Reduced APOBEC3H Variant Anti-Viral Activities Are Associated with Altered RNA Binding Activities

APOBEC3H (A3H) is a member of the APOBEC3 family of proteins with varying activities against retroviruses and retrotransposons. The A3H gene contains several single nucleotide polymorphisms and up to seven haplotypes have been detected in humans. Although variations in anti-viral function among A3H haplotypes are not fully understood, only 15N105R-containing A3H variants are known to have potent activities against Vif-deficient HIV-1. Unique motif RLYY(F/Y)W of APOBEC3G (A3G) and APOBEC3F (A3F) required for 7SL RNA binding and HIV-1 incorporation is also conserved in all A3H variants. Like A3G, A3H HapII also demonstrated high binding affinity to host small RNAs such as 7SL and Y RNAs. Mutation of a critical amino acid, W115A resulted in reduced expression level, decreased affinity for 7SL RNA, impairment of virion packaging and reduced anti-viral activity. By comparison, A3H HapI had lower binding affinities to host small RNAs and reduced efficiency of virion incorporation, resulting in significantly reduced anti-viral activity. The SNP ΔN15 commonly found in A3H HapIII and HapIV abolished their abilities to associate with RNAs, and A3H HapIIΔ15N failed to package into HIV-1 virions or exhibited any anti-viral activity. Finally, we showed that A3H variants had distinct cellular localization patterns, which correlated with their different RNA binding affinities. Thus, Pol-III RNA such as 7SL RNA binding is a conserved feature of potent anti-HIV human APOBEC3 cytidine deaminases.

However, the underlying mechanism of diverse anti-viral activities of A3H variants is still not clear. As for A3G and A3F, its anti-viral effects require efficient packaging into the HIV-1 virion [5,25,26,27,28]. In the absence of Vif, A3G and A3F virion incorporation requires the RNA binding nucleo-capsid (NC) domain of Gag and many studies have found that RNA is required for the interaction between Gag and A3G [25,26,28,29,30]. Some studies have reported viral genomic RNA is required for efficient A3G packaging [31,32], while others argued that cellular RNA, especially 7SL RNA is responsible for mediating A3G and A3F packaging [27,33]. Host 7SL RNA is most abundant in HIV-1 particle and appeared to be specifically enriched in HIV-1 virion [34]. A3G and A3F have high affinity with 7SL RNA and association with 7SL RNA is crucial for antiviral activity as well as accurate viral core targeting of various APOBEC3 proteins [35].
Previous studies have reported that A3H HapII incorporation into HIV-1 requires the Gag NC domain; however, NC is not required for A3H HapI incorporation into virions [23]. Wang, et al [36] showed little virion packaging for HapI, III, IV, and VI. However, these haplotypes were poorly expressed in the cell and it is unknown if these haplotypes have virion packaging ability when expressed to a similar level as A3H HapII. In addition, it is not completely clear how A3H is packaged into HIV-1. Here we report that like A3G, A3H anti-viral activity and virion incorporation are also dependent on RNA binding activity, and that the differences in RNA binding activity determine anti-viral potency and cellular localization.

Immunoprecipitation and immunoblot analysis
293T cells were harvested 48 h after transfection, washed with PBS and lysed in lysis buffer (50 mM Tris, pH 7.5, with 150 mM NaCl, 1% Triton X-100 and Roche Complete Protease Inhibitor Cocktail Tablets) at 4uC for 30 min, followed by centrifugation at 10,0006g for 30 min. For the immunoprecipitation experiment comparing RNA binding activities of A3H HapII and Hap-IID15N, the transfected cell lysates were sonicated to ensure efficient lysis of both A3H HapII and HapIID15N from the nucleus. The HA-tag immunoprecipitation was carried out by mixing lysates of transfected cells from a T25 flask with a 25-ml bead volume of anti-HA antibody-conjugated affinity matrix (Roche) and incubating the mixture at 4uC for 3 h. The samples were washed six times with wash buffer (20 mM Tris, pH 7.5, with 100 mM NaCl, 0.1 mM EDTA and 0.05% Tween-20). The beads were then eluted with protein 26 loading buffer. The eluted materials were then analyzed by SDS-PAGE and immunoblotting with the appropriate antibodies.

