids  at a ratio of 4 μg per 3.0 × 105 cells. The GFP expression vector pEGFP (Clonteck, Palo Alto, CA, USA) was used as a control. Forty-eight hours after transfection, cells were detached from culture plates with Versene (Life Technologies) and prepared for the evaluation of chemokine receptor expression.
Cell-surface expression of chemokine receptors
Receptor expression was determined by flow cytometry analysis as described previously . The antibodies used were as follows: phycoerythrin-coupled anti-CD4 monoclonal antibody (mAb) Leu3a, phycoerythrin- anti-CXCR4 mAb 12G5, isotype IgG2a and FITC anti-CCR5 mAb 2D7, isotype IgG2a from Becton Dickinson (Mountain View, CA, USA). The mean fluorescence intensity (MFI) and the percentage of receptor-expressing cells were calculated using CellQuest software (Becton Dickinson). The results shown represent the mean of at least two experiments plus or minus standard deviation calculated using Microsoft Excel software.
For fluorescence microscopy studies, 48 h after transfection, cells were labelled with anti-CXCR4 mAb (12G5) and a secondary rhodamine-labelled antibody, and analysed in a Nikon Eclipse microscope.
The titres of all viral stocks were determined in peripheral blood mononuclear cells by determining the production of p24 at day 7 post-infection in a p24 enzyme-linked immunosorbent assay. The tissue culture infectious dose (TCID50) was defined as the amount of virus able to produce a detectable amount of p24 in a culture supernatant. The titre of virus stocks (TCID50/ml) was 1.5 × 105 for NL4-3 and 2.3 × 105 for BaL. HIV-1 replication in mock-transfected or siRNA-transfected cells was evaluated following a procedure described previously . Briefly, 48 h after transfection U87-CD4 cells (1.5 × 105) expressing CXCR4 or CCR5 were infected with X4 NL4-3 HIV-1 (multiplicity of infection; m.o.i. 0.006) or R5 BaL (m.o.i. 0.12) HIV-1 and incubated for 24 h. Cells were then washed and fresh Dulbecco's modified essential medium containing 0.5 μg/ml zidovudine (Sigma, Madrid, Spain) was added to avoid further rounds of viral replication. After 48 h, p24 antigen in the culture supernatant was measured using a commercial p24-antigen enzyme-linked immunosorbent assay test (Innogenetics, Madrid, Spain). Alternatively, cells were permeabilized with Fix-and-Perm (Innogenetics) and stained for intracellular p24 antigen with the KC77 anti-p24 mAb (Coultek, Madrid, Spain). To calculate the inhibitory effect of siRNA, p24 antigen of the control (mock-transfected) samples was normalized to 100, and test samples were calculated as the percentage of p24 antigen compared with the control.
Effect of RNA interference on chemokine receptor expression
A first series of experiments was undertaken to evaluate the effect of RNAi on chemokine receptor expression. After transfection with pEGFP and selection with geneticin (G418), a HeLa cell population stably expressing both CXCR4 and GFP was selected. After 48 h, transfection with 200 pmol RNAX42i induced a significant effect on the expression of CXCR4, but did not have an effect on GFP expression or induced changes in cell morphology (Fig. 1). RNAX42i inhibited the MFI of CXCR4 expression by 80% without affecting GFP expression. Similarly, the transfection of U87-CD4 cells expressing CXCR4 with 200 pmol of the corresponding siRNA duplex (RNAX42i) induced a decrease in the number of positive cells (mean of 63%) with a decrease in MFI of 59% when compared with mock-transfected CXCR4 cells (Fig. 2a). Similar results were obtained with the transient expression of CXCR4 in U87-CD4 cells (data not shown). RNAR53i blocks CCR5 expression by 48%, with a reduction of MFI of 56% (Fig. 2b). Conversely, antisense ssRNA corresponding to the negative strands of RNAX42i and RNAR53i did not have a significant effect on CXCR4 or CCR5 (Fig. 2c). The expression of other cell surface markers was not reduced by the transfection of RNA42i or RNA53i. MFI of CD4 expression remained at 38–39 relative units in U87-CD4-CXCR4 cells and 18–20 relative units in U87-CD4-CCR5 cells, regardless of the transfected siRNA.
Furthermore, siRNA directed to HIV genes (Nef and Pol) that were able to block the production of HIV particles in U87-CD4-CXCR4 cells did not suppress the expression of CXCR4 (data not shown). These results suggest that the suppression of CXCR4 or CCR5 gene expression occurred upon the transfection of siRNA in a gene-specific manner.
RNA interference blocks HIV co-receptor function
We next evaluated the functionality of HIV co-receptor expression and the effect of specific RNAi on HIV replication. After 48 h post transfection, U87-CD4 cells were infected with HIV-1 NL4-3 or BaL, which induced a similar level of p24 antigen production, and evaluated for intracellular p24 antigen expression as a measure of HIV-1 entry and for virus production by p24 antigen detection in the supernatant of infected cells.
