Ancient Adaptive Evolution of the Primate Antiviral DNA-Editing Enzyme APOBEC3G

1Mobile genetic elements have been in conflict with host genomes for over a billion years. Our own genomes reveal the remarkable effects of retrotransposition, as about 45% of our genomic DNA results directly from this process. This perennial state of conflict has led eukaryotes to adopt several strategies to curb the proliferation of transposable elements and viruses. These include transcriptional silencing through DNA and histone methylation or RNA interference, and even directed mutagenesis of mobile elements. Despite facing this gauntlet of defense strategies, transposable elements have thrived in eukaryotic genomes (with Neurospora crassa being a notable exception) by evolving suitable countermeasures. Our current understanding of the intracellular interplay between host defenses and the assault of transposable elements suffers from a paucity of cases where both counterstrategies have been clearly identified. This is in contrast to extracellular cases, where interactions between viral proteins and either host immune surveillance or host receptors have been well established. Understanding the nature and evolutionary time-frame of intracellular conflict is key to understanding the current state of eukaryotic genomes. Recent studies of host inhibition of HIV have uncovered mutations introduced by DNA editing as a novel means by which host genomes battle viruses intracellularly. Furthermore, the means by which viruses combat this defense strategy are also identified, thus providing an unprecedented opportunity to study the evolution of intracellular genetic conflict.

2Different human cell lines vary in their susceptibility to HIV infection. The gene responsible for this differential susceptibility was identified as apolipoprotein B–editing catalytic polypeptide 3G (APOBEC3G), whose product targets HIV and simian immunodeficiency virus (SIV) for editing as their genomes undergo reverse transcription in the cytoplasm of host cells. APOBEC3G is a cytidine deaminase that edits cytosines to uracils in the minus strand DNA copied from the viral RNA genome, resulting in promiscuous guanine-to-adenine (G-to-A) hypermutation of the plus (protein-coding) strand of the viral DNA. APOBEC3G is expressed in testes, ovary, spleen, peripheral blood leukocytes, and T-lymphocytes and is packaged in nascent virions and delivered into new host cells along with the viral genome. How this editing reduces the evolutionary fitness of the virus is not well established. The mutations introduced by the editing process may either directly reduce viral fitness, or target the uracil-containing viral DNA for destruction. Before the discovery of APOBEC3G, RNA editing was thought to function solely in the diversification of gene-encoded information. The discovery of viral targeting by APOBEC3G represents a new phase in our understanding of nucleic acid editing in primates.

3APOBEC3G belongs to a family of nine primate genes that catalyze the deamination of cytosine to uracil in DNA and/or RNA (Figure 1). Two other members of this family are known to have important in vivo editing functions. APOBEC1 encodes a protein that site-specifically edits the mRNA of apolipoprotein B (APOB), leading to a truncated form of the APOB lipid-transport protein, which is important for determining levels of low-density lipoprotein production. Another member of this family, activation-induced deaminase (AID), is important for all steps following V(D)J recombination in B lymphocytes, from generating antibody diversity to class-switching events. Significantly, APOBEC1 and AID act within the nucleus, whereas APOBEC3G is exclusively cytoplasmic, which prevents it from mutating “self” DNA molecules. Whereas rodents have a single APOBEC3 gene, humans have at least six, including APOBEC3G. The functions of the other members of this expanded APOBEC3 cluster are unknown, although APOBEC3C has been shown to be catalytically active, exhibiting DNA mutator activity in a bacterial system that is like APOBEC3G. More recently, APOBEC3F has also been associated with anti-HIV biological activity.

4Most lentiviruses encode an accessory gene, virion infectivity factor (Vif), whose product counteracts the antiviral activity of APOBEC3G. Vif interacts with APOBEC3G and targets it for ubiquitination and proteasome-dependent degradation, thus preventing its incorporation into nascent virions. This interaction can be species-specific, as the Vif protein of one lentivirus will counteract APOBEC3G from its host species, but not always the APOBEC3G from a different primate species. Thus, APOBEC3G and Vif are predicted to be under selection to decrease and enhance, respectively, their interaction with one another, each driving rapid change in the other. Genetic conflicts like this one are predicted to result in the rapid fixation of mutations that alter amino acids, specifically those that affect this protein–protein interaction. This scenario is referred to as positive selection and is commonly seen in host–pathogen interactions.

