Inhibition of HIV-1 infection by a unique short hairpin RNA to chemokine receptor 5 delivered into macrophages through hematopoietic progenitor cell transduction

Abstract

Background: We recently expressed a potent and noncytotoxic short hairpin (sh)RNA directed against chemokine (c-c motif) receptor 5 (CCR5) using lentiviral mediated transduction of CD34+ hematopoietic progenitor cells (HPCs) and demonstrated the stable reduction of CCR5 expression in T-lymphocytes.

Methods: In the present study, we further assessed the activity of the shRNA through HPC transduction and differentiation into macrophages derived from fetal liver CD34+ (FL-CD34+) HPCs. Transduced lentiviral vector encoding the human CCR5 shRNA was stably maintained in FL-CD34+ cells and in the terminally differentiated macrophages using macrophage colony-stimulating factor, granulocyte macrophage colony-stimulating factor, interleukin-3 and stem cell factor.

Results: Quantitative real-time polymerase chain reaction for CCR5 mRNA indicated over 90% reduction of CCR5 mRNA levels in CCR5 shRNA-transduced population. The cells with knockdown of CCR5 expression acquired resistance to R5 tropic HIV-1 NFN-SX strain. We also developed a novel approach utilizing a mCherry-CCR5 chimeric reporter to assess the effectiveness of CCR5 target down-regulation in macrophages directly. Both the shRNA and the reporter were maintained throughout HPC differentiation to macrophages without apparent cytotoxicity.

Conclusions: The present study demonstrates a novel method to simply and directly assess the function of small interfering RNA and the effective inhibition of HIV-1 infection by a potential potent shRNA to CCR5 delivered into macrophages derived from HPCs.

Figure 1

CCR5 shRNA can specifically reduce the cell surface expression of CCR5 in CCR5-293T cells. (A) Schematic diagram of the siRNA-expressing lentiviral vector. The shRNA is expressed under the control of a human H1-RNA Pol III promoter (Pol III). The vector also contains a human ubiquitin C promoter (UbiC) driving the EGFP marker gene for tracking transduced cells. (B) CCR5-293T cells (1 × 10⁵) were transduced with lentiviral vectors encoding CCR5 shRNA 1005 (sh1005) or the mutant CCR5 shRNA (muCCR5 shRNA). The cells were harvested 2 days after virus transduction and analysed by flow cytometry with PE-Cy5 conjugated anti-human CCR5 or isotype control antibody staining (isotype). The transduced (EGFP+) and nontransduced (EGFP−) cells were gated based upon their EGFP expression. The number in each quadrant represents the percentage in each population

Figure 2

Phenotypic FACS analysis of macrophages derived from PBMC and FL-CD34+ cells. FL-CD34+ cells (2 × 10⁵) were cultured in the presence of 20 ng/ml of IL-3, 50 ng/ml of SCF and 20 ng/ml of MCSF for 9 days and then with 5 ng/ml of GM-CSF for 5 days to differentiate to macrophages. Monocytes (1 × 10⁶) derived from PBMCs were cultured in the presence of 10 ng/ml of MCSF for 7 days to differentiate to macrophages. The attached cells were gently scraped off the bottom of the plate and 2 × 10⁵ cells were stained with antibodies to CD14, CD68, CD11b and CD206 (macrophage markers), and CD4, CXCR4 and CCR5 (receptors for HIV-1). The expression of these surface markers was analysed by flow cytometry. The results are shown as a histogram. The number in each panel represents the percentage of positive population

Figure 3

shRNA transduction does not grossly affect differentiation to macrophages derived from FL-CD34+ cells. (A) FL-CD34+ cells (2 × 10⁵) were transduced with lentiviral vectors encoding sh1005 or muCCR5 shRNA as a control. Mock are cells without shRNA transduction. The cells were treated with 20 ng/ml of IL-3, 50 ng/ml of SCF and 20 ng/ml of MCSF for 9 days and then with 5 ng/ml of GM-CSF for 5 days to differentiate to the macrophages. The attached cells were gently scraped off the bottom of the plate and 1 × 10⁵ cells were analysed for CD14, CD11b and CD206 expression by flow cytometry. (B) 1 × 10⁵ macrophage cells were incubated with an MOI of 50 Texas Red-conjugated Zymosan particles for 90 min. Cells were then washed with cold PBS for three times and were analysed by fluorescence microscopy and flow cytometry. The flow cytometry results are exhibited as EGFP versus Zymosan bioparticle (Texas Red) dot plots. (C) Surface CCR5 staining of macrophages derived from sh1005 and muCCR5 shRNA-transduced cells.−, isotype control; +, CCR5 staining

