A siRNA targeting the HIV-1 promoter inhibits HIV-1 replication: (A) Location of sequence targeted by the siRNA. The positioning of NF-κB binding sites in the 5′LTR are indicated by red dots; the blue shading represents the target sequence of the siRNA (HIV-prom-A); the underlining indicates the sequence of the tandem NF-κB binding motifs. (B) Effect of short siRNAs on the time course of HIV-1 (NL4-3) production in MAGIC-5 cells: At day 7 siRNAs were transfected into productively infected cells (indicated by arrow). Reverse transcriptase (RT) levels are shown for cultures transfected with HIV-prom-A siRNA (blue triangle) and scrambled siRNA (red circle), mock transfected cells (green square), and uninfected cells (black square). (C) HIV-1 proviral DNA is present in HIV-prom-A siRNA transfected cells. Provirus was detected by PCR amplification of 154bp fragment of HIV-1 gag gene from DNA extracted from MAGIC-5 cells 25 days after infection. NC indicates non-infected MAGIC-5 cells. (D) Abrogation of HIV-1 protein synthesis induced by HIV-prom-A siRNA shown by western blot analysis of HIV-Gag-p24, Gag precursor protein p55 and beta-actin expression in cell lysates of MAGIC-5 cells 10 days after infection with NL4-3. (E) HIV-1 RNA is markedly reduced in the nuclei of cells transfected with HIV-prom-A siRNA. Viral RNA was quantified by real time PCR in the nuclei isolated from MAGIC-5 cells either 10 days or 20 days after infection. HIV viral RNA copy number is shown as copies per 1000 copies of beta-actin. NC indicates non-infected MAGIC-5 cells.

A siRNA inhibits replication of 5 different strains of HIV-1. Graphs show kinetics of viral replication following transfection of HIV-prom-A siRNA into MAGIC-5 cells 5 hours after infection with laboratory strains of HIV-1 (NL 4-3, IIIB/LAI, and RF) or two clinical isolates (CL-1 and CL-2) from patients failing multiple combination anti-retroviral regimens (Kaufmann et al, 2001). HIV infection was allowed to establish during a 5 hour incubation. Thereafter, cells were transfected with HIV-prom-A siRNA. Infection of MAGIC-5 cells was confirmed by HIV DNA-PCR (data not shown). Reverse transcriptase (RT) levels are shown for cultures transfected with HIV-prom-A siRNA (blue triangle), mock transfected cells (red circle), and uninfected cells (black square). Black arrow indicates timing of transfection.

Nuclear run-on assays indicate transcriptional suppression by HIV-prom-A siRNA. Results of a representative assay (n = 3) in which cDNAs were immobilized on nylon membranes, and probed with RNA extracted from isolated the nuclei after run-on transcription with ³²P-labelled UTP (guide strip at bottom; blank: no cDNA). De novo transcription of HIV-1 is evident in the mock-transfected culture and the culture transfected with the scrambled siRNA. HIV-1 transcription is absent from uninfected cells (NC) and cells transfected with HIV-prom-A siRNA.

HIV-prom-A siRNA induces CpG methylation of the HIV-1 LTR and transcriptional suppression. (A) The DNA from HIV-1 provirus in cells transfected with HIV-prom-A, B, and D siRNA is protected from digestion with the methylation-sensitive restriction enzyme HpaII. DNA was extracted from HIV-1 infected cells at 14, 21, and 38 day after infection (day 7, 14, and 31 post-transfection respectively), digested with HpaII and subjected to PCR using primers with binding sites denoted by arrows above the map. Nucleotide numbering is relative to the transcription initiation site. The downstream primer is located outside the LTR, so that this primer set specifically amplifies only the 5′ LTR. The restriction enzyme HpaII is methylation sensitive such that it will not cut if the CpG in the CCGG recognition site is methylated. HpaII cuts this region of HIV-1 at the single CpG site at position −146 (labeled HpaII); PCR amplification will occur only if the DNA between the primer binding sites has not been cut. W/O indicates that DNA has not been subjected to HpaII digestion. Amplification of proviral DNA from mock-transfected cells was abrogated by pre-digestion with HpaII, indicating that it was unmethylated. By contrast amplification from HpaII-digested DNA is successful from the cultures transfected with HIV-prom-A, B, and D siRNA, indicating that the HpaII sites were methylated. (B) Bisulfite allelic sequencing of the 5′ LTR of the HIV-1 provirus at 38 day after infection. Bisulfite-modified genomic DNA was PCR amplified with nested primers specific for the bisulfite-converted sequence (see Methods) and ligated into a plasmid vector; at least 10 individual plasmid clones were sequenced. Methylated cytosines are spared from bisulfite conversion, while unmethylated cytosines are converted to uracils. The figure shows sequences of individual clones as horizontal lines, with the CpGs numbered as in Figure 6A; unfilled boxes are unmethylated CpGs, and filled boxes are methylated CpGs. (C) The demethylating agent 5-azacytidine (5-aza-C) partially restores HIV transcription in cells previously transfected with HIV-prom-A siRNA. Real-time PCR amplification of the gag region from RNA was used to detect productive infection in MAGIC-5 cells. Six days after transfection with siRNA cells were treated with 5-aza-C at the indicated concentrations for 30 hours. The positive control (PC) was a culture in which no siRNA was transfected, and the negative control (NC) was uninfected cells. The results are shown as HIV RNA copy number per 1000 copies of beta-actin.

