Feline Immunodeficiency Virus Evolutionarily Acquires Two Proteins, Vif and Protease, Capable of Antagonizing Feline APOBEC3

Abstract

The interplay between viral and host proteins has been well studied to elucidate virus-host interactions and their relevance to virulence. Mammalian genes encode apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins, which act as intrinsic restriction factors against lentiviruses. To overcome APOBEC3-mediated antiviral actions, lentiviruses have evolutionarily acquired an accessory protein, viral infectivity factor (Vif), and Vif degrades host APOBEC3 proteins via a ubiquitin/proteasome-dependent pathway. Although the Vif-APOBEC3 interaction and its evolutionary significance, particularly those of primate lentiviruses (including HIV) and primates (including humans), have been well investigated, those of nonprimate lentiviruses and nonprimates are poorly understood. Moreover, the factors that determine lentiviral pathogenicity remain unclear. Here, we focus on feline immunodeficiency virus (FIV), a pathogenic lentivirus in domestic cats, and the interaction between FIV Vif and feline APOBEC3 in terms of viral virulence and evolution. We reveal the significantly reduced diversity of FIV subtype B compared to that of other subtypes, which may associate with the low pathogenicity of this subtype. We also demonstrate that FIV subtype B Vif is less active with regard to feline APOBEC3 degradation. More intriguingly, we further reveal that FIV protease cleaves feline APOBEC3 in released virions. Taken together, our findings provide evidence that a lentivirus encodes two types of anti-APOBEC3 factors, Vif and viral protease.IMPORTANCE During the history of mammalian evolution, mammals coevolved with retroviruses, including lentiviruses. All pathogenic lentiviruses, excluding equine infectious anemia virus, have acquired the vif gene via evolution to combat APOBEC3 proteins, which are intrinsic restriction factors against exogenous lentiviruses. Here we demonstrate that FIV, a pathogenic lentivirus in domestic cats, antagonizes feline APOBEC3 proteins by both Vif and a viral protease. Furthermore, the Vif proteins of an FIV subtype (subtype B) have attenuated their anti-APOBEC3 activity through evolution. Our findings can be a clue to elucidate the complicated evolutionary processes by which lentiviruses adapt to mammals.

Keywords: APOBEC3; FIV; Vif; evolutionary arms race; lentivirus; protease.

FIG 1

Less diversity of FIV subtype B. The sequences of the V3-V5 region of FIV env (subtype A, n = 153; subtype B, n = 100; subtype C, n = 24; subtype D, n = 49) were extracted from the GenBank/EMBL/DDBJ sequence database (the accession numbers used are available upon request). (A) Phylogenetic tree of FIV env. This phylogenetic tree was constructed using the ML method and displays an evolutionary relationship among the FIV sequences used in this study. Each line, called a “branch” of the tree, represents one FIV sequence. The longer branch lengths correlate with larger accumulations of mutations in the sequences. The scale bar indicates 0.1 nucleotide substitution per site. The bootstrap values are indicated on each node. (B) Viral genetic diversity. These values were calculated as described in Materials and Methods. Statistical analyses were performed using Bonferroni's multiple-comparison test. *, P < 0.01 versus subtype B.

FIG 2

Attenuated degradation activity of FIV subtype B Vif against feline A3. The expression plasmids for Flag-tagged FIV Vif were cotransfected with or without the expression plasmids for feline A3Z2, A3Z3, and A3Z2Z3 tagged with HA. Representative results of Western blotting assays (A) and FIV reporter assays (B) are shown. * in panel B, P < 0.05 versus no Vif. The assays were independently performed in triplicate. The data represent averages with SDs.

FIG 3

Effect of FIV Vif against feline A3Z3 haplotypes. The expression plasmids for His-tagged Vif of FIV TM2 or FIV Petaluma were cotransfected with or without the expression plasmids for a series of feline A3Z3 haplotypes tagged with HA. Representative results of Western blotting assays (A) and FIV reporter assays (B) are shown. * in panel B, P < 0.05 versus no A3Z3. The assays were independently performed in triplicate. The data represent averages with SDs. Note that the feline A3Z3 used in the other experiments is haplotype I.

