Novel RNA Duplex Locks HIV-1 in a Latent State via Chromatin-mediated Transcriptional Silencing

Abstract

Transcriptional gene silencing (TGS) of mammalian genes can be induced by short interfering RNA (siRNA) targeting promoter regions. We previously reported potent TGS of HIV-1 by siRNA (PromA), which targets tandem NF-κB motifs within the viral 5'LTR. In this study, we screened a siRNA panel with the aim of identifying novel 5'LTR targets, to provide multiplexing potential with enhanced viral silencing and application toward developing alternate therapeutic strategies. Systematic examination identified a novel siRNA target, si143, confirmed to induce TGS as the silencing mechanism. TGS was prolonged with virus suppression >12 days, despite a limited ability to induce post- TGS. Epigenetic changes associated with silencing were suggested by partial reversal by histone deacetylase inhibitors and confirmed by chromatin immunoprecipitation analyses, which showed induction of H3K27me3 and H3K9me3, reduction in H3K9Ac, and recruitment of argonaute-1, all characteristic marks of heterochromatin and TGS. Together, these epigenetic changes mimic those associated with HIV-1 latency. Further, robust resistance to reactivation was observed in the J-Lat 9.2 cell latency model, when transduced with shPromA and/or sh143. These data support si/shRNA-mediated TGS approaches to HIV-1 and provide alternate targets to pursue a functional cure, whereby the viral reservoir is locked in latency following antiretroviral therapy cessation.

Figure 1

Candidate TGS inducing siRNAs targeting the HIV-1 5′LTR region. (a) Panel of siRNAs designed to target the HIV-1 5′LTR. Relative locations of candidate siRNAs are highlighted in yellow. Positions of previously reported siRNA, of which PromA (shown in red) targeting the NF-κB motifs is the most potent suppressor of HIV-1, while PromB, PromC, and PromD are shown in black. Candidate siRNAs designed to target the R region are shown in green. Sequence specificity controls, siPromA-M2 and siPromA-Sc, contain mismatched base pairs to siPromA. (b) Sequences of all siRNAs targeting the HIV-1 5′LTR region and part of the Tat exon. Bolded siRNA sequences boxed in yellow target the U3 region, while bolded siRNA sequence boxed in green and orange target the R region and Tat exon, respectively. PromA (bolded and boxed red), PromB, PromC, and PromD (bolded and boxed black) are shown for comparison. Nucleotide sequence shown is from HIV-1_NL4.3. (c) Flow cytometry analysis of pseudotyped HIV-1 GFP expression in 293T cells transfected with a panel of candidate siRNAs targeting the HIV-1 5′LTR region. GFP expression was measured 48 hours posttransfection of siRNA. Data shown is mean ± SD from three independent experiments. *P ≤ 0.02, **P ≤ 0.008. The effect of the previously identified lead siRNA candidate for suppressing HIV-1 by TGS, PromA, is shown in red. Statistical comparisons were made between the HIV-1 GFP-pseudotyped positive control culture (black) and candidate siRNA-transfected cultures.

Figure 2

Novel candidate siRNAs targeting the HIV-1 5′LTR region. (a) The regions within HIV-1 5′LTR targeted by 71, 143, and PromA siRNAs are highlighted in blue, green, orange, and red, respectively. (b) Effect of siRNAs on the time course of HIV-1_BaL production in MAGIC-5 cells (HeLa cells stably transfected with CD4, CCR5, and CXCR4). (c) Effect of siRNAs on the time course of HIV-1_SF162 production in HeLa T4+ cells. 5 × 10⁴ MAGIC-5 cells or HeLa-T4+ cells were transfected with 80 pmol/l of the appropriate siRNA, then infected using HIV-1_BaL (100 pg of RT/μl) or HIV-1_SF162 (140 pg of RT/μl), respectively. Supernatants were harvested and virus production assessed using levels of RT activity. SiRNAs 71 (blue) and143 (dark green) profoundly suppressed HIV-1_BaL production for >12 days and at levels comparable to the current lead candidate, PromA (red), while only siRNA 143 (dark green) suppressed HIV-1_SF162 production for >14 days to similar levels. Asterisk indicates a single mismatch in siRNA 71 target sequence of HIV-1_SF162. (d) Subtype B HIV-1_BaL and HIV-1_SF162 sequence alignment at targets of lead siRNA candidates. (e) Consensus sequence alignments of siRNAs 143 and PromA across HIV-1 subtypes A, B, C, D, F, G, and U. The 305 HIV-1 subtype sequences analyzed were obtained using QuickAlign Analysis from the Los Alamos National Laboratory HIV sequence database, which then generated the WebLogo 3 sequence logos^, and show both siRNAs target relatively conserved regions across HIV-1 subtypes. Gaps are indicated by dashes. The sequence logos display a graphical representation of the consensus sequence within a multiple sequence alignment of each HIV-1 subtype, with the stack height indicating sequence conservation at that position and the symbol height within the stack indicating relative nucleic acid frequency.^,

