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Adenosine Deaminase Acting on RNA (ADAR1) is a Novel Multitargeted Anti HIV-1 Cellular Protein

Biswas, Nabanita (2011) Adenosine Deaminase Acting on RNA (ADAR1) is a Novel Multitargeted Anti HIV-1 Cellular Protein. Doctoral Dissertation, University of Pittsburgh.

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    Abstract

    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.


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    Item Type: University of Pittsburgh ETD
    Creators/Authors:
    CreatorsEmailORCID
    Biswas, Nabanitanabanita.biswas@gmail.com
    ETD Committee:
    ETD Committee TypeCommittee MemberEmailORCID
    Committee ChairGupta, Phalgunipgupta1@pitt.edu
    Committee MemberAmbrose, Zandreazaa4@pitt.edu
    Committee MemberMontelaro, Ronaldrmont@pitt.edu
    Committee MemberReinhart, Toddreinhar@pitt.edu
    Title: Adenosine Deaminase Acting on RNA (ADAR1) is a Novel Multitargeted Anti HIV-1 Cellular Protein
    Status: Unpublished
    Abstract: 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.
    Date: 23 September 2011
    Date Type: Completion
    Defense Date: 19 July 2011
    Approval Date: 23 September 2011
    Submission Date: 26 July 2011
    Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
    Patent pending: No
    Institution: University of Pittsburgh
    Thesis Type: Doctoral Dissertation
    Refereed: Yes
    Degree: PhD - Doctor of Philosophy
    URN: etd-07262011-204453
    Uncontrolled Keywords: ADAR1; Antiviral; Cellular Protein; HIV-1
    Schools and Programs: Graduate School of Public Health > Infectious Diseases and Microbiology
    Date Deposited: 10 Nov 2011 14:54
    Last Modified: 20 Jan 2012 13:22
    Other ID: http://etd.library.pitt.edu/ETD/available/etd-07262011-204453/, etd-07262011-204453

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