Biochemical and Biophysical Research Communications
APOBEC-1 and AID are nucleo-cytoplasmic trafficking proteins but APOBEC3G cannot traffic
Section snippets
Materials and methods
Plasmid constructions. hA3G and hAID cDNAs were produced from oligo(dT)-primed total cellular RNA using avian myoblastosis virus reverse transcriptase (Promega) from H9 cells, and Raji cells, respectively. hA3G was subsequently subcloned into a pIRES-P [24] vector with N-terminal 6 His and HA tags, respectively. hAID was subcloned into pIRES-P with a C-terminal V5 tag. The N-terminal and C-terminal deletion clones of hA3G were subcloned from full length hA3G described above and were separated
hA3G does not traffic between the nucleus and cytoplasm
Both APOBEC-1 and AID require a bipartite NLS within the first 35 amino acids (aa) and cytoplasmic chaperones to traffic to the nucleus [14], [17], [20]. BLAST analysis of both APOBEC-1 and AID revealed that a bipartite NLS is conserved among nine and eight mammalian species for APOBEC-1 and AID, respectively. However, inspection of hA3G showed it lacks key basic amino acids associated with an NLS (Fig. 1). Immunocytochemistry (ICC) suggested that hA3G was localized only in the cytoplasm (Fig. 2
Discussion
We have shown that hA3G is strongly retained in the cytoplasm and does not undergo nucleo-cytoplasmic trafficking like its paralogs APOBEC-1 and AID. Moreover, hA3Gs cytoplasmic retention was not mediated by the NES-like, leucine-rich regions homologous to those identified in APOBEC-1 and AID [15], [16], [17], [19], [20]. hA3G contains an N-terminal and C-terminal cytidine deaminase domain and both halves of hA3G contain sufficient information to confer cytoplasmic retention. Consistent with
Acknowledgments
We thank J. Reeder for guidance in the use of the fluorescence microscope and imaging software. This work was supported by NIH Grant R21 AI58789 awarded to H.C.S. The authors also acknowledge NIH Training Grant T32 AI49815 for support of R.P.B.
References (44)
- et al.
Messenger RNA editing in mammals: new members of the APOBEC family seeking roles in the family business, Trends Genet.
(2003) - et al.
HIV-1 Vif Blocks the Antiviral Activity of APOBEC3G by Impairing both Its Translation and Intracellular Stability
Mol. Cell
(2003) - et al.
A novel form of tissue-specific RNA processing produces apolipoprotein-B48 in intestine
Cell
(1987) Apolipoprotein B mRNA editing: the sequence to the event
Semin. Cell Biol.
(1993)- et al.
Apobec-1 and apolipoprotein B mRNA editing
Biochim. Biophys. Acta
(1997) - et al.
Activation induced deaminase: the importance of being specific
Trends Genet.
(2004) - et al.
Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme
Cell
(2000) - et al.
A novel nuclear localization signal in the auxiliary domain of apobec-1 complementation factor regulates nucleocytoplasmic import and shuttling
J. Biol. Chem.
(2003) - et al.
Activation-induced cytosine deaminase (AID) is actively exported out of the nucleus but retained by the induction of DNA breaks
J. Biol. Chem.
(2004) - et al.
Antiviral function of APOBEC3G can be dissociated from cytidine deaminase activity
Curr. Biol.
(2005)
Development of a bicistronic vector driven by the human polypeptide chain elongation factor 1alpha promoter for creation of stable mammalian cell lines that express very high levels of recombinant proteins
Biochem. Biophys. Res. Commun.
