Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages - PubMed (original) (raw)

Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages

Erwei Song et al. J Virol. 2003 Jul.

Erratum in

Abstract

Small interfering RNAs (siRNAs) can induce potent gene silencing by degradation of cognate mRNA. However, in dividing cells, the silencing lasts only 3 to 7 days, presumably because of siRNA dilution with cell division. Here, we investigated if sustained siRNA-mediated silencing of human immunodeficiency virus type 1 (HIV-1) is possible in terminally differentiated macrophages, which constitute an important reservoir of HIV in vivo. CCR5, the major HIV-1 coreceptor in macrophages, and the viral structural gene for p24 were targeted either singly or in combination. When transfected 2 days prior to infection, both CCR5 and p24 siRNAs effectively reduced HIV-1 infection for the entire 15-day period of observation, and combined targeting of both genes abolished infection. To investigate whether exogenously introduced siRNA is maintained stably in macrophages, we tested the kinetics of siRNA-mediated viral inhibition by initiating infections at various times (2 to 15 days) after transfection with CCR5 and p24 siRNAs. HIV suppression mediated by viral p24 siRNA progressively decreased and was lost by day 7 posttransfection. In contrast, viral inhibition by cellular CCR5 knockdown was sustained even when transfection preceded infection by 15 days, suggesting that the continued presence of target RNA may be needed for persistence of siRNA. The longer sustenance of CCR5 relative to p24 siRNA in uninfected macrophages was also confirmed by detection of internalized siRNA by modified Northern blot analysis. We also tested the potential of p24 siRNA to stably silence HIV in the setting of an established infection where the viral target gene is actively transcribed. Under these circumstances, long-term suppression of HIV replication could be achieved with p24 siRNA. Thus, siRNAs can induce potent and long-lasting HIV inhibition in nondividing cells such as macrophages.

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Figures

FIG. 1.

FIG. 1.

Efficient delivery of duplex siRNA into macrophages. MDMs were mock transfected or exposed to Cy5-labeled p24 siRNA in the presence or absence of transfection reagent. After 24 h of culture, the cells were removed by trypsinization, stained with anti-CD14-FITC, and analyzed by flow cytometry. The percentage of Cy5+ cells is indicated in each panel.

FIG. 2.

FIG. 2.

CCR5 and p24 siRNAs inhibit HIVBAL infection in MDMs. (a) MDMs were transfected with the indicated doses of CCR5 (▪) or p24 (□) siRNA and infected 2 days later with HIVBAL. Cell-free virus production was measured on day 7 postinfection by p24 ELISA. (b) MDMs were either mock transfected (♦) or transfected with the GFP (▪), p24 (▴), or CCR5 (×) siRNA or with the p24 and CCR5 siRNAs (*) and infected after 2 days with HIVBAL, and virus production was measured by p24 ELISA at the indicated times postinfection. (c) The siRNA-transfected cells described in panel b were stained with anti-p24-FITC 15 days after infection and examined by flow cytometry. The percentage of p24+ cells is shown in each panel. (d) siRNA-transfected and HIVBAL-infected MDMs were probed for HIV-1 RNA by in situ hybridization with a fluorescein-labeled HIV-1 gag-pol oligonucleotide probe cocktail 7 days after infection. Fluorescence microscopy (magnification, ×200) was used to evaluate fluorescence signals for HIV-1 RNA (bottom). At the top are the same cells counterstained with Texas red-X phalloidin.

FIG. 3.

FIG. 3.

CCR5, but not p24, siRNA persists in uninfected MDMs. (a) Modified Northern blot analysis showing levels of internalized CCR5 and p24 siRNAs in MDMs on the indicated days after transfection. Before loading, samples were normalized for total RNA content. The sense strand of each siRNA was end labeled with γ-32P and used as a probe. Lanes loaded with graded amounts of the antisense strand of siRNA and mock-transfected samples served as positive and negative controls, respectively. (b) CCR5 (top) and GFP (bottom) siRNA-transfected MDMs were examined for CCR5 expression over time. Overlay histograms of CCR5-stained mock-transfected (open solid line), control immunoglobulin-stained (open dotted line), and siRNA-transfected (filled) cells are shown. (c) RT-PCR for CCR5 and γ-actin mRNA expression was performed with mock-transfected (lanes 2 to 5) and CCR5 siRNA-transfected (lanes 6 to 9) cells on days 1 (lanes 2 and 6), 4 (lanes 3 and 7), 7 (lanes 4 and 8), and 15 (lanes 5 and 9) after transfection (M, molecular weight marker; lane 1, negative control). CCR5 mRNA was not detected, even after an additional 25 cycles of PCR amplification (data not shown).

FIG. 4.

FIG. 4.

CCR5, but not p24, siRNA confers sustained and uniform protection when MDMs are infected at increasing intervals after transfection. MDMs were transfected with GFP (top), p24 (middle), or CCR5 (bottom) siRNA and infected with HIVBAL at the indicated times after transfection. Cells were analyzed 10 days postinfection for p24 expression by flow cytometry. The percentage of p24+ cells is shown in each panel.

FIG. 5.

FIG. 5.

p24, but not CCR5, siRNA suppresses HIV-1 replication in an established infection. (a) MDMs infected with HIVBAL for 16 days (>90% of the MDMs were p24+; data not shown) were transfected with CCR5 siRNA and examined for p24 expression 3 days later. The percentage of p24+ cells is shown in each panel. (b) MDMs infected for 16 days were transfected with p24 or control siRNA and examined for p24 expression on various days posttransfection.

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