Purchase this article with an account.
Natalia Nikitenko, Anastasia Klimova, Pavel Spirin, Peter Groitl, Thomas Speiseder, Vladimir Prassolov; Targeted Suppression of Human Adenovirus DNA-polymerase Gene by RNA Interference. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2726.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Epidemic keratoconjunctivitis is a hyperacute and highly contagious infection of the eye caused by human adenovirus types within species D, in particular, serotypes D8, D19, and D37. Currently there is no causally directed treatment available that is effective against epidemic keratoconjunctivitis. The E2B region of the adenovirus genome encodes for DNA polymerase (pol) that is required for adenoviral DNA replication. We assume that blocking of pol mRNA translation will stop the adenoviral infection. The purpose of our study was to develop a model system for studying DNA-polymerase inhibitors and elaborate an approach for significant reduction of pol expression by RNA interference (RNAi).
To express the gene pol of human adenovirus D36 (pol-D36) in the model cell lines a set of recombinant lentiviral vectors was constructed, containing pol-D36 and tdTomato (tandem tomato fluorescent protein) cDNAs separated by an IRES sequence. The transgenic cell lines expressing pol-D36 and the fluorescent marker gene were obtained from human lung carcinoma cell lines A549, H1299 transduced with the corresponding recombinant lentiviruses. We designed 3 types of small interfering RNAs (siRNA) complementary to the pol mRNA sequences of serotypes D8, D19, D36, and D37. In addition, based on the sequence of the siRNA with the highest level of gene inhibition, a small hairpin RNA (shRNA) encoding vector was designed.
Inhibition of pol-D36 expression was observed within 48 h post transfection of 3 siRNAs types by flow cytometry and qPCR. The fluorescence signal of tdTomato decreased 2.5, 2.0 and 1.5 fold respectively corresponding to control cells. The amount of pol-D36 mRNA decreased 5.0, 2.5 and 1.7 fold respectively. The progress of the shRNA biological activity was monitored 14 days post transduction of transgenic cells A549, H1299 by qPCR and Western blot. The amount of pol-D36 mRNA decreased 4.5 and 3.5 fold respectively, compared to the control cells transduced with shRNA expressing lentiviral vectors targeting nonspecific sequences. The gene suppression was confirmed by Western blot and reduced steady state protein levels.
We proposed a powerful model system to test viral key factor, i.e. DNA-polymerase, as putative target to develop a highly efficient antiviral therapy based on the inhibitory effects of siRNAs and showed significantly reduced expression levels of the corresponding polymerase.
This PDF is available to Subscribers Only