Analysis of HIV-1 viral infectivity factor-mediated proteasome-dependent depletion of APOBEC3G: correlating function and subcellular localization
J. Biol. Chem.
A short amino acid sequence able to specify nuclear location
Cell
Identification of a specific domain required for dimerization of activation-induced cytidine deaminase
J. Biol. Chem.
Tissue-specific inhibition of apolipoprotein B mRNA editing in the liver by adenovirus-mediated transfer of a dominant negative mutant APOBEC-1 leads to increased low density lipoprotein in mice
J. Biol. Chem.
An anthropoid-specific locus of orphan C to U RNA-editing enzymes on chromosome 22
Genomics
APOBEC3A is a potent inhibitor of adeno-associated virus and retrotransposons
Curr. Biol.
Chronic lymphocytic leukemia B cells expressing AID display dissociation between class switch recombination and somatic hypermutation
Blood
Overexpression of APOBEC-1 results in mooring sequence-dependent promiscuous RNA editing
J. Biol. Chem.
RNA editing enzyme APOBEC1 and some of its homologs can act as DNA mutators
Mol. Cell
A role for activation-induced cytidine deaminase in the host response against a transforming retrovirus
Immunity
The structure of a yeast RNA-editing deaminase provides insight into the fold and function of activation-induced deaminase and APOBEC-1
Proc. Natl. Acad. Sci. USA
Cited by (29)
Engineered deaminases as a key component of DNA and RNA editing tools
2023, Molecular Therapy Nucleic AcidsEndogenous APOBEC3A DNA cytosine deaminase is cytoplasmic and nongenotoxic
2013, Journal of Biological ChemistryCitation Excerpt :We conclude that cells that endogenously express A3A, i.e. CD14+ monocytic cells, are able to control its activity and protect their DNA from this powerful DNA cytosine deaminase by a cytoplasmic retention mechanism. This mechanism may be fundamentally distinct from that previously inferred for A3G as even heterologously expressed full-length A3G and the N-terminal half of A3G are cytoplasmic with the latter being of similar size to A3A (45, 47, 52). The mechanism may also be distinct from that of A3H haplotype II as A3A is cell-wide in HeLa and 293T cells, in contrast to A3H-hapII (53), suggesting that in these cells, the A3A cytoplasmic retention mechanism may be either lacking or defective.
APOBEC3 proteins and reverse transcription
2008, Virus ResearchRNA editing, DNA recoding and the evolution of human cognition
2008, Trends in NeurosciencesNuclear exclusion of the HIV-1 host defense factor APOBEC3G requires a novel cytoplasmic retention signal and is not dependent on RNA binding
2008, Journal of Biological ChemistryCitation Excerpt :It is of interest in this regard that the nuclear paralog hA3B mRNA is expressed at low to no levels in normal tissue types but highly expressed in cancer cell lines (1, 78). Identification of the hA3G CRS is significant because the well studied antiviral activity of hA3G is a cytoplasmic phenomenon (40, 45–48). The block to HIV-1 reverse transcription, hypermutation of ssDNA replicating HIV-1 proviral DNA and assembly with HIV-1 virions all occur in the cytoplasm (33, 34, 37, 57, 64–66).
Measuring Editing Activity and Identifying Cytidine-to-Uridine mRNA Editing Factors in Cells and Biochemical Isolates
2007, Methods in EnzymologyCitation Excerpt :Moreover, hyperphosphorylation of nuclear ACF enhances the assembly of active 27S editosomes (Lehmann et al., 2006a,b), and therefore phosphatase inhibitors such as sodium fluoride (10–50 mM) can be added during extract preparation. On the other hand, APOBEC-1 homologs such as APOBEC3G (Bennett et al., 2006) do not traffic to the nucleus and cytoplasmic extracts contain all of the editing activity for these enzymes. The following protocol has been successfully applied to diverse cell types including McArdle 7777 rat hepatoma, HepG2 human hepatoma, HeLa human cervical carcinoma, Chinese hamster ovary (CHO), COS7 monkey kidney, 293T human embryonic kidney, A172 human astrocytoma and NGP human neurofibroma, H9 and MT2 human T cell lines, and peripheral blood mononuclear cells (PBMC).
- 1
Present address: Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Washington Road, Princeton, NJ 08544, USA.