APOBEC-1 and AID are nucleo-cytoplasmic trafficking proteins but APOBEC3G cannot traffic

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Abstract

Human APOBEC3G (hA3G) is a member of the APOBEC-1 related protein (ARP) family of cytidine deaminases. hA3G functions as a natural defense against endogenous retrotransposons and a multitude of retroviruses, most notably human immunodeficiency virus type 1 (HIV-1). Nothing is known about the cellular function of hA3G, however, upon HIV-1 infection hA3G functions as an antiviral factor by mutating viral single-stranded DNA during reverse transcription. Whereas homologous deaminases such as APOBEC-1 and AID act on RNA and DNA, respectively, in the cell nucleus, hA3G mutagenic activity appears to be restricted to the cytoplasm. We demonstrate that hA3G is not a nucleo-cytoplasmic shuttling protein like APOBEC-1 and AID, but is strongly retained in the cytoplasm through a mechanism that involves both the N and C-terminal regions of the protein.

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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.

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    Present address: Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Washington Road, Princeton, NJ 08544, USA.

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