APOBEC3B and AID Have Similar Nuclear Import Mechanisms

https://doi.org/10.1016/j.jmb.2012.03.011Get rights and content

Abstract

Members of the APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) protein family catalyze DNA cytosine deamination and underpin a variety of immune defenses. For instance, several family members, including APOBEC3B (A3B), elicit strong retrotransposon and retrovirus restriction activities. However, unlike the other proteins, A3B is the only family member with steady-state nuclear localization. Here, we show that A3B nuclear import is an active process requiring at least one amino acid (Val54) within an N-terminal motif analogous to the nuclear localization determinant of the antibody gene diversification enzyme AID (activation-induced cytosine deaminase). Mechanistic conservation with AID is further suggested by A3B's capacity to interact with the same subset of importin proteins. Despite these mechanistic similarities, enforced A3B expression cannot substitute for AID-dependent antibody gene diversification by class switch recombination. Regulatory differences between A3B and AID are also visible during cell cycle progression. Our studies suggest that the present-day A3B enzyme retained the nuclear import mechanism of an ancestral AID protein during the expansion of the APOBEC3 locus in primates. Our studies also highlight the likelihood that, after nuclear import, specialized mechanisms exist to guide these enzymes to their respective physiological substrates and prevent gratuitous chromosomal DNA damage.

Graphical Abstract

Highlights

► A3B is imported actively into the nucleus. ► A3B and AID can interact with several adaptor importin proteins. ► A3B cannot substitute for AID in antibody gene diversification. ► Despite some similarities, A3B and AID have distinct biological functions.

Introduction

Human cells can express up to 11 distinct polynucleotide cytosine deaminases (reviewed in Refs. 1 and 2). AID (activation-induced cytosine deaminase), APOBEC1, APOBEC3A (A3A), APOBEC3B (A3B), APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G (A3G), and APOBEC3H (APOBEC being apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) have demonstrated DNA deaminase activity in one or more biochemical or biological assays (reviewed in Ref. 1). Two other family members, APOBEC2 and APOBEC4, do not appear to have DNA editing activity.3, 4, 5 Genomic cytosine to uracil deamination is potentially dangerous because uracil lesions can template the insertion of adenines and cause C/G-to-T/A transition mutations and, depending on DNA repair fidelity, other types of base substitutions, DNA breaks, and larger-scale chromosome aberrations (reviewed in Refs. 6, 7, 8).

AID is arguably the most ancient family member because all vertebrates use it for adaptive immunity through antibody gene diversification (reviewed in Refs. 1, 9, and 10) (Fig. 1a). APOBEC1 appears to have evolved more recently, most likely from the duplication of an ancestral AID gene prior to the split of species such as birds and lizards from the vertebrate phylogenetic tree11, 12 (Fig. 1a). In most species, the AID and APOBEC1 genes are located adjacent to one another on the same chromosome. Current models posit that these genes provided the substrate to root the APOBEC3 locus prior to the radiation of placental mammals, most likely by another ancestral gene duplication event2, 13, 14 (Fig. 1a). Thus, despite this considerable evolutionary gap, their common origin suggests that some present-day APOBEC3 protein activities and/or regulatory programs may still be conserved with those of AID (in addition to their shared DNA deaminase activity). Indeed, we and others have seen that localization through CRM1 (Exportin-1) mediated nuclear export is a conserved property of AID from a variety of different species15, 16, 17, 18, 19 (Fig. 1b).

