A Naturally Occurring Domestic Cat APOBEC3 Variant Confers Resistance to Feline Immunodeficiency Virus Infection

Abstract

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; A3) DNA cytosine deaminases can be incorporated into progeny virions and inhibit lentiviral replication. On the other hand, viral infectivity factor (Vif) of lentiviruses antagonizes A3-mediated antiviral activities by degrading A3 proteins. It is known that domestic cat (Felis catus) APOBEC3Z3 (A3Z3), the ortholog of human APOBEC3H, potently suppresses the infectivity of vif-defective feline immunodeficiency virus (FIV). Although a recent report has shown that domestic cat encodes 7 haplotypes (hap I to hap VII) of A3Z3, the relevance of A3Z3 polymorphism in domestic cats with FIV Vif has not yet been addressed. In this study, we demonstrated that these feline A3Z3 variants suppress vif-defective FIV infectivity. We also revealed that codon 65 of feline A3Z3 is a positively selected site and that A3Z3 hap V is subject to positive selection during evolution. It is particularly noteworthy that feline A3Z3 hap V is resistant to FIV Vif-mediated degradation and still inhibits vif-proficient viral infection. Moreover, the side chain size, but not the hydrophobicity, of the amino acid at position 65 determines the resistance to FIV Vif-mediated degradation. Furthermore, phylogenetic analyses have led to the inference that feline A3Z3 hap V emerged approximately 60,000 years ago. Taken together, these findings suggest that feline A3Z3 hap V may have been selected for escape from an ancestral FIV. This is the first evidence for an evolutionary "arms race" between the domestic cat and its cognate lentivirus.

Importance: Gene diversity and selective pressure are intriguing topics in the field of evolutionary biology. A direct interaction between a cellular protein and a viral protein can precipitate an evolutionary arms race between host and virus. One example is primate APOBEC3G, which potently restricts the replication of primate lentiviruses (e.g., human immunodeficiency virus type 1 [HIV-1] and simian immunodeficiency virus [SIV]) if its activity is not counteracted by the viral Vif protein. Here we investigate the ability of 7 naturally occurring variants of feline APOBEC3, APOBEC3Z3 (A3Z3), to inhibit FIV replication. Interestingly, one feline A3Z3 variant is dominant, restrictive, and naturally resistant to FIV Vif-mediated degradation. Phylogenetic analyses revealed that the ancestral change that generated this variant could have been caused by positive Darwinian selection, presumably due to an ancestral FIV infection. The experimental-phylogenetic investigation sheds light on the evolutionary history of the domestic cat, which was likely influenced by lentiviral infection.

FIG 1

Characteristics of 7 feline A3Z3 haplotypes. (A) Summary of 7 feline A3Z3 haplotypes. Five amino acids and codons (in parentheses) that differed in 7 haplotypes are summarized. The amino acids and nucleotides that differed in the 7 haplotypes are represented in red. Note that the information presented, including the frequency of each haplotype shown here, is from a recent report by de Castro and colleagues, who surveyed 111 domestic cats in Porto Alegre, the capital city of Rio Grande do Sul state in Brazil (25). Also note that the feline A3Z3 used in the previous studies was hap I (GenBank accession number EU011792). (B and C) Positive selection on feline A3Z3. (B) Log-likelihood values and parameter estimates determined for two pairs of site models in the PAML package. Positive selection was detected to operate on feline A3Z3 at a high significance level (P < 0.001) by both analyses. 2Δl, twice the log likelihood difference between the compared models; df, degrees of freedom. (C) Identification of codons under conditions of positive selection. The dN/dS ratio (top) and posterior probability (bottom) inferred for each codon position by PAML analysis are shown. The positively selected codon identified with high probability (posterior probability, >0.95) is represented in red. The values in parentheses indicate the dN/dS ratio (top) and posterior probability (bottom) for the positively selected codon. The bars shown in orange indicate the codons that differed among the 7 haplotypes (codons 68, 94, 96, and 100). (D to F) Structure modeling of feline A3Z3. Cartoon (D and E) and surface (F) models of the structure of feline A3Z3 hap I are shown. In panel D, the α-helix and β-sheet are shown in green and pale blue, respectively. In panels D and E, Zn²⁺ is represented as a gray sphere. In panels E and F, the positively selected amino acid (residue 65) and the amino acids that differed among the 7 haplotypes (residues 68, 94, 96, and 100) are represented in red and orange, respectively. Note that residue 96 (V) is not localized on the surface of a protein.

