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

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.

Fig. 1

A diagram depicting the two zinc-dependent deaminase (ZDD) domains of human APOBEC3G (hA3G) and the regions homologous to the bipartite NLS and NES regions of hAPOBEC-1 and hAID. The sequence homologies of the bipartite NLS are shown in the boxed regions in hAPOBEC-1 and hAID. The underlined region in hA3G, demonstrates that a bipartite NLS is not conserved in hA3G. The NES homologies show three conserved leucines (boxed) and one leucine not conserved (underlined) in both the N-terminal (hA3G-NT) and C-terminal (hA3G-CT) regions of hA3G corresponding to critical leucines that make up the NES regions of hAPOBEC-1 and hAID, respectively (the human sequences are representative of the respective regions of APOBEC-1, AID and A3G in a multitude of mammalian species). The consensus NLS and NES are indicated below the respective homologies where x means any amino acid and B means basic amino acids lysine and arginine.

Fig. 2

hA3G does not traffic to the nucleus. (A) Cellular distribution of AID. C-terminal V5 tagged hAID transfected into HeLa cells localized to the cytoplasm (−LMB). Upon treatment with 20 ng/mL of LMB for 4 h (+LMB) hAID localized to the nucleus. (B) N-terminal HA tagged hA3G transfected into HeLa cells localized in the cytoplasm with and without LMB treatment (+/− LMB) and (C) NLS-hA3G with a SV40 NLS added to the N-terminus remained cytoplasmic with and without LMB treatment (+/− LMB). The upper and lower panels for A–C are two representative fields of the same treatment group. DAPI staining indicates the position of the cell nuclei in each panel.

Fig. 3

The two NES-like leucine-rich domains in hA3G are nonfunctional. (A) Chicken muscle pyruvate kinase (CMPK) transfected into HeLa cells localized to the cytoplasm. (B) SV40 NLS added to the N-terminus of CMPK enables trafficking to the nucleus. hA3G with three leucine to alanine mutations (boxed in hA3G-NT and hA3G-CT from Fig. 1) in either the N or C-termini (C and E, respectively) or in both regions (G) localized in the cytoplasm when transfected into HeLa cells. An N-terminal SV40 NLS added to the hA3G mutants in C, E, and G, (D, F and H, respectively) were also localized in the cytoplasm when transfected into HeLa cells. The side-by-side panels for A–H are two representative fields of the same treatment group. DAPI staining indicates the position of the cell nuclei in each panel.

Fig. 4

Both halves of hA3G are responsible for cytoplasmic retention. Constructs were designed that split hA3G in the predicted linker region between the two homologous halves of the protein. The C-terminal deletion hA3G mutant, a.a. 1-208 (A), N-terminal deletion hA3G mutant, a.a. 209-384 (C) and each mutant with an N-terminal SV40 NLS (B and D, respectively) localized to the cytoplasm when transfected into HeLa cells with or without (+/−) LMB treatment. The upper and lower panels for A–D are two representative fields of the same treatment group. DAPI staining indicates the position of the cell nuclei in each panel. The dashed lines indicate the missing portions of the deletion mutants.