The physiological functions of AID in antibody diversification and APOBEC1 in APOB mRNA editing necessitate the transport of these enzymes to the nuclear compartment (reviewed in Refs. 1 and 20). Despite < 30 kDa protein sizes, which should allow for passive diffusion through nuclear pores, both enzymes are imported actively.21, 22, 23 At least for AID, import is accomplished through a noncanonical nuclear localization signal (NLS) interacting with the adaptor importins α1, α3, and α5 in concert with the import factor β1.23 AID import may also be affected by interactions with other proteins such as GANP and CTNNBL1 (reviewed in Ref. 24). Similarly, APOBEC1 binds with at least one other cellular protein, ACF, to enter the nuclear compartment and access APOB mRNA substrates.25, 26, 27, 28 However, at steady state, both AID and APOBEC1 appear cytoplasmic due to a strong C-terminal leucine-rich nuclear export signal.15, 16, 29 This nuclear export signal directs export through the CRM1 pathway, which can be blocked by incubating cells with leptomycin B (lepB) (Fig. 1b).30

Curiously, A3B is the only human DNA deaminase family member with an apparent steady-state nuclear localization.31, 32, 33, 34, 35, 36, 37, 38 This property is also evident in rhesus macaque A3B.38 Although human A3B has a putative positively charged NLS spanning residues 206–212, mutagenesis studies showed that these residues are dispensable.36 However, individual domain as well as A3B/G chimera analyses were able to implicate an N-terminal region spanning residues 1–60 in nuclear import.31, 36, 37

Here, we find that A3B actively imports into the nucleus through a noncanonical N-terminal NLS within the same region as the noncanonical NLS of AID. In addition, both A3B and AID interact with adaptor importin proteins. Despite previous work showing that these enzymes can both inhibit retrotransposon replication,31, 32, 33, 39, 40, 41 other aspects of the regulation and function of A3B and AID have clearly bifurcated. For instance, we show that A3B and AID localize differently during mitosis. In addition, overexpression of A3B in primary B cells does not confer an ability to perform class switch recombination, and, unlike A3B, AID demonstrates little or no ability to restrict human immunodeficiency virus type 1 (HIV-1) replication in single-cycle infectivity assays. Since A3B appears to have constitutive access to genomic DNA, and it is actively shuttled to the nuclear compartment, understanding additional levels of A3B regulation will be important for determining whether in certain circumstances it is able, like AID, to mutate genomic DNA and contribute to carcinogenesis.

Section snippets

Relationship between A3B and AID

Consistent with a prior report,23 we noticed that AID-enhanced green fluorescent protein (eGFP) has an import delay in HEK293T cells compared to HeLa cells after lepB treatment (see  15-min time point in Fig. 1c). Similarly, we noticed less nuclear A3B-eGFP in HEK293T in comparison to HeLa cells, an average of 60% versus 70% of measurable fluorescent signal, respectively (Fig. 1d). A3B-eGFP displayed a HeLa-like nuclear concentration in a wide variety of other cell types (e.g., Fig. 1e and

Discussion

AID is a well-studied enzyme with important functions in the adaptive immune response. A3B has not been as extensively studied because it is specific to primates (there are no exact homologs of any specific APOBEC3 in mice2), and it is more difficult to work with (it is lethal to Escherichia coli33). To address the hypothesis that A3B and AID are regulated in the same way, we confirmed that A3B is generally nuclear, and we extended present knowledge by showing that A3B is actively imported into

Constructs

Several constructs used in this article have been reported previously including A3B-eGFP, N-terminal A3B-eGFP, AID-eGFP, zebrafish AID-eGFP, and MLV AID viral constructs.36, 38, 40, 78 The pmCherry vectors used for time-lapse microscopy and the HIV localization experiment are direct derivatives of these constructs. The eGFP-tagged H2B construct was a gift from J. Mueller. The GST-tagged importin α1, α3, and α5 constructs were gifts from the J. Di Noia laboratory.23 The HIVLAI nef∷eGFP construct

Acknowledgements

We thank J. Di Noia, M. Herzberg, J. Mueller, and M. Stevenson for reagents; R. LaRue for the phylogenetic schematic; and J. Lee for technical assistance. This work was supported by National Institutes of Health (NIH) Grants R01 AI064046 and P01 GM091743. L. Lackey was supported in part by a National Science Foundation Pre-doctoral Fellowship and subsequently by a position on the Institute for Molecular Virology Training Grant NIH T32 AI083196. Z.L. Demorest was supported in part by NIH Grant

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