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.
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.
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