Quantitative real-time PCR (qRT-PCR)
RNA obtained from immunoprecipitated samples were treated with DNase I by incubation in 10 ml of diethyl pyrocarbonate (DEPC)-treated water with 1x RQ1 RNase-Free DNase buffer, l ml of RQ1 RNase-free DNase (Promega) and 4 U of RNase inhibitor (New England Biolabs) for 30 min at 37uC. The DNase was inactivated by the addition of 1 ml RQ1 DNase stop solution and incubated at 65uC for 10 min. The RNA was reverse transcribed by using random primers and the Multiscribe reverse transcriptase from the High-Capacity cDNA Archive Kit (Applied Biosystems) according to the manufacturer's instructions. The cDNA was either undiluted or serially diluted in DEPC-treated water before input into the real-time reaction to ensure that the amplification was within the linear range of detection.
The ABI 7000 sequence detection system (Applied Biosystems, Carlsbad, CA) was used for the qRT-PCR amplifications. All primers were synthesized by Invitrogen, and fluorescence-tagged probes were synthesized by Applied Biosystems. Agarose gel analysis was used to verify that each primer pair produced single amplicons, and the identities of the PCR products were verified by cloning and sequencing. qRT-PCR was performed by using SYBR Green. Each 20-ml reaction mixture contained 1 ml forward and reverse specific primers (10 mM each), 10 ml 2x SYBR Green PCR Master Mix, 5 ml RNase-free water and 3 ml template cDNA. The reactions were performed under the following conditions: 50uC for 2 min and 95uC for 10 min, followed by 40 cycles of 95uC for 15 s and 60uC for 1 min, followed by a dissociation protocol. Single peaks in the melting curve analysis indicated specific amplicons. The target sequences were amplified using the following primer pairs: 7SL RNA, forward (59-ATCGGGTGTCCGCACTAAG-

Immunofluorescent staining
Hela cells were transfected with various A3H expression plasmids and were passed onto cover slips 24 h after transfection and allowed to reattach to the cover slip for another 24 h. The cells are then fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton and blocked with 10% BSA. HA-tagged A3H variants were stained with a mouse anti-HA antibody (Sigma, H9658) followed by a Texas Red conjugated anti-mouse antibody. The cell nucleus was stained with DAPI and examined by deconvolution microscopy using a Nikon i90 microscope. A series of Z-stack images were acquired with z-step of 0.2 mm, and raw image sequences were processed with a velocity imaging system and deconvolved.

Modeling
Homology modeling of A3H-HapII was carried out using both the program Modeller [37] and the modeling server I-TASSER [38], which has been ranked as the best server in the recent CASP7 and CASP8 modeling contests [39]. Both Modeller and I-TASSER produced very similar models, suggesting convincing modeling results. Prior to modeling in Modeller, multiple sequence alignment was carried out for APOBEC2, the amino-terminal domain of A3G (A3G-NTD), carboxyl-terminal domain of A3G (A3G-CTD), and A3H-HapII by using the program MUSCLE [40]. The availability of crystal structures of two homologous APOBECs, APOBEC2 [41] and A3G CTD [42,43], helped to validate and manually improve the alignment. The aligned sequences of A3G-NTD/A3H-HapII, APOBEC2, and A3G-CTD were input into Modeller for homology modeling using the two crystal structures as multiple templates, which improved the quality of the modeling. The high degree of homology among the proteins and the similarity of the resulting models impart confidence in the homology modeling, especially for the regions in which the conserved motif RLYYFW and A3H-HapII residues 15N, 105, 121, and 178 are located.