FIG 2

Resistance of feline A3Z3 hap V to FIV Vif-dependent degradation. (A and B) Anti-FIV ability of feline A3Z3 haplotypes. (A) Immunoblot analysis. Representative results for FIV Vif strains Petaluma (top), C36 (middle), and Shizuoka (bottom) are shown. (B) FIV reporter assay. FIV infectivity is shown as the percentage of the value of “no A3.” (C) Correlation of the expression levels of FIV Vif and feline A3Z3. Data are representative of the results of immunoblot analysis. In panel B, asterisks (*) represent a P value of <0.05 (versus “no Vif” by Student's t test). NS, no statistical significance. The assays were independently performed in triplicate. Data represent averages with SD.

FIG 3

Deaminase-dependent anti-FIV ability of feline A3Z3 haplotypes. (A) Mutations in vif-deficient FIV-based reporter provirus. The partial luciferase gene (411 bp) was cloned and sequenced, and the results are summarized in the mutation matrix. The numbers of amplicons sequenced (n) and the percentages of G-to-A mutations in all substitutions are represented. (B and C) Dose and deaminase-dependent anti-FIV ability of feline A3Z3. (B) Representative results of immunoblot analysis. WT, wild type. (C) FIV reporter assay. FIV infectivity is shown as the percentage of the value corresponding to “no A3.” (D) Enzymatic activity of feline A3Z3 haplotypes. Representative results of oligonucleotide cleavage assay (top) and immunoblot analysis (bottom) are shown. In the top panel, the enzymatic activity of feline A3Z3 haplotypes in the 2 different contexts (substrate/product 1 and product 2) was measured. Human (HU) A3A tagged with mycHis was used as a positive control in the assay. S, substrate; P, product; WB, Western blot. (E) Sensitivity of feline A3Z3 hap I and hap V to a FIV IMC in feline CRFK cells. Representative results of immunoblot analysis are shown. In panel C, an asterisk (*) represents P < 0.05 (versus “no A3” by Student's t test). The assays were independently performed in triplicate. Data represent averages with SD.

FIG 4

The effect of feline A3Z3 haplotypes against FeLV, RD-114 virus, and a retrotransposon. FeLV infectivity (A), RD-114 virus infectivity (B), and LINE-1 retrotransposition (C) are shown as percentages of the value corresponding to “no A3.” Representative results of immunoblot analysis are shown in the left panels. In the right panels, each value is shown as the percentage of the value corresponding to “no A3.” The assays were independently performed in triplicate. Data represent averages with SD.

FIG 5

Determinant of feline A3Z3 sensitivity to FIV Vif-mediated degradation. (A) Proteasome-dependent degradation of feline A3Z3 by FIV Vif. The transfected cells were treated with or without 10 μM MG132. Representative results of immunoblot analysis are shown in the top panel, and percentages of the band intensity of A3Z3 compared to hap I without FIV Vif-Flag and MG132 are shown in the bottom panel. This assay was independently performed in triplicate. Data represent averages with SD. (B to D) Determinant responsible for the resistance of feline A3Z3 to FIV Vif-mediated degradation. (B) A representative result of immunoblot analysis. (C and D) The correlations of the percentage of the degradation efficacy of each feline A3Z3 mutant (x axes) with the surface area (y axis in panel C) or the hydrophobicity (y axis in panel D) of residue 65 are shown. The capitalized characters represent the amino acid at position 65. The variants that were resistant and sensitive to FIV Vif are represented in red and black, respectively. The Spearman rank correlation coefficient (r_s) was used to determine statistically significant correlations between the values. The assay was independently performed in triplicate. Data represent averages with SEM. In panel C, the line represents a linear approximation.

FIG 6

Estimation of the age of feline A3Z3 hap V emergence. (A) Maximum clade credibility tree inferred by using BEAST v 1.7.5. Asterisks indicate the nodes with a posterior probability value of >0.5. The lineage detected under conditions of positive selection by the branch-site test (see panel C) is represented in red. (B) Divergence times and posterior probabilities inferred by BEAST. In the column with the heading with the superscript “a,” node numbers are consistent with the numbers in panel A. In the column with the heading with the superscript “b,” HPD represents “highest posterior density” and the units in the column represent years. In the column with the heading with the superscript “c,” the ML bootstrap value is for 1,000 bootstrap replicates. In the entries with the superscript “d,” NA represents “not applicable.” (C) Likelihood ratio test statistics of 7 feline A3Z3 haplotypes under the branch-site models in PAML analysis. In the column with the heading with the superscript “a,” the number in parentheses represents posterior probability. df, degrees of freedom.