Equine infectious anemia virus resists the antiretroviral activity of equine APOBEC3 proteins through a packaging-independent mechanism

Abstract

Equine infectious anemia virus (EIAV), uniquely among lentiviruses, does not encode a vif gene product. Other lentiviruses, including human immunodeficiency virus type 1 (HIV-1), use Vif to neutralize members of the APOBEC3 (A3) family of intrinsic immunity factors that would otherwise inhibit viral infectivity. This suggests either that equine cells infected by EIAV in vivo do not express active A3 proteins or that EIAV has developed a novel mechanism to avoid inhibition by equine A3 (eA3). Here, we demonstrate that horses encode six distinct A3 proteins, four of which contain a single copy of the cytidine deaminase (CDA) consensus active site and two of which contain two CDA motifs. This represents a level of complexity previously seen only in primates. Phylogenetic analysis of equine single-CDA A3 proteins revealed two proteins related to human A3A (hA3A), one related to hA3C, and one related to hA3H. Both equine double-CDA proteins are similar to hA3F and were named eA3F1 and eA3F2. Analysis of eA3F1 and eA3F2 expression in vivo shows that the mRNAs encoding these proteins are widely expressed, including in cells that are natural EIAV targets. Both eA3F1 and eA3F2 inhibit retrotransposon mobility, while eA3F1 is a potent inhibitor of a Vif-deficient HIV-1 mutant and induces extensive editing of HIV-1 reverse transcripts. However, both eA3F1 and eA3F2 are weak inhibitors of EIAV. Surprisingly, eA3F1 and eA3F2 were packaged into EIAV and HIV-1 virions as effectively as hA3G, although only the latter inhibited EIAV infectivity. Moreover, all three proteins bound both the HIV-1 and EIAV nucleocapsid protein specifically in vitro. It therefore appears that EIAV has evolved a novel mechanism to specifically neutralize the biological activities of the cognate eA3F1 and eA3F2 proteins at a step subsequent to virion incorporation.

FIG. 1.

Schematic of the equine APOBEC3 gene locus on chromosome 28. The six equine A3 genes are shown between the flanking genes NPTXR and CBX7. The entire region spans 255,301 bp and the schematic is to scale, as indicated by bars. Transcriptional orientation is indicated by arrows; locations of conserved zinc-binding domains are indicated by asterisks. Gene schematics include exons (filled boxes), introns (lines), and untranslated regions (open boxes). Ψ, the location of a truncated, EcA3A exon 2-like open reading frame. Gene order, size, and exon/intron boundaries are based on the EquCab1 assembly available at NCBI (accession number NW_001799702).

FIG. 2.

Phylogenetic analysis of A3 domains. Neighbor-joining tree with bootstrap values based on A3 CDA domains. For proteins containing two CDA domains, domains are annotated with “N” for the N-terminal CDA domain or “C” for the C-terminal CDA domain. Domain names start with species: human (Hs), horse (Ec), cow (Bt), sheep (Oa), pig (Ss), cat (Fc), mouse (Mm), and rat (Rn). GenBank accession numbers are given in Materials and Methods.

FIG. 3.

Alignment of the predicted amino acid sequences of the full-length HsA3F (hA3F), EcA3F1 (eA3F1), and EcA3F2 (eA3F2) proteins. Critical CDA domain residues are indicated by asterisks. The sequences of EcA3F1 and EcA3F2 have been deposited in GenBank under NCBI accession numbers FJ174662 and FJ174663, respectively.

FIG. 4.

Detection of eA3F1 and eA3F2 transcripts in equine cells and tissues. Total RNA isolated from the indicated tissues or cells was amplified by RT-PCR using eA3F1- and eA3F2-specific primers. Amplified products were visualized following agarose gel electrophoresis and ethidium bromide staining. Specificity of each primer set is shown using peA3F1-HA and peA3F2-HA plasmid templates. β-actin was used as an internal control for input RNA.

FIG. 5.

Biological activity of double-CDA-domain eA3 proteins. (A) This assay measures the abilities of A3 proteins to enhance mutagenesis levels in bacteria. Plasmids encoding the indicated proteins were introduced into bacteria, and their expression was induced. The level of mutagenesis was then assessed by plating the bacteria on medium containing rifampin and counting the number of Rif^r colonies. The average of eight experiments with the standard deviation is indicated. (B) Western blotting of A3 protein expression in the bacterial strain analyzed in panel A using an HA-tag-specific antibody. The propensity of hA3G to give rise to a truncated, carboxy-terminal form in bacteria has been previously noted (8). Size markers are indicated. (C) 293T cells were cotransfected with 200 ng of the VSV-G expression plasmid pHIT/G, 125 ng of a plasmid encoding the indicated hA3 or eA3 protein, or the parental pcDNA3 plasmid, together with either 2 μg of a self-packaging retroviral luciferase expression plasmid (pNL-Luc-HXBΔVif or pSIV-AGM-LucΔVif) or 1 μg each of a retroviral packaging plasmid and a cognate luciferase expression vector (pNCS and pFB-Luc or pEV53B and pUNC-SIN6.1CLW-1). At 44 h posttransfection, released retroviral virions were collected and used to infect naïve 293T cells. A further 24 h later, the infected cells were lysed and luciferase expression levels were determined. Data are presented relative to the control culture, cotransfected with the parental pcDNA3 plasmid, which is set to 100 for each virus. The average of three independent experiments with the standard deviation is indicated. Neg., no APOBEC3 protein expressed.

