The antiviral APOBEC3 proteins block the replication of viruses by altering the sequence of viral DNA, changing cytidines (C) to uridines (U). Humans express seven different APOBEC3 proteins and several of them have been shown to inhibit the growth of lentiviruses such as HIV-1 in cell culture models. The APOBEC3C (A3C) protein, on the other hand, only weakly restricted viral replication. Given the strong antiviral activity of the other APOBEC3 proteins, former graduate student Cristina Wittkopp in the Emerman Lab (Human Biology and Basic Sciences Divisions) aimed to investigate the evolutionary history of A3C and illuminate the mechanisms of antiviral gene evolution. In her recent publication in PLOS Pathogens, she reports that A3C, despite its apparent weak activity, shows evolutionary evidence of having evolved to fight pathogens such as HIV-1. She also identifies that a specific variation in the A3C gene sequence, called a polymorphism, that is present in some humans confers increased antiviral activity toward HIV-1 replication in a cell culture model.
Wittkopp and her colleagues performed a phylogenetic analysis to compare the A3C gene sequence in 22 primate species of Old World monkeys and hominoids. They found statistical evidence that the gene as a whole is under positive selection and identified several primate lineages with a large number of mutations that change the protein sequence (dN/dS > 1), which is an established signature of positive selection.
Image provided by the Emerman Lab
A rapidly evolving A3C sequence suggested that the variation has been under selection by pathogens. With this in mind, Wittkopp et al analyzed human genetic variation in the A3C gene. They noted that there is a single polymorphism, changing the serine at position 188 to isoleucine (S188I), that is present in 2.44% of the human population according to data from the 1000 Genomes Project. Specifically, this allele, or version of the gene, is found in 0.72% of American populations and 8.85% of African populations, but appears absent from Asian and European populations.
When they compared the sequence of A3C to the other APOBEC3 genes, they found that all of the other ten genes had an isoleucine at the position equivalent to S188 in A3C. Hypothesizing that having an isoleucine at that position is critical for antiviral efficacy, they directly compared the ability of A3C S188 versus A3C I188 to restrict the replication of HIV-1 in cell culture. They found that A3C I88 inhibited HIV-1 replication 10-fold more than A3C S188 and the protein enzymes were expressed at equivalent levels in cells.
The scientists measured the ability of purified A3C S188 and I188 enzyme to modify fluorescently labeled single-stranded DNA in vitro. They found that A3C I188 had a 10-fold higher specific activity (measured in pmol/ug/min) than A3C S188. Additionally, A3C I188 induced a higher mutation rate than A3C S188 in a reverse transcription assay. These results suggest that A3C I188 is more effective as an antiviral protein because its enzymatic activity towards mutating the viral genome is higher.
Interestingly, the scientists also found that A3C I188 can form a dimer (a complex of two proteins) while S188 A3C cannot and only appears as a monomer in solution. They reasoned that I188 may allow A3C to form a dimer and that this is important for antiviral potency. To test whether it was dimerization or I188 that was important for antiviral activity, they genetically fused two A3C S188 proteins and expressed this artificial tandem domain protein in cells to measure its ability to restrict HIV-1. They found that the artificial tandem of A3C S188 inhibited replication of HIV-1 even better than A3C I188, approximately 100 fold better than monomeric A3C S188.
"Our work highlights a new restriction factor for HIV that is found in some, but not most people," explains principal investigator Michael Emerman. "The gain-of-function polymorphism is not found in chimpanzees or gorillas, but is the ancestral allele in other primates. Thus, it has likely been regained in ancient human history. It is possible that some humans may be afforded some level of additional protection from lentiviruses by a more active antiviral version of this protein."
Wittkopp CJ, Adolph MB, Wu LI, Chelico L, Emerman M. 2016. “A Single Nucleotide Polymorphism in Human APOBEC3C Enhances Restriction of Lentiviruses.” PLOS Pathogens.
This research was funded by the National Institutes of Health and the Canadian Institutes of Health Research.