Figure 4

Specific knockdown of mCherry-CCR5 chimera expression by sh1005 in 293T cells. 293T cells (1 × 10⁵) were cotransfected with lentiviral vectors expressing sh1005 or the muCCR5 shRNA and either mCherry-CCR5 chimera or mCherry encoding lentiviral vector by FuGENE. The cells were then cultured for 2 days and the mCherry expression was analysed by fluorescence microscopy (A) and flow cytometry (B). The results are exhibited as side scatter versus mCherry dot plots. The quadrant lines were defined by mock 293T cells (data not shown) and the percentage numbers are indicated

Figure 5

Specific knockdown of mCherry-CCR5 target expression by sh1005 in 293T cells. 293T cells (1 × 10⁵) were cotransfected with lentiviral vectors expressing sh1005 or muCCR5 shRNA and either mCherry-CCR5 target or mCherry encoding lentiviral vector by FuGENE. The cells were then cultured for 2 days and the mCherry expression was analysed by flow cytometry. The results are exhibited as side scatter versus mCherry dot plots. The quadrant lines were defined by mock 293T cells (data not shown) and the percentage numbers are indicated

Figure 6

Specific knockdown of mCherry-CCR5 target expression by sh1005 in FL-CD34+ cells. FL-CD34+ cells (2 × 10⁵) were transduced with lentiviral vectors expressing sh1005 or Luc shRNA. The cells were harvested 3 days after virus transduction, sorted by the EGFP expression, and further transduced with the lentiviral vector encoding mCherry-CCR5 target sequence at an MOI of 0.5. The mCherry expression was monitored by flow cytometry 3 days after the transduction. The results are exhibited as mCherry dot plots versus EGFP dot plots. The number in each quadrant represents the percentage in each population

Figure 7

Specific knockdown of mCherry-CCR5 target expression by sh1005 in macrophages derived from FL-CD34+ cells. FL-CD34+ cells (2 × 10⁵) were transduced with lentiviral vectors expressing sh1005 or Luc shRNA. The cells were harvested 3 days after virus transduction, sorted by the EGFP expression, and further transduced with the lentiviral vector encoding mCherry-CCR5 target sequence at an MOI of 0.5. FL-CD34+ cells were then differentiated to macrophages with 20 ng/ml of IL-3, 50 ng/ml of SCF and 20 ng/ml of MCSF for 9 days and then with 5 ng/ml of GM-CSF for 5 days. The effect of shRNA was monitored by the expressions of mCherry by florescence microscopy and by flow cytometry. The results are exhibited as mCherry dot plots versus EGFP dot plots. The number in each quadrant represents the percentage in each population. The numbers on right side of each panel show the mean fluorescent intensity of the mCherry expression

Figure 8

sh1005 can effectively reduce the levels of endogenous CCR5 mRNA in macrophages derived from FL-CD34+ cells. FL-CD34+ cells (2 × 10⁵) were transduced with lentiviral vectors expressing sh1005 or Luc shRNA. The cells were harvested 3 days after virus transduction and sorted by the EGFP expression. The cells were then differentiated to macrophages with 20 ng/ml of IL-3, 50 ng/ml of SCF and 20 ng/ml of MCSF for 9 days and then with 5 ng/ml of GM-CSF for 5 days. Total RNA were isolated from macrophages using the Qiagen RNeasy extraction kit and treated with DNase I. Quantification of mRNA was performed using IQ5 with iScript one-step RT-PCR kit. RNA standards for CCR5 mRNA quantification were made by serial dilution of in vitro transcribed human CCR5 RNA using T7 RNA polymerase

Figure 9

Stable knockdown of CCR5 expression on macrophages derived from FL-CD34+ cells rendered resistance to R5 tropic HIV-1 NFN-SX infection. FL-CD34+ cells (2 × 10⁵) were transduced with lentiviral vectors expressing sh1005 or muCCR5 shRNA. The cells were harvested 3 days after virus transduction, sorted by the EGFP expression. FL-CD34+ cells (1 × 10⁵) stably expressed sh1005 or the muCCR5 shRNA were treated with 20 ng/ml of IL-3, 50 ng/ml of SCF and 20 ng/ml of MCSF for 9 days and then with 5 ng/ml of GM-CSF for 5 days to differentiate to macrophages. The cells were then infected with either wild-type NFN-SX or VSV-G pseudotyped NFN-SX_ΔEnv, and cell free culture supernatant (1.0 ml) was harvested every 7 days post-infection for 21 days. Virus replication was monitored by measuring p24 antigen in culture supernatants. Mock, cells not infected by NFN-SX; −, nontransduced cells