5In this report, we directly test this prediction by studying the paleontology of selective pressures that have acted on APOBEC3G in the primate lineage, to ask whether APOBEC3G has been subject to positive selection, and to date the origins of this genetic conflict. We find that APOBEC3G has been under remarkably strong positive selection, and has undergone several episodes of adaptive evolution throughout the history of primates. Unexpectedly, we find that the positive selection acting on APOBEC3G predates modern lentiviruses, indicating that a more ancient, and perhaps ongoing, conflict has shaped its evolution. We also report evidence for strong positive selection acting on a majority of the APOBEC genes, suggesting that this family of genes may have expanded in primate genomes for genome defense via RNA/DNA editing.

6To determine what selective pressures have shaped APOBEC3G evolution, we sequenced the APOBEC3G gene from a panel of primate genomes representing 33 million years of evolution. We sequenced the complete APOBEC3G coding sequence (approximately 1,155 bp) from ten primate species, including four hominids (other than human), four Old World monkeys (OWMs), and two New World monkeys (NWMs) (Figure 2). A phylogeny constructed using either complete APOBEC3G sequences or individual exons (unpublished data) is congruent to the widely accepted primate phylogeny, indicating that all sequences isolated by our PCR strategy are truly orthologous.

7The hallmark of positive selection is an excess of non-synonymous substitutions (which alter the amino acid being encoded) relative to synonymous substitutions (which retain the encoded amino acid). Because non-synonymous changes are more likely to be deleterious, they are typically culled out by selection (referred to as purifying or negative selection). Therefore, in protein-coding open reading frames, the number of observed changes per synonymous site (Ks) usually exceeds the number of observed changes per non-synonymous site (Ka). In the case of the APOBEC3G, however, we found that a majority of branches of the phylogeny (including internal branches) show evidence of positive selection (defined as Ka/Ks [ω] greater than one; see Figure 2). This implies that the APOBEC3G has been subject to positive selection throughout the history of primate evolution. In support of this conclusion, all pairwise comparisons of the entire APOBEC3G gene between the various primates have ω greater than one (unpublished data). Maximum likelihood analysis using the PAML (phylogenetic analysis by maximum likelihood) suite of programs also finds strong evidence that the full-length APOBEC3G gene has been subject to positive selection (p < 10–13). Numbers in parenthesis in Figure 2 indicate the actual number of non-synonymous and synonymous changes (R:S) that have occurred along each branch.

8The average Ks in APOBEC3G is not unusually low; it is about 0.09 between hominids and OWMs and 0.26 between hominids and NWMs, compared to 0.08 and 0.15 respectively for comparisons of various intronic and noncoding regions of primate genomes. Thus, we can rule out the possibility that selection has led to deflated Ks values in APOBEC3G that lead to artificially high ω ratios. Indeed, these high ω ratios can be explained only by a significantly higher rate of non-synonymous substitutions. Of the primates analyzed, lentiviral infections have been observed only in the African monkeys, chimpanzees, and humans. HIV/SIV-infected species are indicated with asterisks in Figure 2. Estimating the age of lentiviruses is difficult because of their rapid rate of evolution and frequent cross-species transfer, but it has been suggested that primate lentiviruses are no older than 1 million years. The presence of modern lentiviruses appears to bear no correlation to either the presence or the strength of positive selection. For instance, the lineage leading to hominids has a ω of 3.3, the highest overall. The positive selection acting on APOBEC3G thus appears to predate modern lentiviruses, and interactions with lentiviral Vif proteins are not likely to be a major cause of this unusually strong signal of positive selection. In support of this conclusion, HIV has not been in the human population long enough to account for the positive selection of APOBEC3G specific to the human lineage (a 7:0 R:S ratio) arguing that, although the positive selection of Vif may be explained in large part by that of APOBEC3G, the reverse is certainly not the case.

9We wanted to identify the specific domains in APOBEC3G that were subject to positive selection, because this might suggest the driving evolutionary force. For instance, the positive selection in the major histocompatibility complex proteins is confined to only small segments of the protein that constitute the antigen-recognition site, because only these sites participate in protein–protein interactions subject to genetic conflict. All members of the APOBEC family contain a similar domain organization (see Figure 1B) that consists of an active site that encodes a zinc-dependent cytidine deaminase domain with the HXE, PCXXC (H, histidine; X, any amino acid; E, glutamic acid; P, proline; C, cysteine) signature, a linker peptide, and a pseudoactive domain. The active and pseudoactive domains are believed to have originated from a gene duplication event followed by degeneration of the catalytic activity of the pseudoactive domain. APOBEC3G and some other APOBEC genes have also undergone a second gene duplication/fusion event.

