ADAR1 is an RNA editing enzyme which acts on completely or partially double-stranded RNA. Since HIV-1 RNA has such secondary structures, we have examined whether ADAR1 exhibits antiviral activity against HIV-1. Our results indicated that ADAR1 inhibited viral replication and infectious HIV-1 production in various cell lines including 293T, HeLa and Jurkat T cells, and was active against a number of X4- and R5-tropic HIV-1 of different clades. Analysis of the level of intracellular HIV-1 RNA showed no change in levels of intracellular gag, pol, and env RNA in the presence of ADAR1 despite a significant inhibition of intracellular and virion associated HIV-1 protein production. Furthermore mutational analysis showed that ADAR1 introduced most of the A-to-G mutations in the first exon of rev at positions 5998, 6011, 6017, and 6036 and in the Rev Response Element (RRE) binding region (positions 8413 and 8438) of rev and env RNA. In elucidating the mechanism of ADAR1 inhibition of HIV-1, we observed that A-G mutations in rev have a significant negative effect on the expression of Rev. However, all mutations could be complemented by wild type Rev.Furthermore, these A-G mutations in the RRE binding region of rev inhibited the binding of Rev to the RRE region in env and inhibited transport of primary transcripts like gag, pol and env from the nucleus to the cytoplasm. Introduction of these specific mutations in rev of an infectious molecular clone of HIV-1 by site directed mutagenesis abolished the replication capacity of HIV-1 by inhibiting viral protein synthesis without any effect on viral RNA synthesis, a phenotype exhibited by HIV-1-infected cells exposed to ADAR1. ADAR1 induced mutations in env further attenuated viral infectivity. ADAR1, thus, constitutes a novel class of cellular antiviral proteins with multiple targets in the viral genome thereby providing a new avenue of exploration for therapeutic drugs benefitting public health.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-07262011-204453 |
| Date | 23 September 2011 |
| Creators | Biswas, Nabanita |
| Contributors | Professor Phalguni Gupta, Dr. Zandrea Ambrose, Professor Todd Reinhart, Professor Ronald Montelaro |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-07262011-204453/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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