As seen in Fig. 3, RNAX42i and RNAR53i displayed a significant effect (mean 55% and 33% inhibition, respectively) on HIV-1 entry, as evaluated by intracellular p24 antigen. This reduction in the number of p24-positive cells was strongly reflected in X4 or R5 HIV-1 production. In fact, the quantification of p24 in the supernatant of infected cells showed that RNAX42i and RNAR53i inhibited virus production from 21 ± 4 to 7 ± 4.6 ng/ml (67%) for HIV-1 NL4-3 and 75 ± 14 to 15.5 ± 4 ng/ml (79%) for HIV-1 BaL, respectively. As expected, a complete inhibition of HIV entry and replication is obtained with the CCR5 antagonist TAK-779 (2 μM)  or the CXCR4 antagonist AMD3100 (1 μg/ml) . However, the transfection of an irrelevant siRNA (GFPi, Fig. 3) did not have a significant effect on HIV-1 NL4-3 or HIV-1 BaL entry or replication, suggesting a specific effect of RNAX42i and RNAR53i on co-receptor gene expression.
The effect observed with RNAX42i on HIV-1 X4 or RNAR53i on HIV-1 R5 replication was similar regardless of the m.o.i. employed (0.003–0.024 for HIV-1 NL4-3 and 0.03–0.24 for HIV-1 BaL) (Fig. 4), confirming that co-receptor expression in target cells is a rate-limiting step for HIV replication.
The discovery that siRNA may silence gene expression in mammalian cells opens the possibility to generate a potent approach for gene therapy. Most evaluations of RNAi have consisted of the inhibition of transiently expressed genes or the intracellular expression of cellular genes. Here, we show that the inhibition of cell-surface chemokine receptor expression can be achieved by RNAi of chemokine receptor genes. The transfection of specific siRNA blocked CXCR4 and CCR5 expression, respectively, without blocking other cell surface expression markers i.e. CD4 or the expression of intracellular markers, i.e. GFP. A small but not significant increase in CXCR4 expression was noted in cells that were transfected with RNAR53i or antisense RNAX42i. Quantitative fluorescence cell-sorting assays that allow for a refined evaluation of CXCR4 expression  should determine if there is a relevant biological effect of antisense RNA on cell surface co-receptor expression.
Partial inhibition may be ascribed to the inefficient transfection of target cells. Nevertheless, the inhibitory effect could be maintained in cells cultured for up to 6 days, and in some experiments an increased effect (up to 82% inhibition) was noted with time (data not shown). At present, the kinetics of RNAi in mammalian cells are unknown. It has been shown that silencing is transmitted between cells [14,15], suggesting a mechanism for amplification and maintenance of the gene- silencing effect within the cell or into neighbouring cells. Furthermore, nucleotide fragments that originate after the initial siRNA-directed cleavage of ssRNA should amplify the silencing effect.
siRNA-mediated gene silencing offers a potentially powerful tool to inhibit replication at a number of stages in the life cycle of HIV by targeting both viral and cellular genes. The in-vivo suppression of genes such as CD4  may be limited by its role in normal immune function. HIV-1 co-receptors provide more attractive alternatives for targeting host proteins. The effect of siRNA directed to CXCR4 or CCR5 was sufficient to induce a clear inhibitory effect on the entry and replication of X4 or R5 HIV-1. The suppression of co-receptor gene expression thus appears to be a valid target for the inhibition of HIV replication.
Besides their clear role in HIV infection , chemokine receptors are involved in a number of pathological processes [17–19]. Unlike CXCR4 , CCR5 may be dispensable for normal life . Nevertheless, potent antagonists of CXCR4  and CCR5 [11,22] are being considered as potential candidates for anti-HIV intervention. The interference of gene expression of CXCR4 or CCR5 may have an advantage if cells are specifically targeted for the stable expression of dsRNAi without affecting their normal function in bystander cells. DNA vectors for the stable suppression of gene expression by RNAi are already being developed [4,23], and RNAi is being evaluated as a means to block HIV replication [16,24,25,27,28]. We thus envisage that selective viral vectors targeting CXCR4 and CCR5 in CD4 T cells could be employed to alter their role in disease, or as shown herein, to block HIV replication especially if RNAi targeting both CXCR4 and CCR5 could be employed in combination to suppress X4, R5 and dual tropic virus replication. Viruses may have developed mechanisms to counteract the virus-specific RNAi cell-defence mechanism . Consequently, targeting cellular and not viral genes for RNAi could have an advantage. Our results are the first demonstration that RNA interference of gene expression may be a valid approach to suppress gene expression of chemokine receptors CXCR4 and CCR5, with the consequent block of receptor/co-receptor function in disease. Improved transfection methods that allow for the evaluation of siRNA in primary T cells are required to confirm our results in cells that are more relevant to HIV-1 infection. This approach will also be valuable to identify those genes that, after the suppression of chemokine receptor gene expression, may be directly or indirectly involved in cell homeostasis.
U87-CD4 cells, and chemokine receptor plasmids were received from the NIH AIDS Research and Reference Reagent Program.
Sponsorship: This work was partly supported by the Spanish MCyT project BFM2000-1382, FIS project 01/1116 and 01/0067-02 and FIPSE projects 3014/99, 36207/01 and 36293/02.
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Co-receptors; CXCR4; CCR5; gene silencing; RNA interference
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