10Representative examples of pairwise (sliding window) comparisons of Ka/Ks ratios between two hominids, two OWMs, and two NWMs suggest that the same domain of APOBEC3G has not been subject to positive selection throughout primate evolution (Figure 3A–3C). In both the hominid and NWM comparisons, the second half of the gene shows evidence of positive selection (Figure 3A and 3C), but in an OWM comparison, it is the first half that is under positive selection (Figure 3B). When the APOBEC3G gene is divided into structural domains, we find that all domains, including the active site domains, have undergone multiple distinct episodes of positive selection (Figure S1). This highly unusual pattern suggests that the genetic conflicts that have shaped APOBEC3G evolution have involved episodic protein–protein interactions with different parts of the entire APOBEC3G protein.

11We also employed a maximum-likelihood approach (see Materials and Methods), using the PAML suite of programs to identify the specific residues that have been repeatedly subject to positive selection in primates. These analyses (in the best fit model) identify 30% of the codons as having evolved under stringent purifying selection (ω of approximately zero). These include the catalytically important residues that are invariant throughout all APOBECs. The same analysis also identifies approximately 30% of the codons as having evolved under positive selection with an average ω of nearly 3.5 (residues that are evolving without selective constraint would be expected to have an average ω of one). Even among adaptively evolving proteins, this is an unusually high proportion of sites, once again implicating a large number of residues in APOBEC3G as having participated in some kind of genetic conflict. Of these, several residues are identified as being under positive selection with high confidence (posterior probability greater than 0.95, inverted triangles in Figure 3D). In simulations using datasets with comparable levels of sequence divergence and strength of positive selection to our APOBEC3G dataset (tree length = 1.59), PAML analyses were found to be highly accurate in identifying residues subject to positive selection.

12The schematic in Figure 3D highlights the region where Vif is believed to interact with human APOBEC3G. It also highlights the single amino acid residue (cross symbol in linker 1) that is responsible for the species-specific interactions seen between Vif and APOBEC3G in African green monkeys (SIV) and humans (HIV). There is a noticeable lack of correlation between the sites on APOBEC3G that are important for Vif interaction and those sites that are identified by PAML with high confidence, supporting our earlier conclusion that Vif interactions have played only a small role in dictating the positive selection of APOBEC3G.

13The discovery that APOBEC3G is involved in host defense was predicated on the tissue-specific inhibition of HIV. Other studies have investigated a possible inhibitory role of other APOBEC genes but found that only APOBEC3G and APOBEC3F exert an antiviral defense against HIV. An unbiased look at selective pressures among other APOBEC genes could reveal clues to their function. We calculated whole-gene Ka/Ks ratios for other members of the human APOBEC family, using orthologs from the chimpanzee genome project (Table 1, second column). This analysis reveals strong evidence of purifying selection acting on AID and APOBEC3A but positive selection acting on APOBEC3B and APOBEC3DE (as well as APOBEC3D and APOBEC3E alone) in addition to APOBEC3G. There is no expression evidence for APOBEC3E, and it is unclear whether it occurs as a stand-alone gene, but its ω ratio of 5.6 is among the highest seen for any human–chimp comparison and argues strongly that it is a functional gene and an active participant in some form of genetic conflict. Whole-gene analyses are notoriously poor at identifying specific domains of positive selection, especially when the rest of the gene is subject to purifying selection. We therefore performed a sliding window Ka/Ks test, which also reveals positive selection acting on APOBEC3F (amino acids [aa] 117–250).

14The limited divergence between human and chimp genomes leads to some comparisons not being informative enough to detect selection (APOBEC1 and APOBEC3C), and there was insufficient chimpanzee sequence available in one case (APOBEC2). To gain further information about these genes, we sequenced them from either orangutan or gorilla (Table 1, third column). These comparisons reveal that strong purifying selection has acted on APOBEC2, but positive selection can be detected in both APOBEC1 (aa 1–100; also see Figure 4) and APOBEC3C (aa 34–133). Although we might have expected APOBEC1 to be evolving only under purifying selection based on its important editing of APOB mRNA, our analysis suggests that APOBEC1 has also participated in some kind of genetic conflict involving its first active site, and suggests that the rapid evolution of APOBEC1 seen previously in mouse–rat comparisons may also be due to positive selection. Figure 4 shows representative sliding window analyses of genes undergoing gene-wide purifying (APOBEC2) and positive (APOBEC3E) selection. These findings greatly extend the current understanding of the APOBEC family, and implicate a majority of APOBEC genes as participants in host defense. They also raise the possibility of other editing systems being involved in genome defense; for instance, hepatitis delta virus is known to be edited by adenosine deaminase.