FIG. 6.

Dose-response analysis of the inhibition of HIV-1 and EIAV infectivity. This experiment was performed as described in the legend to Fig. 4, except that three different levels of each A3 expression plasmid (62 ng, 125 ng, or 250 ng) were analyzed. The upper panel shows the level of each A3 protein expressed in the EIAV-producing 293T cells, as determined by Western blot analysis. The middle panel shows the level of infectivity in each virus sample, given as a percentage of the level of transduced luciferase gene expression seen in the control culture cotransfected with the pcDNA3 parental expression plasmid. The average of three independent experiments with the standard deviation is indicated. The lower two panels show the relative level of production of the EIAV or HIV-1 Gag polyprotein in the producer cells, measured by Western blot analysis. A representative experiment is shown. α-HA, anti-HA.

FIG. 7.

Editing of HIV-1 reverse transcripts by hA3G or eA3F1. Infectious HIV-1 viral particles were produced in 293T cells cotransfected with pNL-Luc-HXBΔVif and either pcDNA3, phA3G-HA, or peA3F1-HA. These viruses were collected and used to infect naïve 293T cells, and the infected cells were then lysed 24 h later. Total DNA was isolated, and a segment of the virally encoded luciferase gene was amplified by PCR, cloned, and sequenced. (A) These boxes show differences between the predicted luciferase DNA sequence and the observed DNA sequence, given the total number of specific mutations observed in the absence of any exogenous A3 protein (none) or in the presence of hA3G or eA3F1. The number below each box shows the total number of bases sequenced. (B) The G to A mutations compiled in panel A were analyzed to reveal that the sequence context of the dC residue on the proviral minus strand was edited to dU by either hA3G or eA3F1.

FIG. 8.

Analysis of packaging of eA3F1 and eA3F2 into HIV-1 and EIAV virions. 293T cells were cotransfected with either the HIV-1 proviral vector pNL4-3ΔVifΔEnv, or the EIAV vectors pEV53B and pUNC-SIN6.1CLW-1, together with phA3A-HA, phA3G-HA, peA3F1-HA, or peA3F2-HA. At 48 h posttransfection, supernatant media were collected and virions were isolated by centrifugation through a sucrose cushion. (A) Western blot analysis of lysed HIV-1 producer cells and HIV-1 virions using an anti-HA (α-HA) antibody or an anti-HIV-1 p24 (α-p24) capsid antiserum. (B) Western blot analysis of lysed EIAV producer cells and EIAV virions using an anti-HA antiserum or an anti-EIAV Gag antiserum that recognizes the p26 capsid protein. The mobility of protein size markers is indicated.

FIG. 9.

Specific interaction of eA3F1 and eA3F2 with both the HIV-1 and EIAV NC protein. Recombinant bacterial GST, or GST-HIV NC or GST-EIAV NC fusion proteins, were mixed with HA-tagged hA3A, hA3G, eA3F1, or eA3F2 from overexpressing 293T cells, and GST protein complexes were collected by incubation with glutathione-agarose beads. Bound (25% of total) and input (5% of total) proteins were then visualized by Western blot analysis using anti-GST (α-GST) or anti-HA (α-HA) antisera as previously described.

FIG. 10.

Analysis of the effect of eA3F1 and eA3F2 on retrotransposon mobility. HeLa cells were transfected with either pcDNA3, which contains an intact neo gene, or with retrotransposition indicator constructs based on MusD or IAP, which can confer Neo^r only after undergoing retrotransposition. The cells were also cotransfected with pK-based vectors expressing the indicated A3 proteins or with pK itself. At 72 h posttransfection, to allow retrotransposition to occur, the transfected cells were selected for Neo^r, and resistant colonies were stained and counted 12 days later. (A) These data summarize four independent experiments analyzing the effect of the indicated A3 proteins on the mobility of MusD or IAP. The pcDNA3 plasmid represents a control for nonspecific toxicity. Data are given relative to the pK control vector and show the observed standard deviation. (B) The expression of the indicated A3 proteins in the cotransfected HeLa cells was analyzed by Western blot analysis using an anti-HA antiserum. A representative experiment is shown.

FIG. 11.

Subcellular localization of eA3F1 and eA3F2 in transfected cells. HeLa cells were transfected with either peA3F1-HA or peA3F2-HA, and the subcellular localization of each protein was determined at 44 h posttransfection by immunofluorescence. Nuclei were identified in parallel using Hoechst stain, as previously described (8).