FIG 4

Conservation of reduced degradation activity of FIV subtype B Vif against feline A3. The expression plasmids for Flag-tagged FIV Vif were cotransfected with or without the expression plasmids for feline A3Z2Z3 tagged with HA. Representative results of Western blotting assays (A) and FIV reporter assays (B) are shown. FIV infectivity is shown as the percentage of the value for no A3Z2Z3. * in panel B, P < 0.05 versus Petaluma. The assays were independently performed in triplicate. Data represent averages with SDs. (C) Co-IP assay. The expression plasmids for His-tagged FIV Vif were cotransfected with or without the expression plasmids for feline A3Z2Z3 tagged with HA, and the co-IP assay was performed as described in Materials and Methods. Representative results from co-IP assays using an anti-HA antibody (top) and cell lysate (bottom) are shown.

FIG 5

The responsive residue determines the ability of FIV Vif to degrade feline A3. (A) Schematic depicting the FIV Vif derivatives used. (B and C) Expression plasmids for a series of FIV Vif derivatives tagged with Flag were cotransfected with or without the expression plasmids for feline A3Z2Z3 (B) or A3Z3 (C) tagged with HA. Representative results of Western blotting assays are shown. (D) VESPA analysis. The percent amino acid sequence conservation was analyzed using VESPA (http://www.hiv.lanl.gov/content/sequence/VESPA/vespa.html) (34). The accession numbers of the FIV Vif sequences (subtype A, n = 5; subtype B, n = 8; subtype C, n = 10; subtype D, n = 4) are listed in Table 1. In the bottom panel, residues 167 and 242 to 243 are indicated in red, while the (T/S)LQ motif (residues 200 to 202) is indicated in blue. In the top panel, the amino acids in these positions are indicated as logo plots. n, the number of sequences used.

FIG 6

Evolution of FIV Vif. (A) Reconstructed phylogenetic tree of FIV vif. A red dot indicates the ancestral node of FIV vif. (B and C) Expression plasmids for Flag-tagged FIV Vif were cotransfected with or without the expression plasmids for feline A3Z2, A3Z3, and A3Z2Z3 tagged with HA. Representative results of Western blotting assays (B) and FIV reporter assays (C) are shown. * in panel C, P < 0.05 versus no A3. The assays were independently performed in triplicate. Data represent averages with SDs. (D) Phylogenetic trees of FIV genes. The phylogenetic trees of gag, pol, and env are shown. n, the number of sequences used. The bootstrap values are as follows: >50% (*) and >80% (**). An FIVpco sequence (GenBank accession no. KF906157) was specified as the root of these trees. (E) Schematic of putative scenario for FIV Vif evolution.

FIG 7

Cleavage of feline A3Z2Z3 by FIV PR. (A) Expression plasmids for feline A3Z2Z3 tagged with HA were cotransfected with pFP93 derivatives. Representative results of Western blotting assays are shown. Cleaved feline A3Z2Z3 (cA3Z2Z3) in the released viral particle is indicated with red arrowheads. (B) Schematic of the feline A3Z2Z3 derivatives used in the assay whose results are presented in panel C. The putative cleavage site is indicated with a vertical broken red line. (C) Expression plasmids for feline A3Z2Z3 derivatives tagged with HA were cotransfected with pFP93. Representative results of Western blotting assays are shown. (D to F) Expression plasmids for feline A3Z2Z3 WT or HSLH/IIII mutant derivatives (0, 200, 400, 800, 1,200, and 1,800 ng) were cotransfected with pFP93 and FIV-based virus plasmids. Representative results of Western blotting assays (D) and FIV reporter assays (E) are shown. * in panel E, P < 0.05 versus A3Z2Z3 WT. The assays were independently performed in triplicate. The data represent averages with SDs. The lane numbers in panel D correlate with those in panel E. (F) A correlation between the amount of A3Z2Z3 and cleaved A3Z3 (cA3Z3) in the released viral particle (x axis) and the viral infectivity (y axis) is shown. The circles with error bars represent averages with SDs (n = 3), and the lines represent exponential approximation. AU, arbitrary units.