A3H HapII binds host RNA with high affinity
Potent anti-HIV-1 cytidine deaminases possess unique RNAbinding abilities which contribute to HIV-1 packaging and antiviral activity [35]. A3G and A3F both contain a conserved motif, RLYY(F/Y)W, demonstrated to be crucial for host RNA binding and HIV-1 virion incorporation [27,35]. Among all known A3H variants, A3H HapII has the most potent anti-viral activity and can be packaged into virions efficiently [14,22]. To determine the level of conservation of this RLYYF/YW motif, we aligned amino acid sequences from various human AID/APOBEC proteins. As shown in Figure 1, the RLYY(F/Y)W motif is conserved in A3B, A3G and A3F. Interestingly, the RLYYHW sequence was found in all A3H haplotypes.
A3G binds with high affinity to many cellular RNAs, notably 7SL RNA and Y RNAs [27,44]. Indeed, binding to 7SL RNA has been proposed to be essential for A3G incapsidation in the HIV-1 viral core [27,35,45]. To test whether A3H HapII has similar RNA binding activity, 293T cells were transfected with either empty vector, A3G-HA or A3H HapII-HA constructs. The cell lysates were harvested 48 h post-transfection, and the A3 proteins were immunoprecipitated. RNAs bound to the immunoprecipitated A3 proteins were extracted and reverse transcribed. The levels of 7SL, Y1, Y3 and Y4 RNAs, as well as GAPDH RNA were then measured as fold increase compare to control using qRT-PCR. RNA binding activities of A3G and A3H HapII, relative to control, are presented in Figure 2. Similar to previous report [27], more than 100 fold 7SL RNA were co-precipitated Figure 5. A3H HapI had a reduced ability to associate with 7SL and Y RNAs. Single mutation G105R restored its RNA binding activity. A) To compare RNA binding activities between A3H HapII and HapI, 293T cells were transfected with HA-tagged A3H HapII and HapI, and the cell lysates were immunoprecipitated using anti-HA affinity matrix agarose beads. Immunoprecipitated RNA were purified and reverse transcribed, and the relative amounts of 7SL, Y RNAs, GAPDH, b-actin and 5s RNA were measured using qRT-PCR. B) Relative RNA binding activities of A3H HapI and HapI105R compared to HapII. doi:10.1371/journal.pone.0038771.g005 with A3G-HA compared to control, while more than 300 fold of various Y RNA were associated with A3G-HA. A3H HapII demonstrated similar high binding affinities to 7SL, Y1, Y3 and Y4 RNAs. In particular, more than 300 folds of 7SL RNA were co-precipitated with A3H-HA compared to control.

A3H HapII packaging in HIV-1 virions is dependent on host RNA binding
A previous report demonstrated that mutating A3G at amino acid 127 W to A within the RNA binding motif RLYYFW abolished its ability to bind 7SL and Y RNAs as well as reduced its antiviral activity against HIV-1 NL4-3DVif [45]. To determine if this motif, RLYYHW, is also important for A3H HapII RNA binding, we constructed a vector to express A3H HapII W115A and tested its RNA binding and anti-viral activities. 293T cells were co-transfected with pNL4-3Dvif and various amounts of A3H HapII or A3H HapII W115A expression plasmids. As shown in Figure 3A, A3H HapII demonstrated strong suppressive activity against the Dvif virus, while HapIIW115A lost the ability to suppress viral replication. A3H HapIIW115A was also expressed at lower levels compared to wild-type A3H HapII ( Figure 3B, compare lanes 5 and 2). However, when expressed at equal levels ( Figure 3B, lanes 3 and 5), A3H HapII W115A still could not suppress viral replication as efficiently as wild-type A3H HapII. In addition, A3H HapII W115A was barely detectable in the viral lysate ( Figure 3B, lane 5), indicating that A3H HapII W115A lost its viral packaging ability. We next examined whether A3H HapII W115A may also have lost the ability to bind host RNAs. As shown in Figure 3C, compared to A3H HapII wild type, A3H HapIIW115A had significantly reduced RNA binding activities to Pol III RNAs such as 7SL, Y1 and Y4 RNAs. Interestingly, unlike A3G-W127A, which maintained its ability to associate with mRNAs such as GAPDH, A3H HapIIW115A also showed a reduced ability to associate with GAPDH mRNA, indicating that the RLYYHW motif plays a crucial role in general RNA binding  and as a consequence, it is absolutely required for virion incorporation.

A3H HapI has reduced ability to be packaged into HIV-1 virions and decreased anti-viral activity
Compared to A3H HapII, the A3H HapI protein is less stable and has weaker anti-viral activity [14,16,18,22]. However, when expressed at high levels, A3H HapI can also elicit potent anti-viral activity in vitro and is resistant to Vif degradation [20]. A recent study reported a negative association between genotype HapII and A3F/H DNA editing signature on HIV-1 DNA sequences from early infected patients, suggesting that Vif-resistant haplotypes could contribute to HIV-1 diversification in vivo [46]. To investigate if A3H HapI is efficiently packaged into virions compared to A3H HapII, we co-transfected 293T cells with pNL4-3DVif along with various amounts of A3H vectors. Infectivity assays were performed using MAGI cells to measure anti-viral activities of A3H HapI and HapII. As shown in Figure 4A and B, A3H HapII had strong anti-viral activity and was efficiently packaged into the virus ( Figure 4B, lanes 1, 2, and  3). When expressed at a high level, A3H HapI also suppressed virus replication up to about 60%. Interestingly, even when expressed at a similar or higher level in comparison to A3H HapII ( Figure 4B, compare lane 5 to lanes 3 and 4), HapI had lower antiviral activities and the level of A3H HapI in the purified virus was significantly lower compared to A3H HapII.

