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      Characterization of the Deamination Coupled with Sliding along DNA of Anti-HIV Factor APOBEC3G on the Basis of the pH-Dependence of Deamination Revealed by Real-Time NMR Monitoring

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          Abstract

          Human APOBEC3G (A3G) is an antiviral factor that inactivates HIV. The C-terminal domain of A3G (A3G-CTD) deaminates cytosines into uracils within single-stranded DNA (ssDNA), which is reverse-transcribed from the viral RNA genome. The deaminase activity of A3G is highly sequence-specific; the third position (underlined) of a triplet cytosine (CC C) hotspot is converted into CCU. A3G deaminates a CCC that is located close to the 5′ end of ssDNA more effectively than ones that are less close to the 5′ end, so-called 3′ → 5′ polarity. We had developed an NMR method that can be used to analyze the deamination reaction in real-time. Using this method, we previously showed that 3′ → 5′ polarity can be explained rationally by A3G-CTD's nonspecific ssDNA-binding and sliding direction-dependent deamination activities. We then demonstrated that the phosphate backbone is important for A3G-CTD to slide on the ssDNA and to exert the 3′ → 5′ polarity, probably due to an electrostatic intermolecular interaction. In this study, we investigate the pH effects on the structure, deaminase activity, and 3′ → 5′ polarity of A3G-CTD. Firstly, A3G-CTD was shown to retain the native structure in the pH range of 4.0–10.5 by CD spectroscopy. Next, deamination assaying involving real-time NMR spectroscopy for 10-mer ssDNA containing a single CCC revealed that A3G-CTD's deaminase activity decreases as the pH increases in the range of pH 6.5–12.7. This is explained by destabilization of the complex between A3G-CTD and ssDNA due to the weakened electrostatic interaction with the increase in pH. Finally, deamination assaying for 38-mer ssDNA having two CCC hotspots connected by a long poly-adenine linker showed that A3G-CTD retains the same pH deaminase activity preference toward each CCC as that toward the CCC of the 10-mer DNA. Importantly, the 3′ → 5′ polarity turned out to increase as the pH decreases in the range of 6.5–8.0. This suggests that A3G-CTD tends to continue sliding without abortion at lower pH, while A3G-CTD tends to dissociate from ssDNA during sliding at higher pH due to the weakened electrostatic interaction.

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          Most cited references36

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          How to study proteins by circular dichroism.

          Circular dichroism (CD) is being increasingly recognised as a valuable technique for examining the structure of proteins in solution. However, the value of many studies using CD is compromised either by inappropriate experimental design or by lack of attention to key aspects of instrument calibration or sample characterisation. In this article, we summarise the basis of the CD approach and its application to the study of proteins, and then present clear guidelines on how reliable data can be obtained and analysed.
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            The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA.

            High mutation frequency during reverse transcription has a principal role in the genetic variation of primate lentiviral populations. It is the main driving force for the generation of drug resistance and the escape from immune surveillance. G to A hypermutation is one of the characteristics of primate lentiviruses, as well as other retroviruses, during replication in vivo and in cell culture. The molecular mechanisms of this process, however, remain to be clarified. Here, we demonstrate that CEM15 (also known as apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G; APOBEC3G), an endogenous inhibitor of human immunodeficiency virus type 1 (HIV-1) replication, is a cytidine deaminase and is able to induce G to A hypermutation in newly synthesized viral DNA. This effect can be counteracted by the HIV-1 virion infectivity factor (Vif). It seems that this viral DNA mutator is a viral defence mechanism in host cells that may induce either lethal hypermutation or instability of the incoming nascent viral reverse transcripts, which could account for the Vif-defective phenotype. Importantly, the accumulation of CEM15-mediated non-lethal hypermutation in the replicating viral genome could potently contribute to the genetic variation of primate lentiviral populations.
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              Retroviral restriction by APOBEC proteins.

              A powerful mechanism of vertebrate innate immunity has been discovered in the past year, in which APOBEC proteins inhibit retroviruses by deaminating cytosine residues in nascent retroviral cDNA. To thwart this cellular defence, HIV encodes Vif, a small protein that mediates APOBEC degradation. Therefore, the balance between APOBECs and Vif might be a crucial determinant of the outcome of retroviral infection. Vertebrates have up to 11 different APOBEC proteins, with primates having the most. APOBEC proteins include AID, a probable DNA mutator that is responsible for immunoglobulin-gene diversification, and APOBEC1, an RNA editor with antiretroviral activities. This APOBEC abundance might help to tip the balance in favour of cellular defences.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                28 April 2016
                2016
                : 7
                : 587
                Affiliations
                [1] 1Institute of Advanced Energy, Kyoto University Kyoto, Japan
                [2] 2Graduate School of Energy Science, Kyoto University Kyoto, Japan
                Author notes

                Edited by: Toshinori Sato, Keio University, Japan

                Reviewed by: Ryuichi Sugiyama, Yokohama City University School of Medicine, Japan; Yoshiyuki Tanaka, Tokushima Bunri University, Japan

                *Correspondence: Takashi Nagata nagata.takashi.6w@ 123456kyoto-u.ac.jp ;

                This article was submitted to Virology, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2016.00587
                4848395
                27199921
                6e99101e-2191-4a34-bcb6-b47a26f94f2d
                Copyright © 2016 Kamba, Nagata and Katahira.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 20 January 2016
                : 11 April 2016
                Page count
                Figures: 5, Tables: 4, Equations: 5, References: 42, Pages: 11, Words: 7505
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                apobec3g,nmr,monitoring,enzyme,hiv-1
                Microbiology & Virology
                apobec3g, nmr, monitoring, enzyme, hiv-1

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