Cytidine deaminase efficiency of the lentiviral viral restriction factor APOBEC3C correlates with dimerization.

The seven APOBEC3 (A3) enzymes in primates restrict HIV/SIV replication to differing degrees by deaminating cytosine in viral (-)DNA, which forms promutagenic uracils that inactivate the virus. A polymorphism in human APOBEC3C (A3C) that encodes an S188I mutation increases the enzymatic activity of the protein and its ability to restrict HIV-1, and correlates with increased propensity to form dimers. However, other hominid A3C proteins only have an S188, suggesting they should be less active like the common form of human A3C. Nonetheless, here we demonstrate that chimpanzee and gorilla A3C have approximately equivalent activity to human A3C I188 and that chimpanzee and gorilla A3C form dimers at the same interface as human A3C S188I, but through different amino acids. For each of these hominid A3C enzymes, dimerization enables processivity on single-stranded DNA and results in higher levels of mutagenesis during reverse transcription in vitro and in cells. For increased mutagenic activity, formation of a dimer was more important than specific amino acids and the dimer interface is unique from other A3 enzymes. We propose that dimerization is a predictor of A3C enzyme activity

Cytidine deaminase efficiency of the lentiviral viral restriction factor APOBEC3C correlates with dimerization.

The seven APOBEC3 (A3) enzymes in primates restrict HIV/SIV replication to differing degrees by deaminating cytosine in viral (-)DNA, which forms promutagenic uracils that inactivate the virus. A polymorphism in human APOBEC3C (A3C) that encodes an S188I mutation increases the enzymatic activity of the protein and its ability to restrict HIV-1, and correlates with increased propensity to form dimers. However, other hominid A3C proteins only have an S188, suggesting they should be less active like the common form of human A3C. Nonetheless, here we demonstrate that chimpanzee and gorilla A3C have approximately equivalent activity to human A3C I188 and that chimpanzee and gorilla A3C form dimers at the same interface as human A3C S188I, but through different amino acids. For each of these hominid A3C enzymes, dimerization enables processivity on single-stranded DNA and results in higher levels of mutagenesis during reverse transcription in vitro and in cells. For increased mutagenic activity, formation of a dimer was more important than specific amino acids and the dimer interface is unique from other A3 enzymes. We propose that dimerization is a predictor of A3C enzyme activity

In vitro characterization of APOBEC3C S188 and I188.

<p>(Top) The specific activity of APOBEC3C S188 and I188 was determined by incubating the enzyme with a 118 nt ssDNA substrate with an internal fluorescein label (yellow star) and 2 possible sites for cytidine deamination (marked as “C”). Single deaminations of the 5'C and 3'C are detected as the appearance of fluorescently labeled 100 nt and 81 nt fragments, respectively; double deamination of both C residues on the same molecule results in a 63 nt labeled fragment. Substrate usage is quantified for below each lane of the gels. (Bottom) The substrate usage during a 60 min time course was plotted from three independent experiments (bottom left) and used to calculate the specific activity of the enzymes (bottom right).</p

A Single Nucleotide Polymorphism in Human APOBEC3C Enhances Restriction of Lentiviruses

<div><p>Humans express seven human APOBEC3 proteins, which can inhibit viruses and endogenous retroelements through cytidine deaminase activity. The seven paralogs differ in the potency of their antiviral effects, as well as in their antiviral targets. One APOBEC3, APOBEC3C, is exceptional as it has been found to only weakly block viruses and endogenous retroelements compared to other APOBEC3s. However, our positive selection analyses suggest that APOBEC3C has played a role in pathogen defense during primate evolution. Here, we describe a single nucleotide polymorphism in human APOBEC3C, a change from serine to isoleucine at position 188 (I188) that confers potent antiviral activity against HIV-1. The gain-of-function APOBEC3C SNP results in increased enzymatic activity and hypermutation of target sequences when tested <i>in vitro</i>, and correlates with increased dimerization of the protein. The I188 is widely distributed in human African populations, and is the ancestral primate allele, but is not found in chimpanzees or gorillas. Thus, while other hominids have lost activity of this antiviral gene, it has been maintained, or re-acquired, as a more active antiviral gene in a subset of humans. Taken together, our results suggest that APOBEC3C is in fact involved in protecting hosts from lentiviruses.</p></div

APOBEC3C is rapidly evolving in primates.