The low A3H HapI binding affinities to host RNAs are enhanced by the G105R mutation
Since RLYY(Y/F/H)W is also conserved in A3H HapI, we asked if A3H HapI was also associated with host RNAs, as was the case with A3H HapII. We performed immunoprecipitation of A3H HapI and HapII and examined a panel of HapII-associated RNAs, including Pol III RNAs 7SL, Y1, Y3, Y4, 5s RNA as well as GAPDH and b-actin mRNA. Because different amounts of proteins were pulled down, we measured the intensities of the bands and normalized them to A3H HapII. Interestingly, we saw little difference in the RNA binding for GAPDH, b-actin, and 5s RNA which are not specifically packaged into the HIV-1 virion ( Figure 5A). By contrast, A3H HapI showed reduced binding affinities to Y RNAs and 7SL RNA, the latter which is found abundantly in the virus [34] (Figure 5A). Therefore, the reduced Figure 8. Cellular localization of A3H variants. Hela cells were transfected with various A3H variant constructs, fixed, permeabilized and stained with a mouse anti-HA antibody followed by a secondary Texas Red conjugated anti-mouse antibody. The nucleus was stained with DAPI. The cells were examined by deconvolution microscopy as described in Methods. A series of Z-stack images were acquired with z-step of 0.2 mm and raw image sequences were processed with velocity imaging system and deconvolved. Various cellular localization patterns of the A3H variants were observed. The numbers of cells observed in each location were counted and summarized as percentages of total cells in the table. doi:10.1371/journal.pone.0038771.g008 ability of A3H HapI to bind to 7SL RNA may explain its less efficient and accurate packaging into the virion compared to A3H HapII.
A3H HapI and HapII differ by only 3 amino acids: 105G/R, 121K/E/D and 178E/D. Previous reports demonstrated that amino acid 105G/R determines protein stability while 121 K/E/ D determines A3H binding to Vif and its sensitivity to Vif-induced degradation [7,14,16,18,22,23]. Interestingly, single mutation G105R was shown to not only restore the expression level of A3H HapI, but also increase its anti-viral activity and the ability to be packaged [14,17,22,23]. Accordingly, we were interested in determining if G105R could also restore the ability of A3H HapI to interact with 7SL and Y RNAs. After transfecting 293T cells with empty vector, A3H HapI, HapII or HapI105R plasmids, the expressed A3H proteins in the cell lysates were subsequently immunoprecipitated and their association with RNAs compared. As expected, the single point mutation G105R in A3H HapI restored its ability to bind to 7SL and Y RNAs at levels similar to that of A3H HapII ( Figure 5B).

A3H HapIID15N does not have anti-viral activity and cannot be packaged into the virus
A3H HapIII and HapIV both differ from HapII at position 15(D or N). Both A3H HapIII and HapIV were reported to have weak or no anti-viral activities, and A3H D15N has greatly reduced protein stability [22,24], which results in the loss of antiviral activity. However, whether A3H HapIID15N can be packaged in virions and has anti-viral activity when expressed at a similar level to A3H HapII are unknown. To test this, we cotransfected 293T cells with pNL4-3Dvif and decreasing amounts of A3H HapII or HapIID15N expression constructs. As expected, A3H HapII showed strong antiviral activity and could be efficiently incorporated into virions ( Figure 6A). In contrast to A3H HapI, which was present in purified virions and had low levels of anti-viral activity, A3H HapIID15N, even when expressed at high levels similar to A3H HapII, was not detectable in the viral lysate and did not display any anti-viral activity ( Figure 6B, compare lanes 5 and 6 to lanes 2,3,4).