15The antiviral activity of APOBEC3G and the excess of non-synonymous changes specific to human APOBEC3G (see Figure 2) implicate non-synonymous polymorphisms as being functionally very important. Because binding by Vif inhibits APOBEC3G's antiviral ability, we might predict that APOBEC3G should be subject to overdominant selection (heterozygous individuals being at a selective advantage), especially in populations with a high incidence of HIV infection, since different alleles of APOBEC3G may have different susceptibility to various viral strains. The action of APOBEC3G on viral evolution could also be complex because, although it is ineffective as an antiviral mechanism in the presence of Vif, its action could also result in an increased likelihood of adaptive changes and viral diversity in the host due to the introduced G-to-A hypermutations. Polymorphisms in APOBEC3G may thus have direct impact on the progression time from initial HIV infection to AIDS, and should be investigated as such.

16The evidence for positive selection of APOBEC3G does not identify the biological step that exerts this selective pressure. Formally, this step could be the yet-undefined mechanism by which APOBEC3G is packaged into virions, the interaction of APOBEC3G with Vif-like destruction proteins encoded by other viruses, and/or its interaction with the proteasome machinery. APOBEC3G may indeed interact with other viruses, because G-to-A hypermutation—a hallmark of the single-stranded DNA–editing activity of APOBEC3G-like enzymes—has been observed in some nonlentivirus viruses, and because APOBEC3G has recently been shown to inhibit the replication of the hepatitis B virus upon deliberate coexpression. However, this inhibition of hepatitis B is not correlated with G-to-A hypermutation, suggesting that APOBEC3G may also inhibit viral replication independent of its catalytic activity.

17The ancient, constant pressure of positive selection on APOBEC3G in primates raises the possibility that at least some of its evolution may be explained by a struggle not in the lymphocytes, but in the germline, where APOBEC3G is also abundantly expressed, and where genome-restricted mobile genetic elements need to transpose to ensure survival. Of the three main classes of eukaryotic mobile elements, only two are active in humans and, most likely, other primate genomes. The first and major class includes the LINE1 (long interspersed element–1) non-LTR (long terminal repeat) retroposons that are not a likely target for APOBEC3G, because they carry out their reverse transcription in the nucleus (APOBEC3G is restricted to the cytoplasm). A second class, the LTR-bearing human endogenous retroviruses (HERVs), is identical in many aspects of its life cycle to retroviruses. While the selective disadvantage to an individual organism conferred by endogenous retroviruses may pale in comparison to that of pathogenic viruses, over time the steady retrotransposition of endogenous retroviruses is likely to be more detrimental to a species than scattered, episodic interactions with viruses. Thus, the constant efforts of HERVs to jockey for evolutionary dominance may provide a more likely explanation for the positive selection of APOBEC3G and other APOBEC genes in primate genomes.

18Genomic DNA was obtained from Coriell (Camden, New Jersey, United States). Species and Coriell repository numbers are: Pan troglodytes (chimpanzee) (NAO3448A), Pan paniscus (bonobo) (NGO5253), Gorilla gorilla (gorilla) (NG05251B), Pongo pygmaeus (orangutan) (NAO4272), Macaca nigra (Celebes crested macaque) (NG07101), Macaca fascicularis (crab-eating macaque) (NA03446), Erythrocebus patas (patas monkey) (NG06254), Lagothrix lagotricha (common woolly monkey) (NG05356), and Saguinus labiatus (red-chested mustached tamarin) (NG05308). Papio anubis (baboon) DNA was a personal gift from Dr. Trent Colbert. The APOBEC3G, APOBEC1, APOBEC2, and APOBEC3C genes were amplified exon-by-exon from genomic DNA with PCR Supermix High Fidelity (Invitrogen, Carlsbad, California, United States), and PCR products were sequenced directly. PCR and sequencing primers are shown in Table S1. The human APOBEC3G sequence was obtained from the Ensembl database of the human genome project (ENSG00000100289). The Chlorocebus aethiops (African green monkey) APOBEC3G sequence (GenBank AY331714.1) is missing the last 21 bp of the coding sequence because it was sequenced from mRNA in a previous study. Exon–intron boundaries are conserved, except in APOBEC3G from NWMs (woolly monkey and tamarin) where the “AG” directly 5′ of the eighth coding exon is missing. Sequences have been deposited in GenBank under the following accession numbers: APOBEC3G (AY622514–AY622593), APOBEC3C (AY622594–AY622597), APOBEC2 (AY622598–AY622599), APOBEC1 (AY622600–AY622604).

