20th International AIDS Conference - Melbourne, Australia

Abstract

TUPDA0104 - Poster Discussion


RNA targeting the promoter region potently inhibits HIV-1 activation from latently infected cells

Presented by Kazuo Suzuki (Australia).

K. Suzuki1, T. Tsukamoto2, K. Marks2, D. Cooper2, A. Kelleher2


1St Vincent's Centre for Applied Medical Research, The Kirby Institute, Faculty of Medicine, University of NSW, Darlinghurst, Australia, 2The Kirby Institute, Darlinghurst, Australia

Background: We reported previously that certain RNA targeting the HIV-1 promoter region can induce transcriptional gene silencing (TGS), which is associated with heterochromatin formation in the HIV-1 promoter region to suppress HIV-1 transcription. We investigated whether or not the RNA based TGS approach is able to inhibit reactivation of HIV-1 in latently infected cells.
Methods: We transduced HIV-1 latently infected U1 cells, with a VSV-g pseudotyped lentiviral delivery system to express short-hairpin-RNA homologous to a conserved sequence in the HIV promoter (shPromA). We also transduced U1 cells with a 2-based mismatched variant (shPromA-M2), and scrambled (shPromA-Sc) as specificity control. These transduced U1 cells were then cultured in the presence of various stimuli, proinflammatory cytokine (TNF-α, 10ng/ml), histone deacetylase inhibitor (Trichostatin-A, 0.5µM), hematopoietic growth factor (GM-CSF, 1ng/ml) for 3 days. RT assays were used to determine the extent of newly related virus in the culture supernatants following activation.
Results: RT data indicated that massive amount of de-novo HIV-1 was produced from original latently infected U1 cells with TNF-α. RT levels were reduced by over 100 fold in culture supernatant of shPromA transduced-U1 cells, while shPromA-M2 transduced-U1 and shPromA-Sc transduced-U1 cells failed to suppress HIV-1 production from activated U1 cells. Similarly the shPromA transduced-U1 cells inhibited production of HIV-1 in the presence of other stimuli, Trichostatin-A and GM-CSF.
Conclusions: These findings suggested that shRNA targeting promoter region was able to maintain viral latency in U1 cells even in the presence of the strong activating stimuli. The induced suppression mediated by the shRNA was sequence specific. The shPromA seems to maintain the heterochromatin that characteristically associated with the promoter of the latent pro-virus preventing viral activation. TGS induced by shRNAs targeting the HIV-1 promoter region may be a useful strategy to sustain HIV latency and provide a pathway to a functional cure in future.


Back to the Programme-at-a-Glance