A3H HapII D15N does not associate with cellular RNAs
We performed immunoprecipitation procedures as above to determine if A3H HapIID15N could bind RNA, and the abilities of A3H HapID15N and HapII to associate with cellular RNAs were compared using qRT-PCR. A3H variant protein band intensities were measured and normalized to A3H HapII. Surprisingly, The A3H D15N deletion not only significantly impaired the ability of A3H HapII to bind to 7SL RNA and abolished its ability to bind to Y RNAs, it also greatly reduced its associations with other RNAs such as GAPDH ( Figure 7A).
The loss of anti-viral activity and viral packaging of A3H HapIID15N observed was very similar to that of the mutation HapIIW115A in the RLYYHW motif. To understand the role of 15N in relation to RLYYHW RNA motif, homology modeling of the A3H HapII was carried out based on the crystal structures of both APOBEC2 and the carboxyl-terminal domain of A3G. The result predicted that N15 is close in space to the RLYYHW region ( Figure 7B).

RNA binding activities are correlated with the cellular localizations of A3H variants
Previous report from Ohainle, et al showed that subcellular localization of A3H has changed during primate evolution: while rhesus macaque A3H is mainly cytoplasm, chimpanzee A3H and human A3H HapI is mostly nucleus [16]. A recent study that followed up on the localization of human A3H revealed that in contrast to A3H HapI, HapII localizes predominantly in the cytoplasm [47]. Using HapI and HapII fusion proteins, the study also suggested that HapI enters the nucleus through passive diffusion while HapI is being actively retended in the cytoplasm [47]. However, the localization of other haplotypes of A3H are unknown. To examine whether A3H haplotypes vary in their subcellular localization, we transfected Hela cells with the Cterminally HA-tagged A3H HapI, HapII, HapIID15N and HapII115A constructs. Forty-eight hours post-transfection, the cells were fixed and stained with a mouse anti-HA antibody followed by a secondary Texas Red anti-mouse antibody for examination by deconvolution microscopy. As shown in figure 8, consistent with previous results, 92% of A3H HapII localized predominantly in the cytoplasm while for A3H HapI, a range of different localization patterns were observed: in some cells (,64%) it localized both in the nucleus and cytoplasm, while in others (,36%) it was predominantly in the cytoplasm. Surprisingly, A3H HapIID15N and HapII115A, which has lost RNA binding activity, were found to be localized predominantly in the nucleus. The red offset signals were increased for A3H HapI, HapIID15N, and HapII115A which was expressed at lower levels compared to A3H HapII. Our results showed that subcellular localizations of the A3H variants were highly diverse, and the presence of A3H in the cytoplasm appeared to correlate with the RNA binding activity of this protein.

