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Transcriptional modulation of HIV-1C LTR promoter
BMC Infectious Diseases volume 12, Article number: O6 (2012)
All current anti-HIV1 therapies target the viral proteins or RNA; however targeting HIV1 at the transcriptional level of the integrated provirus has been less explored. In India, AIDS is commonly caused by HIV-1C compared to HIV-1B in developed countries. HIV1-5’LTR acts as a promoter and shows sequence variation among different clades. Transcriptional gene silencing (TGS) is a method wherein dsRNA targeting the promoter/enhancer of a gene are used to down regulate its expression.
We used SiHa cell line stably expressing a bi-cistronic reporter system (5’LTR-SEAP-IRES-EGFP), in which secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein (EGFP) are expressed under 5’LTR of HIV-1B/C. The cell line was transfected with different dsRNAs (S1-S6) targeting the core promoter/enhancer of HIV-1C LTR to induce TGS. Screening for decreased transcription was done using real-time PCR (mRNA expression of SEAP and EGFP), fluorescence microscopy (EGFP) and flow cytometry (EGFP).
After single or multiple (thrice) transfection of dsRNAs, we identified one dsRNA (S4) which showed consistent and significant down regulation of both SEAP (44% & 68% respectively) and EGFP (40% & 65%) (p<0.001 in both cases) mRNA levels. This reporter down regulation was also confirmed by studying EGFP expression using fluorescence microscopy and flowcytometry which also showed a significant fall after S4 transfection.
TGS usually involves epigenetic modifications like DNA methylation/histone methylation at the targeted region and induces long term suppression of gene expression. So targeting of the HIV-1C LTR by dsRNA can be used as a therapeutic modality in the future.
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Singh, A., Ramalingam, P., Palanichamy, J.K. et al. Transcriptional modulation of HIV-1C LTR promoter. BMC Infect Dis 12, O6 (2012). https://doi.org/10.1186/1471-2334-12-S1-O6
- Alkaline Phosphatase
- Green Fluorescent Protein
- Fluorescence Microscopy
- Viral Protein
- Enhance Green Fluorescent Protein