19Human sequences were obtained from the Ensembl or GenBank databases: APOBEC1 (ENSG00000111701), APOBEC2 (ENSG00000124701), AID (ENSG00000111732), APOBEC3A (ENSG00000128383), APOBEC3B (NM_004900.3), APOBEC3C (ENSG00000179750), APOBEC3DE (ENSG00000179007), and APOBEC3F (ENSG00000128394). Transcripts for both APOBEC3D (NM_152426) and APOBEC3DE (BC017022.1) exist in the database. Chimp sequences were obtained from orthology to human genes assigned on the University of California at Santa Cruz Genome Bioinformatics Website (http://www.genome.ucsc.edu). All orthologous chimp exons were checked for AG and GT flanking the 5′ and 3′ boundaries, respectively, an indication that human splice sites are conserved. The mouse APOBEC3 protein sequence can be found in GenBank (NP_084531.1).

20DNA sequences were aligned using Clustal_X, with hand alignment of small indels based on amino acid sequence. Changes along each lineage (see Figure 2) were assigned using parsimony and counted by hand. Changes at 18 positions could not be unambiguously assigned as non-synonymous or synonymous and were excluded from the R:S ratios. Ka and Ks for pairwise comparisons (Figures 3A–3C and 4; Table 1), as well as their confidence values, were calculated using the K-estimator software package. For confidence values, simulations were carried out under the condition where Ka equals Ks and compared to actual Ka from that region, and multiple parameters for transition:transversion ratios were simulated.

21Maximum likelihood analysis was performed with the PAML software package. Global ω ratios for the tree (see Figure 2) were calculated by a free-ratio model, which allows ω to vary along different branches. To detect selection, the multiple alignments were fitted to either the F3×4 or F61 models of codon frequencies. We then compared the log-likelihood ratios of the data using different NSsites models: model 1 (two-state, neutral, ω > 1 disallowed) to model 2 (similar to model 1 but ω >1 allowed), and model 7 (fit to a beta distribution, ω > 1 disallowed) to model 8 (similar to model 7 but ω >1 allowed). In both cases, permitting sites to evolve under positive selection gave a much better fit to the data (p < 10−13) with a significant fraction of the sites (more than 30%) predicted to evolve at average ω ratios greater than 3.5 (see Figure S2 for details). These analyses also identified certain amino acid residues with high posterior probabilities (greater than 0.95) of having evolved under positive selection (Figures 3D and S2).

22The GenBank (http://www.ncbi.nlm.nih.gov/) and Ensembl (http://www.ensembl.org/) accession numbers for the genes and gene products discussed in this paper are as follows. GenBank: APOBEC1 (AY622600–AY622604), APOBEC2 (AY622598–AY622599), mouse APOBEC3 (NP_084531.1), human APOBEC3B (NM_004900.3), APOBEC3C (AY622594–AY622597), APOBEC3D (NM_152426), APOBEC3DE (BC017022.1), APOBEC3G (AY622514–AY622593), and African green monkey APOBEC3G (AY331714.1). Ensembl (all human sequences): APOBEC1 (ENSG00000111701), APOBEC2 (ENSG00000124701), AID (ENSG00000111732), APOBEC3A (ENSG00000128383), APOBEC3C (ENSG00000179750), APOBEC3DE (ENSG00000179007), APOBEC3F (ENSG00000128394), and APOBEC3G (ENSG00000100289).

23Coriell (http://www.coriell.undmj.edu/) repository numbers for primate genomic DNAs are Pan troglodytes (chimpanzee) (NAO3448A), Pan paniscus (bonobo) (NGO5253), Gorilla gorilla (gorilla) (NG05251B), Pongo pygmaeus (orangutan) (NAO4272), Macaca nigra (Celebes crested macaque) (NG07101), Macaca fascicularis (long-tailed macaque) (NA03446), Erythrocebus patas (patas monkey) (NG06254), Lagothrix lagotricha (common woolly monkey) (NG05356), and Saguinus labiatus (red-chested mustached tamarin) (NG05308).