Figure 3

SiRNAs targeting the U3 region of the HIV 5'LTR promoter have limited PTGS activity. (a) Map of the HIV-1 3′LTR under control of the immediate early CMV promoter, with the location of sequences targeted by the selected candidate siRNAs (143/143T; green box, PromA; red box) and positive control siRNAs, PolyA and Nef366; black boxes). Arrows indicate the position of PCR primers used for the detection of HIV-LTR mRNA. (b) Assessment of the extent gene silencing contributed through PTGS following transfection of HeLa T4+ cells, stably expressing CMV-3LTR1-4, with the selected candidate siRNA panel. RT-PCR data are shown as a relative reduction in HIV-mRNA levels normalized to the mock transfection control. Data were normalized to a GAPDH control and statistical comparisons were made between the mock-transfected cultures and siRNA-transfected cultures. Data shown are from three independent experiments (mean ± SEM). *P = 0.028; **P = 0.009.

Figure 4

Drug Reactivation of HIV-1 transcription was observed in HIV-1 cultures suppressed by siRNA candidates. (a) Following HIV-1_SF162 infection (140 pg of RT/μl) for 6 days and then transfection with 80 pmol/l siRNAs 143,143T, PromA, and PromA-M2, HIV infected DMSO control untreated cultures transfected with siRNA 143, and PromA showed suppressed levels of gag mRNA normalised to GAPDH expression compared to specificity control siRNAs 143T and siPromA-M2. Following established virus suppression for 24 hours with siRNAs (b) 143 (dark green), (c) PromA (red), (d) 143T (light green), and (e) M2 (gray), cultures were treated with TSA (50 nmol/l), SAHA (2.5 μmol/l), or TNF (10 ng/ml), or a combination of SAHA (2.5 μmol/l) and TNF (10 ng/ml) for 24 hours, then analyzed for intracellular viral RNA levels by RT-PCR. DMSO was added to untreated cultures at an equivalent concentration to drug treated cultures. Data are shown as a relative reduction in HIV-gag mRNA levels normalized to the HIV infected siRNA transfected DMSO control. Data shown are from three independent experiments (mean ± SEM). *P = 0.026, **P ≤ 0.002, ***P = 0.0002, ****P < 0.0001. All statistical analyses were performed using a Mann–Whitney test comparing the siRNA alone control with the drug activation cultures.

Figure 5

Heterochromatin markers were assessed in candidate siRNA-transfected cultures. (a) Arrows indicate the location of PCR primers used for SYBR-Green–based PCR amplification of DNA isolated by the ChIP process. Enrichment of heterochromatin markers (b) H3K27me3, (c) H3K9me3, and (d) Ago1 was found in siRNA 143, 143T, and PromA-transfected cultures compared to 48 hours mock-transfected cultures following 3 days of HIV-1_SF162 infection. (e) Reduction of H3K9Ac expression was found in siRNA 143, 143T, and PromA-transfected cultures compared to mock-transfected cultures. Data shown are from three independent experiments (mean ± SEM). *P ≤ 0.05, ****P < 0.0001. Percentage of input was calculated, and data were normalized to the mock-transfected cultures for H3K27me3, H3K9me3, Ago1, and H3K9Ac.