Discussion
Up to seven haplotypes have been identified to circulate in the human population for the A3H gene [24], having different combinations of 4 SNPs (18R/L, 105R/G, 121K/D, 178E/D) and one codon deletion (15N/D). In the absence of the Vif protein, only A3H variants that carry the combination of amino acids 15N and 105R (A3H HapII, V and VII) demonstrate strong anti-HIV-1 activity. A3H HapI (105G) and other haplotypes that carry D15N were reported to have weak or no anti-viral activity [14,16,17,18,22,23,24]. Why A3H variants differ in their anti-viral capabilities is currently unclear. Here we show that the anti-viral activity of human A3H correlated with its ability to bind host RNA.
It is critical for APOBEC3 proteins to be incorporated into virion particles in order to elicit a strong anti-viral response [27,35,45]. 7SL RNA binding activity has been proposed to be directly associated with the strength of APOBEC3-mediated antiviral suppression [35]. Consistent with previous findings [36], we found that the RNA binding motif RLYY(Y/F/H)W that is conserved on A3G and A3F and in all A3H haplotypes is required for their effective packaging into the virion. We observed similar RNA binding activity of A3H HapII as that found with A3G. Previous study suggests 115W to A mutation on this motif on HapII abolish A3H packaging [36]. We found that mutation of 115W not only impaired A3H HapII virion packaging but also abolished its RNA binding activity, arguing for the importance of RNA binding for virion packaging of A3H. This finding is also in agreement with a previous report that the NC domain of Gag is required for A3H HapII packaging [23].
A3H HapI, differing in only 3 amino acids from A3H HapII, was expressed at a lower level when introduced into 293T and HeLa cells and has reduced anti-viral activity [14,16,18,22,23,24]. However, A3H HapI appears to have relatively greater anti-viral activity when expressed at a higher level [20]. In this study, we showed that even at similar expression levels, A3H HapI had significantly reduced anti-viral activity compared to A3H HapII and could not package into virions efficiently. Furthermore, although A3H HapI maintained similar RNA binding activity to non-specific RNAs such as GAPDH and b-actin, it associated with significantly less 7SL and Y RNAs compare to HapII. Previous studies have shown that the G105R mutation restores anti-viral activity of A3H HapI [14,17,23,24]. Here we determined that introducing an R to G mutation at 105 restored the A3H HapI RNA binding activity. Interestingly, when expressed at a high level, A3H HapI was detected in the virion, indicating that it was packaged via other mechanisms, possibly mediated by the MA-CA region of Gag [23].
A3H HapIII differs from A3H HapII only at one amino acid (15D/N), while A3H HapIV also has a deletion at position 15 as well as an additional mutation (18L/R). It was reported that D15N destabilizes A3H [16], but it is not clear if A3H HapIII and HapIV possess any anti-viral activity at all. Here we were able to express A3H HapIID15N at a similar level to that of HapII, and strikingly, this deletion completely abolished A3H packaging into the virion as well as anti-viral activity. Remarkably, A3H HapIID15N also lost almost all RNA binding activity. Our homology modeling of A3H HapII based on the APOBEC1 and APOBEC3G C-terminal domain showed that 15N sits closely to the putative RLYYHW RNA binding motif. We hypothesize that 15N is either directly involved in RNA binding or at least is important for maintaining the structure of the RNA binding pocket of A3H.
Members of the APOBEC3 family have diverse anti-viral and anti-retrotransposition activities [1,2,48,49,50,51]. Here we showed that similar to A3F and A3G, A3H HapII had strong anti-viral activities while A3H HapI, like A3C, had modest antiviral activity. Meanwhile, A3H HapIII/IV, like A3A, exhibited no anti-viral activity. The potency of A3H anti-viral activity was directly correlated with its ability to associate with cellular RNAs such as 7SL RNA and its ability to efficiently package into HIV-1 viral particles. We have identified a putative RNA binding site RLYYHW for A3H HapII and showed that 105R and 15N are essential for RNA binding activity.
Besides having diverse anti-viral activities, APOBEC3 proteins also show distinct features in cellular localization [4,23,52,53,54]. A3H HapII localized predominantly in the cytoplasm, while A3H HapI was found in both the cytoplasm and nucleus and A3H HapIID15N primarily in the nucleus. Strikingly, we found that the localization of A3H variants correlated with their RNA binding activities; that is, A3H proteins that had higher affinities to 7SL and Y RNAs localized in the cytoplasm. Indeed, A3H Hap-IIW115A which lost its RNA binding activity also localized to the nucleus. Interestingly, a previous report had identified A3G amino acids 113-128 that includes the RNA binding RLYYFW motif as a cytoplasmic retention signal [52]. The authors concluded that RNA binding is not important for cytoplasmic retention by showing that the N-terminus of A3G is not associated with radiolabeled RNA but is still localized in the cytoplasm [52]. However, more sensitive detection methods have been developed, and a recent study concluded that the N-terminal A3G domain is actually responsible for association with specific host RNAs such as 7SL RNA [35]. Moreover, Stenglein et al. also reported that F126S and W127A as critical residues determining A3G cytoplasmic localization [55]. Therefore, it is likely that RNA binding is involved in regulating cytoplasmic localization of A3G. In the case of A3H, we have observed an association of RNA binding and cytoplasmic localization, suggesting that RNA is also involved in regulating A3H localization. Previous study has reported that both HapI and HapII associate with similar molecular mass complex [47], we therefore speculate that RNA binding may either play a role in cytoplasmic retention or mask critical amino acids responsible for nuclear proteins to interact with A3H. However, the specific mechanism responsible for A3H cellular localization requires further study.
It is striking that natural amino acid changes within A3H can give rise to variants that are highly diverse in anti-viral activities and cellular localizations. Distinct cellular localizations suggest that A3H variants interact with different proteins and/or nucleic acids and may have distinct functions within the cell. Previous reports have demonstrated that A3H variants distribute differently in geographically distinct human populations. For example, A3H HapII, III and IV are most commonly found in African populations, while HapI is the most common haplotype among Asians and Europeans [7,14,16,22,23,24]. Different selection pressures may have driven A3H into having distinct functions, and it will be intriguing to study the inhibitory activities of A3H variants against other viruses.