Prom-A siRNA inhibits syncytia formation and host cell death. (A) HIV-prom-A siRNA reduces syncytium formation in infected MAGIC-5 cells (Phase-contrast microscopy of MAGIC-5 cells at 200X magnification). The time points indicate days after infection (see Fig. 1B). The prominent syncytia seen in the mock-transfected cells are absent from the HIV-prom-A siRNA transfected culture. (B) Caspase-3 activity is reduced to normal levels in HIV-1 infected cells by transfection of HIV-prom-A siRNA. Caspase-3 activity (red) is detected in MAGIC-5 cells transfected with siRNA 7 days earlier. DAPI staining (blue) outlines the nuclei, and cell structure was highlighted by Vimentin staining (green). NC indicates non-infected MAGIC-5 cells.

Specific suppression achieved after transfection of prom-A siRNA. (A) The promoter regions of HIV-1, HIV-2, and ICAM-1 are aligned. Sequence corresponding to HIV-prom-A siRNA highlighted in blue. Non-homologous bases in HIV-2 and ICAM-1 are indicated in red. Underlining indicates the NF-κB binding sites. (B) HIV-prom-A siRNA does not inhibit HIV-2 infection. Histogram shows RT levels 8 days after transfection with siRNAs, 8hours after infection of MAGIC-5 cells with laboratory stain HIV-2 CBL-20. No marked difference was observed among cultures transfected with HIV-prom-A, scrambled siRNA or mock transfection. NC indicates non-infected MAGIC-5 cells. (C) ICAM-1 gene expression is not affected by HIV-prom-A siRNA transfection. MAGIC-5 cells were infected with HIV-1 molecular clone NL4-3 for 5 hours followed by transfection with siRNAs. Expression levels of HIV-1 gag and ICAM-1 mRNA at day 3 post infection are shown in the histograms normalized to 10³ copies of β-actin mRNA. Mean and standard deviations were obtained from three different experiments. Mock: transfection with lipofectamine only, NC: non-infected Magic-5 cells.

Effect of six short siRNAs on productive HIV-1 infection including 4 siRNAs targeting HIV-1 promoter region and siRNA HIV-gag, which is known act as PTGS. (A) The sequences within HIV-1 5′LTR targeted by HIV-prom-A-D siRNAs are indicated by highlighting. HIV-prom-B siRNA target sequence includes CpG sites at −159 and −146, HIV-prom-C siRNA target sequence includes CpG at −120, and HIV-prom-D siRNA target sequence includes CpG at −48. CpGs within the 5′LTR of HIV-1 are indicated by emboldened text. Nucleotide numbering is relative to the transcription start site and sequence is that of HIV-1 strain NL4-3. Each siRNA contains at least one CpG site. (B) Effect of siRNAs on the time course of HIV-1 (NL4-3) production in MAGIC-5 cells. At day 7 post-infection siRNAs were transfected into the cells (indicated by arrow). Reverse transcriptase (RT) levels are shown for cultures transfected with HIV-prom-A, B, C, D, HIV-1-gag, and scrambled siRNAs as indicated in the figure legend. (C) HIV-1 proviral DNA is present in cultures transfected with HIV-prom siRNAs regardless of whether infection is productive or not. PCR amplification of proviral DNA extracted from MAGIC-5 cells 38 days after infection (Figure 6B). The amplified region is a 154bp fragment of the HIV-1 gag gene. NC indicates non-infected MAGIC-5 cells.