<p>(A) Twenty-two primate APOBEC3C coding sequences were obtained by PCR or from the NCBI sequence database. A phylogeny of APOBEC3C, indicating the branch analysis results of the positive selection tests. The ratio of rate of nonsynonymous changes (dN) and the rate of synonymous changes (dS) that occurred along each branch are shown above each branch. For dN/dS values = ∞, the total number of non-synonymous changes (N) and synonymous changes (S) are shown (N:S) below the branch. The red asterisks mark the branches where the dN/dS is significantly greater than 1 across the entire gene. (B) Maximum likelihood tests for positive selection, with 2lnl values indicating twice the log difference between the model that allows for positive selection (M8) and the model that does not allow for positive selection (M8a), as well as a P-value to indicate whether the M8 model better fits the data than the M8a model. (C) Sites under positive selection in APOBEC3C are shown in a cartoon diagram, comparing these sites to the Vif binding domain and the cytidine deaminase enzymatic domain (CD). Red triangles depict sites with a posterior probability >0.99 (red triangle). The structure of APOBEC3C[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005865#ppat.1005865.ref031" target="_blank">31</a>] is represented, with the Vif binding domain[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005865#ppat.1005865.ref031" target="_blank">31</a>] shown in blue. Two of the seven positively selected sites (PP > 0.99) overlap with this domain, are shown with arrows. The cytidine deaminase domain is shown in green.</p

APOBEC3C is targeted by Vif.

<p>(A) Restriction of HIV-1 Δ<i>vif</i> by APOBEC3C S188 (A3C S188), APOBEC3C I188 (A3C I188), and full recovery of infectivity by the presence of HIV-1 (LAI strain) and HIV-2 (ROD strains) <i>vif</i>. 0.4μg of APOBEC3 plasmid was used for each condition, and 0.6μg of each provirus was used. Infectivity of each virus is set to 100% for infection with No APOBEC3 (No A3) present. Error bars represent standard deviation of three independent transfections and infections. Black bars indicate no Vif, orange bars indicate HIV-1 (LAI) Vif, and green bars indicate HIV-2 (ROD) Vif (B) Vif degradation of APOBEC3C S188 and I188 was detected by Western blot analysis. 0.4μg of APOBEC3C and 0.6μg of an HIV-1 provirus (either Δ<i>vif</i>, lane labelled as (-)) or containing HIV-1 <i>vif</i> or containing HIV-2 <i>vif</i>, lanes labelled as HIV-1 or HIV-2) were used to transfect 293T cells and lysates were probed for APOBEC3C expression. Tubulin was used as a loading control.</p

Purified APOBEC3C I188 forms dimers in solution.

<p>(A) Size exclusion chromatography profiles of the APOBEC3C S188 and I188 from a 10mL G200 Superdex column were used to calculate the oligomerization states of the enzymes. Molecular weights were calculated by comparing to a calibration curve (see inset on right). When APOBEC3C S188 and APOBEC3C I188 were loaded onto the column, APOBEC3C S188 was a monomer in solution (apparent MW 21 kD in peak fraction), whereas APOBEC3C I188 could form dimers (apparent MW 42 kD in peak fraction) in addition to monomers (apparent MW 21 kD in peak fraction). Chromatograms were made using the integrated gel band intensities from three independent experiments of each protein fraction after resolution by SDS-PAGE. A representative Western blot of the size exclusion chromatography fractions is shown. (B) A3C S188 or I188 were incubated in the absence or presence of 10 μM bis(sulfosuccinimidyl)suberate (BS3) crosslinker (indicated as -BS3 or +BS3) and then visualized through SDS-PAGE and Western blotting. The Western blot demonstrates that A3C S188 remained monomeric in the presence of crosslinker, whereas A3C I188 was both monomeric and dimeric in the presence of the crosslinker. Molecular weight standards are indicated.</p

APOBEC3C SNP Isoleucine 188 confers increased antiviral activity.

<p>(A) Infectivity of HIV-1 Δ<i>vif</i> in the absence of APOBEC3 (No A3), APOBEC3G (A3G), APOBEC3C S188 (A3C S188) and APOBEC3C I188 (A3C I188). 0.3μg of HA-tagged APOBEC3 was expressed in virus-producing cells, and viruses were collected and use for infection. Infectivity in the absence of APOBEC3 is set to 100%. Error bars indicate the standard deviation of triplicate transfections and infections, and this experiment was repeated four times with similar results. Intracellular expression of APOBEC3 was measured by Western Blot using an anti-HA antibody. A section of the blot was probed with an anti-tubulin antibody as a loading control. (B) Dose-response analysis showing restriction of HIVΔ<i>vif</i> in the presence of two-fold dilutions of transfected APOBEC3C S188, or APOBEC3C plasmids I188 along with Western blot analysis of APOBEC3C S188, and APOBEC3C I188 protein expression during virus production. This experiment was performed three times, and a representative result is shown. (C) Infectivity of Simian Immunodeficiency virus SIVagmΔ<i>vif</i>, in the presence of APOBEC3C S188, APOBEC3C I188. Infectivity is set to 100% for infection with No APOBEC3 present. Error bars indicate the standard deviation of three independent experiments.</p