Figure 6

SiRNA 143 mimetic targeting the HIV-1 5'LTR does not induce off target effects. (a) HeLa T4+ cells were transfected with appropriate siRNAs for 48 hours. The mRNA expression levels of four IFNα response genes, ISG20, Viperin, IFIT1, and OAS1 were detected by SYBR-Green–based quantitative real-time PCR assays as described. ISG20, Viperin, IFIT1, and OAS1 were not elevated in cells transfected with candidate siRNAs. The fold change for each mRNA is shown. Mean ± SEM are plotted from triplicate experiments, and the data was normalized to the HeLa T4+ mock-transfected control. **P < 0.006, ***P = 0.003, ****P < 0.0001. (b) Activation of phosphorylated PKR was not detected in cells transfected with novel candidate siRNA for 48 hours. Phosphorylated dsRNA-dependent protein kinase R (P-PKR) was detected as described. Immunoblots were incubated with mouse anti-β-actin (A5441 clone AC-15, Sigma) or rabbit polyclonal antiphosphorylated PKR (pT451, BioSource, Invitrogen, Mulgrave, Australia), developed using Immuno-Star HRP chemiluminescent reagents (Bio-Rad) and visualized using a G:Box chemiluminescence imager and software (SynGene). β-Actin was used as a loading control. The positive controls were cells treated with IFNα (500 IU/ml) for 24 hours or cells treated with Poly(I:C) (10 ng/ml) for 6 h. (c) HeLa-T4+ cells transfected with candidate siRNA mimetics (143; dark green, 143T; light green, PromA; red and M2; black line overlay) display no change in expression of CD4 or CXCR4 when compared to mock-transfected cultures (gray area) at 48 hours posttransfection.

Figure 7

Combining si/shRNAs PromA and 143 suppresses HIV in HeLa cells and diminishes reactivation in J-Lat 9.2 cells. (a) HeLa T4+ cells were infected with HIV-1_SF162, then co-transfected with siRNAs PromA and 143 or single siRNA treatments. RT activity was measured over an 18-day time course. (b) Structure of self-inactivating (SIN) lentivirus vector. SIN vector contains central polypurine tract (cPPT), U6 promoter (U6P), short hairpin RNA (shRNA), Simian virus 40 promoter (SV40), and mCherry reporter gene (mCherry). The mutant woodchuck promoter response element (WPREmt) and modified LTR, allow integration but not expression of viral genome. We utilized U6 promoter-driven shRNA PromA, 143 and control expression SIN lentivirus vectors with a mCherry reporter. (c) Reactivation of J-Lat 9.2 cells stably transduced with lentivirus vectors carrying shPromA, sh143, shPromA, and sh143 lentivirus vectors or control, treated with SAHA at various concentrations for 48 hours, show increased GFP expression upon activation. (d) Reactivation of J-Lat 9.2 cells stably transduced with lentivirus vectors carrying shPromA, sh143, shPromA, and sh143 lentivirus vectors or control, treated with TNF at various concentrations for 48 hours, show increased GFP expression upon activation. (e) Reactivation of J-Lat 9.2 cells stably transduced with lentivirus vectors carrying shPromA, sh143, shPromA, and sh143 lentivirus vectors or control, treated with a combination of SAHA and TNF at various concentrations for 48 hours, showed increased GFP expression upon activation.

Figure 8

shPromA induces potent HIV-1 suppression in monocyte-derived macrophages. Monocyte derived macrophages (MDMs) were purified from PBMCs and differentiated for 7 days prior to being transduced with lentiviral vector expressing shPromA (red bar), shPromA-M2 (gray bar) or mock-transduced (black bar). MDM cultures were transduced for 5 days prior to infection with HIV-1_JRFL for 8 days. (a) RT activity was measured 8 days postinfection and showed potent virus suppression in shPromA-transduced MDM cultures. (b) HIV-1 mRNA levels were analyzed 8 days postinfection and also showed potent virus suppression in shPromA-transduced MDM cultures. **P ≤ 0.02, *P = 0.05.

Figure 9

Schematic model of transcription factors and targeted sites of siRNA mimetics 143 and PromA in the HIV-1 5′LTR. Nucleosomes are comprised of histone 1 linker (gray) and four histone pairs (shown in green, orange, purple, and pink) that generate the histone octamer. Activator protein 1 (AP-1), COUP-TF, upstream stimulatory factor 1/2 (USF 1/2), nuclear factor of activated T cells (NFAT), glucocorticoid receptor (GR), transcription factor II-I (TF II-I), yin yang 1 (YY1), NF-κB, specificity protein 1 (SP1), late SV40 factor (LSF), specificity protein 3 (SP3).