Designed zinc finger protein interacting with the HIV-1 integrase recognition sequence at 2-LTR-circle junctions

Integration of HIV-1 cDNA into the host genome is a crucial step for viral propagation. Two nucleotides, cytosine and adenine (CA), conserved at the 3′ end of the viral cDNA genome, are cleaved by the viral integrase (IN) enzyme. As IN plays a crucial role in the early stages of the HIV-1 life cycle...

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Main Authors: Supachai Sakkhachornphop, Supat Jiranusornkul, Kanchanok Kodchakorn, Sawitree Nangola, Thira Sirisanthana, Chatchai Tayapiwatana
Format: Journal
Published: 2018
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http://cmuir.cmu.ac.th/jspui/handle/6653943832/59337
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Institution: Chiang Mai University
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spelling th-cmuir.6653943832-593372018-09-10T03:14:01Z Designed zinc finger protein interacting with the HIV-1 integrase recognition sequence at 2-LTR-circle junctions Supachai Sakkhachornphop Supat Jiranusornkul Kanchanok Kodchakorn Sawitree Nangola Thira Sirisanthana Chatchai Tayapiwatana Biochemistry, Genetics and Molecular Biology Integration of HIV-1 cDNA into the host genome is a crucial step for viral propagation. Two nucleotides, cytosine and adenine (CA), conserved at the 3′ end of the viral cDNA genome, are cleaved by the viral integrase (IN) enzyme. As IN plays a crucial role in the early stages of the HIV-1 life cycle, substrate blockage of IN is an attractive strategy for therapeutic interference. In this study, we used the 2-LTR-circle junctions of HIV-1 DNA as a model to design zinc finger protein (ZFP) targeting at the end terminal portion of HIV-1 LTR. A six-contiguous ZFP, namely 2LTRZFP was designed using zinc finger tools. The designed motif was expressed and purified from E. coli to determine its binding properties. Surface plasmon resonance (SPR) was used to determine the binding affinity of 2LTRZFP to its target DNA. The level of dissociation constant (Kd) was 12.0 nM. The competitive SPR confirmed that 2LTRZFP specifically interacted with its target DNA. The qualitative binding activity was subsequently determined by EMSA and demonstrated the aforementioned correlation. In addition, molecular modeling and binding energy analyses were carried out to provide structural insight into the binding of 2LTRZFP to the specific and nonspecific DNA target. It is suggested that hydrogen-bonding interactions play a key role in the DNA recognition mechanisms of the designed ZFP. Our study suggested an alternative HIV therapeutic strategy using ZFP interference of the HIV integration process. Published by Wiley-Blackwell. © 2009 The Protein Society. 2018-09-10T03:14:01Z 2018-09-10T03:14:01Z 2009-11-01 Journal 1469896X 09618368 2-s2.0-70350511438 10.1002/pro.233 https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=70350511438&origin=inward http://cmuir.cmu.ac.th/jspui/handle/6653943832/59337
institution Chiang Mai University
building Chiang Mai University Library
country Thailand
collection CMU Intellectual Repository
topic Biochemistry, Genetics and Molecular Biology
spellingShingle Biochemistry, Genetics and Molecular Biology
Supachai Sakkhachornphop
Supat Jiranusornkul
Kanchanok Kodchakorn
Sawitree Nangola
Thira Sirisanthana
Chatchai Tayapiwatana
Designed zinc finger protein interacting with the HIV-1 integrase recognition sequence at 2-LTR-circle junctions
description Integration of HIV-1 cDNA into the host genome is a crucial step for viral propagation. Two nucleotides, cytosine and adenine (CA), conserved at the 3′ end of the viral cDNA genome, are cleaved by the viral integrase (IN) enzyme. As IN plays a crucial role in the early stages of the HIV-1 life cycle, substrate blockage of IN is an attractive strategy for therapeutic interference. In this study, we used the 2-LTR-circle junctions of HIV-1 DNA as a model to design zinc finger protein (ZFP) targeting at the end terminal portion of HIV-1 LTR. A six-contiguous ZFP, namely 2LTRZFP was designed using zinc finger tools. The designed motif was expressed and purified from E. coli to determine its binding properties. Surface plasmon resonance (SPR) was used to determine the binding affinity of 2LTRZFP to its target DNA. The level of dissociation constant (Kd) was 12.0 nM. The competitive SPR confirmed that 2LTRZFP specifically interacted with its target DNA. The qualitative binding activity was subsequently determined by EMSA and demonstrated the aforementioned correlation. In addition, molecular modeling and binding energy analyses were carried out to provide structural insight into the binding of 2LTRZFP to the specific and nonspecific DNA target. It is suggested that hydrogen-bonding interactions play a key role in the DNA recognition mechanisms of the designed ZFP. Our study suggested an alternative HIV therapeutic strategy using ZFP interference of the HIV integration process. Published by Wiley-Blackwell. © 2009 The Protein Society.
format Journal
author Supachai Sakkhachornphop
Supat Jiranusornkul
Kanchanok Kodchakorn
Sawitree Nangola
Thira Sirisanthana
Chatchai Tayapiwatana
author_facet Supachai Sakkhachornphop
Supat Jiranusornkul
Kanchanok Kodchakorn
Sawitree Nangola
Thira Sirisanthana
Chatchai Tayapiwatana
author_sort Supachai Sakkhachornphop
title Designed zinc finger protein interacting with the HIV-1 integrase recognition sequence at 2-LTR-circle junctions
title_short Designed zinc finger protein interacting with the HIV-1 integrase recognition sequence at 2-LTR-circle junctions
title_full Designed zinc finger protein interacting with the HIV-1 integrase recognition sequence at 2-LTR-circle junctions
title_fullStr Designed zinc finger protein interacting with the HIV-1 integrase recognition sequence at 2-LTR-circle junctions
title_full_unstemmed Designed zinc finger protein interacting with the HIV-1 integrase recognition sequence at 2-LTR-circle junctions
title_sort designed zinc finger protein interacting with the hiv-1 integrase recognition sequence at 2-ltr-circle junctions
publishDate 2018
url https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=70350511438&origin=inward
http://cmuir.cmu.ac.th/jspui/handle/6653943832/59337
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