Nuclear Exclusion of the HIV-1 Host Defense Factor APOBEC3G Requires a Novel Cytoplasmic Retention Signal and Is Not Dependent on RNA Binding

Abstract

Human APOBEC3G (hA3G) is a host factor that defends against HIV-1 as well as other exogenous retroviruses and endogenous retroelements. To this end, hA3G is restricted to the cytoplasm of T lymphocytes where it interacts with viral RNA and proteins to assemble with viral particles causing a post-entry block during reverse transcription. hA3G also exhibits a mechanism to inhibit the reverse transcription of retroelements by RNA binding and sequestration into mRNA processing centers in the cytoplasm. We have determined that the molecular basis for this specialized property of hA3G is a novel cytoplasmic retention signal (CRS) that is necessary and sufficient to restrict wild-type hA3G and chimeric constructs to the cytoplasm. The CRS resides within amino acids 113-128 and is embedded within a basic flanking sequence and does not require RNA binding to retain hA3G in the cytoplasm. Paralogs of hA3G that have nuclear or cytoplasmic distributions differ from hA3G within the region encompassing the CRS motif with respect to charge and amino acid composition. We propose that the CRS enables hA3G to interact with cytoplasmic factors, and thereby enables hA3G to serve in host cell defense by restricting an antiviral sentinel to the cytoplasm. The CRS lies in a region involved in both Gag and Vif interactions; therefore, identification of this motif has important implications for the design of therapeutics that target HIV-1 while maintaining antiviral and cellular functions.Cytidine deaminases of the APOBEC 3 family (ApoB mRNA editing catalytic subunit) have one or more zinc-dependent deaminase (ZDD) signature motifs of the form (C/H)XEX n -PCXXC that is characteristic of enzymes that use RNA or single-stranded DNA (ssDNA) as substrates for C to U or dC to dU deamination (1, 2). APOBEC-1, activation-induced deaminase (AID), and APOBEC3G are the most extensively characterized members of this family. In mammals APOBEC-1 carries out site-specific editing of apoB and NF1 mRNAs to produce nonsense codons that lead to truncated proteins with altered functional properties (3-5). Though not observed under physiological conditions, APOBEC-1 can carry out dC to dU ssDNA mutation when expressed under selection in an Escherichia coli-based DNA mutator assay (6). In contrast, the physiological function of AID in germinal center B cells is to carry out multiple dC to dU mutations on ssDNA regions within the variable region and switch regions of immunoglobulin genes as an essential mechanism for somatic hypermutation and class switch recombination, respectively (7,8).The activities of APOBEC-1 and AID are regulated through their tissue-specific and temporal expression during cell differentiation (4, 8 -12). Both enzymes have cytoplasmic and nuclear distributions within cells, but the RNA and ssDNA editing activity of APOBEC-1 and AID are restricted to the cell nucleus (13-15). Each enzyme has a nuclear localization signal (NLS) and a nuclear export signal (NES) (16 -19) and their interaction with cytoplasmic chaperones is essential for ...

Endogenous APOBEC3A DNA Cytosine Deaminase Is Cytoplasmic and Nongenotoxic

“…CD14 ϩ 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 sim- ilar 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.…”

Six Classes of Nuclear Localization Signals Specific to Different Binding Grooves of Importin α

“…S3. In this analysis, however, we cannot exclude the possibility that some of the observed nuclear and cytoplasmic phenotypes of the GFP fusions could be due to nuclear export signals or nuclear/cytoplasmic retention signals potentially contained in fused peptides (31,32). Since these signal sequences are generally short, some screened and mutated NLS sequences could overlap with either of these signal sequences to affect their NLS activities.…”

Active nuclear import and cytoplasmic retention of activation-induced deaminase

“…AID dimerization through the β2 strand has been proposed based on the structure of A2 33 . We tested the effect of perturbing the predicted AID β2, residues [40][41][42][43][44][45][46][47][48][49][50][51][52][53] in our model, on oligomerization by introducing one (F46A), two (F46A/Y48A) 33 or four (F46A/Y48A/R50G/N51A, named AID FYRN) mutations. The ability of each of these mutants to interact with wt AID was monitored by comparing their efficiency in coimmunoprecipitating with AID-Flag.…”

“…The stoichiometry and architecture of the biologically relevant AID molecule is unknown but many indirect evidences suggest that it will have quaternary structure 33,35,42,47,48 as all cytidine deaminases including the APOBECs do 33,49,50 . Predicting AID dimerization through β2 (ref.…”