Intensity of Deoxycytidine Deamination of HIV-1 Proviral DNA by the Retroviral Restriction Factor APOBEC3G Is Mediated by the Noncatalytic Domain

Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
Journal of Biological Chemistry (Impact Factor: 4.57). 02/2011; 286(13):11415-26. DOI: 10.1074/jbc.M110.199604
Source: PubMed


APOBEC3G is a single-stranded (ss) DNA deaminase that restricts replication of HIV-1 by inducing viral genome mutagenesis through deamination of cytosine to uracil on HIV-1 cDNA. APOBEC3G has polydisperse oligomeric states and deaminates ssDNA processively through jumping and sliding. APOBEC3G has a catalytically inactive N-terminal CD1 domain that mediates processivity and an active C-terminal CD2 domain that catalyzes deaminations. Here, we assess the determinants of APOBEC3G deamination efficiency mediated by the CD1 domain by comparing native APOBEC3G and two CD1 mutants, a monomeric mutant (F126A/W127A) and a clinical mutant associated with high viral loads (H186R). Biochemical assays on ssDNA or partially dsDNA and with a reconstituted HIV replication system demonstrate that both mutants of Apo3G have altered DNA scanning properties in either jumping (F126A/W127A) or sliding (H186R), which results in decreased abilities to induce mutagenesis during reverse transcription. The data reveal a functionality for Apo3G oligomers in deamination and provide the first biochemical characterization of the clinical mutant H186R. The data demonstrate that the balance between the jumping and sliding of Apo3G is needed for efficient mutational inactivation of HIV-1.

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Available from: Linda Chelico, Sep 19, 2014
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    • " August 2014 | Volume 5 | Article 450 | 15 movements (Feng and Chelico, 2011 "
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    ABSTRACT: The APOBEC3 restriction factors are a family of deoxycytidine deaminases that are able to suppress replication of viruses with a single-stranded DNA intermediate by inducing mutagenesis and functional inactivation of the virus. Of the seven human APOBEC3 enzymes, only APOBEC3-D, -F, -G, and -H appear relevant to restriction of HIV-1 in CD4+ T cells and will be the focus of this review. The restriction of HIV-1 occurs most potently in the absence of HIV-1 Vif that induces polyubiquitination and degradation of APOBEC3 enzymes through the proteasome pathway. To restrict HIV-1, APOBEC3 enzymes must be encapsidated into budding virions. Upon infection of the target cell during reverse transcription of the HIV-1 RNA into (-)DNA, APOBEC3 enzymes deaminate cytosines to form uracils in single-stranded (-)DNA regions. Upon replication of the (-)DNA to (+)DNA, the HIV-1 reverse transcriptase incorporates adenines opposite to the uracils thereby inducing C/G to T/A mutations that can functionally inactivate HIV-1. APOBEC3G is the most studied APOBEC3 enzyme and it is known that Vif attempts to thwart APOBEC3 function not only by inducing its proteasomal degradation but also by several degradation-independent mechanisms, such as inhibiting APOBEC3G virion encapsidation, mRNA translation, and for those APOBEC3G molecules that still become virion encapsidated, Vif can inhibit APOBEC3G mutagenic activity. Although most Vif variants can induce efficient degradation of APOBEC3-D, -F, and -G, there appears to be differential sensitivity to Vif-mediated degradation for APOBEC3H. This review examines APOBEC3-mediated HIV restriction mechanisms, how Vif acts as a substrate receptor for a Cullin5 ubiquitin ligase complex to induce degradation of APOBEC3s, and the determinants and functional consequences of the APOBEC3 and Vif interaction from a biological and biochemical perspective.
    Full-text · Article · Aug 2014 · Frontiers in Microbiology
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    • "A3G can surpass a double-stranded barrier by ''hopping'' over several nucleotides to gain access to another region of the same ssDNA molecule (Chelico et al., 2006). The noncatalytic NTD motif 126 FW 127 has been shown to be crucial for hopping activities (Feng and Chelico, 2011). In contrast, A3A displays significantly reduced processivity during deamination of synthetic ssDNA substrates in vitro (Love et al., 2012). "
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    ABSTRACT: Human APOBEC3 (A3) proteins are host-encoded intrinsic restriction factors that inhibit the replication of many retroviral pathogens. Restriction is believed to occur as a result of the DNA cytosine deaminase activity of the A3 proteins; this activity converts cytosines into uracils in single-stranded DNA retroviral replication intermediates. A3 proteins are also equipped with deamination-independent means to restrict retroviruses that work cooperatively with deamination-dependent restriction pathways. A3 proteins substantially bolster the intrinsic immune system by providing a powerful block to the transmission of retroviral pathogens; however, most retroviruses are able to subvert this replicative restriction in their natural host. HIV-1, for instance, evades A3 proteins through the activity of its accessory protein Vif. Here, we summarize data from recent A3 structural and functional studies to provide perspectives into the interactions between cellular A3 proteins and HIV-1 macromolecules throughout the viral replication cycle.
    Preview · Article · Mar 2014 · Structure
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    • "Chelico et al. (2006, 2008, 2010) have used an in vitro system to demonstrate that A3G has a 3′ to 5′ catalytic orientation specificity for the deamination of naked ssDNA (Feng and Chelico, 2011). The preferred asymmetric direction for A3G catalysis likely yields an approximately 30-nt “dead” zone located at the 3′ end of ssDNA that is much less efficiently deaminated by A3G (Chelico et al., 2008, 2010). "
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    ABSTRACT: The human APOBEC3 (A3) family (A, B, C, DE, F, G, and H) comprises host defense factors that potently inhibit the replication of diverse retroviruses, retrotransposons, and the other viral pathogens. HIV-1 has a counterstrategy that includes expressing the Vif protein to abrogate A3 antiviral function. Without Vif, A3 proteins, particularly APOBEC3G (A3G) and APOBEC3F (A3F), inhibit HIV-1 replication by blocking reverse transcription and/or integration and hypermutating nascent viral cDNA. The molecular mechanisms of this antiviral activity have been primarily attributed to two biochemical characteristics common to A3 proteins: catalyzing cytidine deamination in single-stranded DNA (ssDNA) and a nucleic acid-binding capability that is specific to ssDNA or ssRNA. Recent advances suggest that unique property of A3G dimer/oligomer formations, is also important for the modification of antiviral activity. In this review article we summarize how A3 proteins, particularly A3G, inhibit viral replication based on the biochemical and structural characteristics of the A3G protein.
    Full-text · Article · Jul 2012 · Frontiers in Microbiology
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