Endogenous APOBEC3B Restricts LINE-1 Retrotransposition in Transformed Cells and Human Embryonic Stem Cells

Gladstone Institute of Virology and Immunology, University of California, San Francisco, California 94158, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2011; 286(42):36427-37. DOI: 10.1074/jbc.M111.251058
Source: PubMed


Members of the APOBEC3 (A3) family of cytidine deaminase enzymes act as host defense mechanisms limiting both infections by exogenous retroviruses and mobilization of endogenous retrotransposons. Previous studies revealed that the overexpression of some A3 proteins could restrict engineered human Long INterspersed Element-1 (LINE-1 or L1) retrotransposition in HeLa cells. However, whether endogenous A3 proteins play a role in restricting L1 retrotransposition remains largely unexplored. Here, we show that HeLa cells express endogenous A3B and A3C, whereas human embryonic stem cells (hESCs) express A3B, A3C, A3DE, A3F, and A3G. To study the relative contribution of endogenous A3 proteins in restricting L1 retrotransposition, we first generated small hairpin RNAs (shRNAs) to suppress endogenous A3 mRNA expression, and then assessed L1 mobility using a cell-based L1 retrotransposition assay. We demonstrate that in both HeLa and hESCs, shRNA-based knockdown of A3B promotes a ∼2-3.7-fold increase in the retrotransposition efficiency of an engineered human L1. Knockdown of the other A3s produced no significant increase in L1 activity. Thus, A3B appears to restrict engineered L1 retrotransposition in a broad range of cell types, including pluripotent cells.

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    • "The APOBEC3 group comprises seven proteins (APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3DE, APOBEC3F, APOBEC3G, and APOBEC3H) that play important roles in the innate immune system by acting on the host defense against endogenous retroelements and exogenous viruses [1] [2] [3]. APOBEC3B, in particular, is involved in the defense against retroelements and viruses, such as the hepatitis B virus (HBV), human T cell leukemia virus (HTLV), human papillomavirus (HPV), simian immunodeficiency virus (SIV), and human immunodeficiency virus (HIV) [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]. 1386-6532/© 2015 Elsevier B.V. All rights reserved. "
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    ABSTRACT: Background: Data regarding the influence of the APOBEC3B deletion on infectious diseases remain limited and shown discrepancies. Objectives: To characterize the APOBEC3B deletion polymorphism status and its association with prevalence of co-infection with blood-borne pathogens in Indonesian HIV-infected individuals. Materials and methods: A total of 597 HIV-positive blood samples were tested for the hepatitis B virus (HBV), hepatitis C virus (HCV), Torque Teno virus (TTV), GB virus-C (GBV-C), and Toxoplasma gondii. Nucleic acid was extracted from plasma samples and used for the molecular detection of HIV RNA, HBV DNA, HCV RNA, TTV DNA, and GBV-C RNA, whereas HBsAg, anti-HCV, IgM and IgG anti-T. gondii were detected through serological testing. The APOBEC3B deletion polymorphism was genotyped by polymerase chain reaction (PCR). Results: The deletion genotype was associated with HCV viremia (p<0.001) as well as elevated IgG anti-T. gondii (adjusted OR [aOR]=3.4). The deletion genotype was also associated with decreased levels of HBsAg (aOR=0.03), and anti-HCV (aOR=0.1). D/D was frequently found in HIV-infected individuals with CD4+T cells<14% (aOR=5.8). The intact genotype was associated with a reduced likelihood of a CD4+T cell count<200 cells/μL (aOR=0.2) but a higher prevalence of TTV co-infection (aOR=8.6). Conclusions: The APOBEC3B deletion polymorphism was found to be associated with HBV, HCV, TTV, and T. gondii co-infection in Indonesian HIV-infected individuals.
    Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 08/2015; 70:67-71. DOI:10.1016/j.jcv.2015.07.009 · 3.02 Impact Factor
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    • "Like the Vif protein of HIV-1 subtype B, Vif proteins of many other HIV-1 subtypes and of simian immunodeficiency virus (SIV) also need CBF-β to degrade their respective Vif-sensitive APOBEC3 proteins [8]. CBF-β, a non-DNA binding subunit, heterodimerizes with Runx proteins to form the CBF family of transcription factors, which are important for cell differentiation and proliferation, hematopoiesis and bone development [9], [10]. Recent studies have shown that CBF-β increases the stability of HIV-1 Vif [7], [11], controls its binding to CUL5 by specifically interacting with Vif [1], as well as increases the solubility of Vif when co-expressed in vitro [12]. "
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    ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) Vif requires core binding factor β (CBF-β) to degrade the host APOBEC3 restriction factors. Although a minimum domain and certain amino acids of HIV-1 Vif, including hydrophobic residues at the N-terminal, have been identified as critical sites for binding with CBF-β, other regions that potentially mediate this interaction need to be further investigated. Here, we mapped two new regions of HIV-1 Vif that are required for interaction with CBF-β by generating a series of single-site or multiple-site Vif mutants and testing their effect on the suppression of APOBEC3G (A3G) and APOBEC3F (A3F). A number of the mutants, including G84A/SIEW86-89AAAA (84/86-89), E88A/W89A (88/89), G84A, W89A, L106S and I107S in the 84GxSIEW89 and L102ADQLI107 regions, affected Vif function by disrupting CBF-β binding. These Vif mutants also had altered interactions with CUL5, since CBF-β is known to facilitate the binding of Vif to CUL5. We further showed that this effect was not due to misfolding or conformational changes in Vif, as the mutants still maintained their interactions with other factors such as ElonginB, A3G and A3F. Notably, G84D and D104A had stronger effects on the Vif-CUL5 interaction than on the Vif-CBF-β interaction, indicating that they mainly influenced the CUL5 interaction and implying that the interaction of Vif with CUL5 contributes to the binding of Vif to CBF-β. These new binding interfaces with CBF-β in HIV-1 Vif provide novel targets for the development of HIV-1 inhibitors.
    PLoS ONE 05/2014; 9(5):e95738. DOI:10.1371/journal.pone.0095738 · 3.23 Impact Factor
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    • "HeLa cells do not express endogenous A3A (Wissing et al., 2011), and therefore provide a system to measure directly the effects of ectopically expressed A3A on L1 retrotransposition. We co-transfected HeLa cells with retroelement expression constructs and plasmids encoding WT A3A or a deaminase-deficient mutant, A3A-C106S (Chen et al., 2006). "
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    ABSTRACT: Long INterspersed Element-1 (LINE-1 or L1) retrotransposition poses a mutagenic threat to human genomes. Human cells have therefore evolved strategies to regulate L1 retrotransposition. The APOBEC3 (A3) gene family consists of seven enzymes that catalyze deamination of cytidine nucleotides to uridine nucleotides (C-to-U) in single-strand DNA substrates. Among these enzymes, APOBEC3A (A3A) is the most potent inhibitor of L1 retrotransposition in cultured cell assays. However, previous characterization of L1 retrotransposition events generated in the presence of A3A did not yield evidence of deamination. Thus, the molecular mechanism by which A3A inhibits L1 retrotransposition has remained enigmatic. Here, we have used in vitro and in vivo assays to demonstrate that A3A can inhibit L1 retrotransposition by deaminating transiently exposed single-strand DNA that arises during the process of L1 integration. These data provide a mechanistic explanation of how the A3A cytidine deaminase protein can inhibit L1 retrotransposition. DOI:
    eLife Sciences 04/2014; 3(3):e02008. DOI:10.7554/eLife.02008.001 · 9.32 Impact Factor
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