APOBEC3H Subcellular Localization Determinants Define Zipcode for Targeting HIV-1 for Restriction

Abstract

APOBEC enzymes are DNA cytosine deaminases that normally serve as virus restriction factors, but several members, including APOBEC3H, also contribute to cancer mutagenesis. Despite their importance in multiple fields, little is known about cellular processes that regulate these DNA mutating enzymes. We show that APOBEC3H exists in two distinct subcellular compartments, cytoplasm and nucleolus, and that the structural determinants for each mechanism are genetically separable. First, native and fluorescently tagged APOBEC3Hs localize to these two compartments in multiple cell types. Second, a series of genetic, pharmacologic, and cell biological studies demonstrate active cytoplasmic and nucleolar retention mechanisms, whereas nuclear import and export occur through passive diffusion. Third, APOBEC3H cytoplasmic retention determinants relocalize APOBEC3A from a passive cell-wide state to the cytosol and, additionally, endow potent HIV-1 restriction activity. These results indicate that APOBEC3H has a structural zipcode for subcellular localization and selecting viral substrates for restriction.

Keywords: APOBEC3H; DNA deamination; cancer mutagenesis; cytoplasmic retention; nuclear import; retrovirus restriction; subcellular localization.

FIG 1

Evidence for cytoplasmic and nucleolar A3H. (A) Representative images of native A3H and mCherry-tagged (mCh) splice variants. Scale bar, 10 μm. (B) Representative images of mCh-A3H with 1, 2, or 3 copies of mCherry coexpressed with fibrillarin-eGFP (FBL-eGFP). Arrows indicate regions of interest (ROI). (C and D) Representative images of a time course treatment with either leptomycin B (LepB) (40 ng/ml) (C) or actinomycin D (AMD) (0.001 ng/ml) (D). (E) Representative images of the indicated mCh-A3H constructs coexpressed with FBL-eGFP in HeLa cells. ROI are indicated by arrows. Scale bars in panels B to E, 5 μm.

FIG 2

A predicted nucleolar targeting sequence in loop 1 is required for nucleolar localization. (A) Alignment of A3H, A3A, and A3B C-terminal domain amino acid sequences flanking loop 1 (dashed line). The predicted nucleolar localization sequence of A3H is indicated by the solid line. (B) Quantification of nucleolar fluorescence intensity of the indicated A3H constructs (n = 50 nucleoli). The dashed lines highlight ±SEM from wild-type quantification. ns, no significance; ***, P < 0.001 by the unpaired Student t test. (C and D) Representative images of the indicated mCh-A3H constructs coexpressed with FBL-eGFP. ROI are indicated by arrows. Basic-to-Ala/Glu refers to amino acids K16 to R21 replaced by either alanine or glutamate. All scale bars, 5 μm.

FIG 3

A3H loop 1 is not sufficient for redistribution of A3A to the nucleolus. (A) Structural overlay of A3H (PDB 6B0B) and A3A (PBD 4XXO), highlighting the positioning of loop 1. (B) Representative images of A3H loop 1 swapped into mCh-A3A. Scale bar, 5 μm.

FIG 4

Loop 1 of A3H in combination with either loop 7 or α-helix 6 can relocalize A3A to the nucleus through an active import mechanism. (A and C) Structural representation of A3H overlaid with A3A to highlight the orientations of loop 1, α-helix 6, and loop 7 within the RNA binding interface. (B and D) Representative images of cells expressing the indicated mCh-A3A construct. Scale bars, 5 μm. (E) Representative images of the indicated construct with either 1× or 3× mCherry. (F) Representative images of A3A chimeras with loop 1 exchanged from either A3H or A3B alone or in combination with A3H loop 7 or α-helix 6. Scale bars, 10 μm.

FIG 5

Loop 3 of A3H contributes to nucleolar retention along with the RNA binding interface. (A) Alignment of wild-type A3H, A3A, and an A3A_A3H-RNA chimera (A3A_A3H-RNA has loop 1, loop 7, and α-helix 6 exchanged from A3H) amino acid sequences that highlight loop 3 (boxed sequence). (B) Structural representation of A3H overlaid with A3A to highlight the proximity of loop 3 to loop 1. (C to E) Representative images of the indicated A3A and A3H constructs. ROI are indicated by arrows. (F) Quantification of nucleolar fluorescence intensity of the indicated A3H constructs (n = 50 nucleoli). The dashed lines highlight ±SEM from wild-type quantification. ns, no significance; ***, P < 0.001 by the unpaired Student t test. All scale bars, 5 μm.

FIG 6

Relocalization of A3A to the cytoplasm confers antiviral activity against Vif-deficient HIV-1. (A) Amino acid alignment of A3H and the A3A_A3H-RNA+L3 chimera. Asterisks indicate amino acids in analogous A3Z3s that have similar biophysical properties. Dashed boxes indicate ROI. (B) Representative images of the indicated mCh-A3 chimeric proteins. Scale bar, 10 μm. (C) Top, Vif-deficient HIV-1 restriction assay with the indicated A3 constructs (“+” indicates that α-helix 1, loop 9, and the RNA binding interface are exchanged along with the indicated regions). A representative image of each enzyme is shown above the corresponding viral infectivity results. Bottom, immunoblots demonstrating expression of each construct in the cell lysate and the ability of the A3 protein to package into Vif-deficient HIV-1 particles. The asterisk indicates a nonspecific band in the viral particle blot. Scale bar, 5 μm.

FIG 7

Computational modeling predicts that the duplexed RNA phosphate backbone interacts with loop 3 of A3H. (A) Structural model of the A3H/RNA cocrystal structure with 5 uracil nucleobases computationally added onto on the RNA chain that extends toward the enzymatic active site (additional residues are colored orange). Loop 1 and loop 3 are highlighted in blue. (B) Predicted model of how A3H maintains steady-state localization in two distinct subcellular compartments. RNP, ribonucleotide protein complex.