We present evidence of the evolutionary history of APOBEC3H in humans in which two independent mutations arose during recent human evolution and gave rise to an unstable APOBEC3H protein with no detectable antiviral activity. This is in contrast to our finding of stable expression and antiviral activity of other hominoid APOBEC3H proteins. Both of the inactivating mutations occur in distinct regions of APOBEC3H, but similarly act to decrease the half-life of the APOBEC3H protein. Reconstruction of the ancestral human APOBEC3H gene suggests that recent human ancestors encoded a very powerful anti-retroviral and anti-retroelement version of this gene. While most humans possess unstable APOBEC3H alleles, we found that a minor fraction of humans still encode a stable allele of APOBEC3H that still retains activity and can be targeted by the HIV Vif protein.
Our finding of an APOBEC3H
allele with variable activity among individuals may have important consequences for the interaction of the human APOBEC3
locus with HIV. First, in those individuals homozygous or heterozygous for the active APOBEC3H
haplotype, there may be an additional APOBEC-mediated block to infection of HIV target cells in addition to the activities of APOBEC3F and APOBEC3G. However, we must point out that although APOBEC3H mRNA is present in the appropriate cell types (T cells), (OhAinle et al., 2006
)) it is still unclear if the APOBEC3H protein is present. Second, our finding that HIV Vif can inhibit the active APOBEC3H
allele argues that HIV Vif may have specifically evolved to counteract the active APOBEC3H protein in addition to the other active APOBEC3 proteins. The mechanism by which HIV Vif recognizes and inhibits APOBEC3H may or may not overlap with the mechanisms of inhibition of other APOBEC3 proteins. Therefore, successful replication of HIV in individuals with active APOBEC3H
alleles may further challenge Vif evolution even though HIV-1 Vif may be sufficient to counteract any potential benefit of active APOBEC3H in humans. Significant differences in HIV-1 progression have been shown to be correlated with APOBEC3G variation (An et al., 2004
), however, we do not yet know if individuals heterozygous or homozygous for active APOBEC3H are more resistant to successful HIV replication. On the other hand, since the stable APOBEC3H proteins appear to be able to inhibit multiple retroelements (against both LINE-1, Alu elements and retroviruses), it is also possible that APOBEC3H served an ancient role and has no modern impact on HIV-1.
In light of the multiple protective functions served by human APOBEC3H (against both LINE-1, Alu elements and retroviruses), it is puzzling that a majority of humans have lost this activity and even more puzzling that loss of activity happened twice in recent human evolution. This suggests the interesting possibility that non-functional APOBEC3H
alleles have been actively selected for in humans. The “less-is-more” hypothesis suggests that loss of function mutations can be maintained at high frequency during a shift of environmental pressures under which they may be selectively advantageous, particularly if the functional allele presents a cost to the host (Olson, 1999
). For example, there may be a significant cost associated with maintaining active APOBEC3 proteins at high levels in the cell as APOBEC3 proteins present a risk to the host if they were to deaminate the wrong targets, such as cellular mRNA or genomic DNA. Consistent with this hypothesis, APOBEC3H was recently shown to hypermutate human papillomavirus, a double-stranded DNA virus thought to closely resemble nuclear DNA (Vartanian et al., 2008
). We speculate that APOBEC3H may be particularly toxic relative to other APOBEC3 family members as no duplications of the APOBEC3H-like domain have been tolerated in any mammalian genome, in striking contrast to the multiple duplications of the non-APOBEC3H domain (Conticello et al., 2005
; OhAinle et al., 2006
). Further, the expression of APOBEC3H
in germline tissues (OhAinle 2006
) poses a mutagenic threat with serious evolutionary consequences and, therefore, could be particularly costly to the host. In this context, APOBEC3H may be especially 'costly' in humans and chimps, compared to other primates, because nuclear localization of the APOBEC3H protein occurred only recently during the evolution of the protein in hominoids, corresponding with loss of the C-terminal domain in the human/chimpanzee ancestor. Therefore, the nuclear localization and potential risk of increased genomic DNA editing are greater for humans and chimpanzees than for other primates. The benefits of maintaining stable and active APOBEC3H alleles (due to significant selective pressure from an active exogenous viral infection or from active endogenous retroviruses or retrotransposons) might outweigh the costs associated with the stable APOBEC3H protein in chimpanzees but not in humans. This is plausible given that retroviral infections and retroelement amplifications are known to be distinct among even these two closely-related species (Jern et al., 2006
; Polavarapu et al., 2006
; Yohn et al., 2005
Taken together, our findings suggest that there may be significant evolutionary pressure to downregulate levels of APOBEC3H protein in the cell in times of decreased pathogenic pressure. Under this scenario, maintaining a stable and active APOBEC3H protein capable of retroelement inhibition would have to be balanced with the costs associated with maintaining high doses of this protein in the cell. Other APOBEC3 proteins that are localized to the nucleus (and therefore may also contribute to higher genomic mutation) might also display evidence of this increased cost. Consistent with this model, APOBEC3B
has also been lost in a significant fraction of human populations (Kidd et al., 2007
), while the start codon of the APOBEC3A
gene is missing from chimpanzees (unpublished data). These debilitating events appear specific to the APOBEC3 genes that have a nuclear localization, supporting the 'costly defense' selective scenario for the acquisition of destabilizing mutations in human APOBEC3H.
Nonetheless, there are important differences between the loss of APOBEC3B and APOBEC3H activities in humans. While APOBEC3B has been lost due to a genomic deletion, APOBEC3H still appears to be expressed but encodes an unstable protein. We cannot rule out the possibility that instability, rather than loss of APOBEC3H function, is what has been selected. Even the unstable APOBEC3H alleles may still be sufficient to carry out some antiviral function that we have not assayed. A simple ‘loss-of-function’ hypothesis might be insufficient to explain the high prevalence of the unstable alleles (~50%) even in the African populations, where APOBEC3H activity still appears to be selectively retained. Instead, our findings suggest that the two unstable alleles appear to be independently under selection, based on the discordance in their allele frequencies in different human populations.
Independent of the true nature of selective pressures shaping the human APOBEC3 locus, the consequences of these pressures on the antiviral repertoire are significant. Combining the presence of two 'loss of stability' mutations in APOBEC3H and the loss of the APOBEC3B gene in humans, we can conclude that at least two APOBEC3 antiretroviral proteins are maintained at higher frequencies in African populations and have subsequently been lost to varying degrees in European and Asian populations. This is consistent with a model in which a lower pathogenic burden outside Africa may allow for the accumulation of inactivating mutations in antiviral genes outside of Africa (either due to genetic drift or due to selection). This implies both that a relatively recent (and perhaps ongoing) pathogenic pressure is maintaining APOBEC3H and APOBEC3B activity in African populations, and further that these populations might have an associated higher risk of genomic mutations. Regardless of the cause, it is clear that much of the redundancy of an expanded APOBEC3 locus that built up over primate evolution to defend the genome against the deleterious effects of retrotransposition or unknown retroviruses has been lost from a substantial fraction of the human species.