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J Virol. Sep 2009; 83(18): 9474–9485.
Published online Jul 8, 2009. doi:  10.1128/JVI.01089-09
PMCID: PMC2738220

Defining APOBEC3 Expression Patterns in Human Tissues and Hematopoietic Cell Subsets[down-pointing small open triangle]

Abstract

Human APOBEC3 enzymes are cellular DNA cytidine deaminases that inhibit and/or mutate a variety of retroviruses, retrotransposons, and DNA viruses. Here, we report a detailed examination of human APOBEC3 gene expression, focusing on APOBEC3G (A3G) and APOBEC3F (A3F), which are potent inhibitors of human immunodeficiency virus type 1 (HIV-1) infection but are suppressed by HIV-1 Vif. A3G and A3F are expressed widely in hematopoietic cell populations, including T cells, B cells, and myeloid cells, as well as in tissues where mRNA levels broadly correlate with the lymphoid cell content (gonadal tissues are exceptions). By measuring mRNA copy numbers, we find that A3G mRNA is ~10-fold more abundant than A3F mRNA, implying that A3G is the more significant anti-HIV-1 factor in vivo. A3G and A3F levels also vary between donors, and these differences are sustained over 12 months. Responses to T-cell activation or cytokines reveal that A3G and A3F mRNA levels are induced ~10-fold in macrophages and dendritic cells (DCs) by alpha interferon (IFN-α) and ~4-fold in naïve CD4+ T cells. However, immunoblotting revealed that A3G protein levels are induced by IFN-α in macrophages and DCs but not in T cells. In contrast, T-cell activation and IFN-γ had a minimal impact on A3G or A3F expression. Finally, we noted that A3A mRNA expression and protein expression are exquisitely sensitive to IFN-α induction in CD4+ T cells, macrophages, and DCs but not to T-cell activation or other cytokines. Given that A3A does not affect HIV-1 infection, these observations imply that this protein may participate in early antiviral innate immune responses.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins are polynucleotide cytidine deaminases that can edit and/or inhibit the replication of a range of retroviruses, retrotransposons, and DNA viruses (15, 30, 42). Most notable among susceptible substrates is human immunodeficiency virus type 1 (HIV-1), whose infectivity is profoundly inhibited by APOBEC3G (A3G) and APOBEC3F (A3F) in the absence of the viral Vif protein (6, 43, 69, 82, 87). In addition, APOBEC3B (A3B) and APOBEC3DE (A3DE) can suppress HIV-1 infectivity to comparatively modest extents, although A3B appears not to be regulated by Vif (6, 20, 23, 64). Most recently, one particular allelic variant of APOBEC3H, haplotype II, has also been shown to inhibit HIV-1, although uncertainty exists concerning this protein's sensitivity to Vif (19, 58).

APOBEC3 proteins, and especially A3G, are effective mutators of HIV-1 sequences (6, 15, 30, 46, 47, 49, 86). Mutation of HIV-1 sequences occurs through the encapsidation of APOBEC3 proteins into assembling virus particles and the ensuing deamination of cytidine residues to uridines in nascent (mostly) minus-strand reverse transcripts. When fixed in viral DNA, these mutations register as guanosine-to-adenosine (G-to-A) changes in the viral plus strand and are often termed hypermutations when occurring at excessive levels. In addition to these editing effects, a growing amount of literature indicates that APOBEC3 proteins also diminish the accumulation of HIV-1 reverse transcripts in infected cells, most likely by interfering with the process of reverse transcription (3, 5, 8, 26, 29, 31, 45, 47, 49, 51). The relative contributions of these functions to viral suppression under differing infection conditions remain to be determined (40). HIV-1 Vif overcomes the activity of APOBEC3 proteins by recruiting them to a cullin 5-elongin B/C-Rbx ubiquitin ligase complex and inducing polyubiquitylation, proteasomal degradation, and exclusion from viral particles (18, 44, 50, 52, 70, 73, 85).

There is persuasive evidence indicating that the APOBEC3 proteins, in particular A3G and A3F, encounter and influence HIV-1 during the course of natural infection in humans: first, all primate immunodeficiency viruses carry vif genes, which counteract their relevant A3G and A3F proteins (9, 25, 48, 68, 83); second, Vif expression is essential for pathogenic simian immunodeficiency virus infection (21) and is implicated as an important factor governing cross-species transmission of primate immunodeficiency viruses (25, 27, 71); and third, G-to-A hypermutated sequences are found frequently in HIV-1-infected individuals, with the targeted sequences matching the consensus substrate sequences for A3G, predominantly, and A3F (24, 32, 37, 38, 41, 59, 81). In addition to the inhibitory effects of the APOBEC3 proteins, it is also possible that low levels of activity could be beneficial to HIV-1 by providing an additional mechanism for acquiring sequence variation through sublethal levels of editing. Such mutations could facilitate immune evasion, drive phenotypic changes, or accelerate the development of drug resistance (34, 36, 54).

Accordingly, it has been anticipated that fluctuation in the balance between A3G and/or A3F and Vif expression or function could influence the course of natural HIV-1 infection or the rate of viral transmission. Such variation could be provided through differences in A3G and/or A3F or Vif protein sequences, expression levels, or regulation of protein function. Indeed, a single nucleotide polymorphism in A3G that encodes the H186R change has been associated with an accelerated progression of HIV-1 disease in African Americans (1), a noncoding polymorphism in intron 4 of A3G has been associated with an increased risk of infection in Caucasians (79), and a polymorphism in CUL5 (which encodes cullin 5) has been linked to more rapid disease progression (2). Conversely, another study was unable to find an association between the H186R or other A3G polymorphisms and disease progression (22). Furthermore, HIV-1-exposed seronegative individuals were previously reported to show higher A3G mRNA expression levels than HIV-1-infected or uninfected controls, suggesting that A3G can modulate susceptibility to HIV-1 infection (4). More recently, it was reported that HIV-1-seropositive individuals with low viral set points have higher A3G and A3F mRNA expression levels than HIV-1-seropositive individuals with high viral set points (35, 78), yet others have been unable to discern a relationship between A3G and A3F mRNA expression levels and viral load (17). Finally, Vif sequence variability has been documented in vivo and was shown in cultured cells to significantly impact its neutralizing activity against A3G and/or A3F (72).

In sum, although several studies indicated that variation in A3G and/or A3F expression levels can influence disease progression after infection by HIV-1, data from previously published reports in this area are not in good general agreement with each other. To investigate such issues with precision, it is important to define the cell types and tissues in which APOBEC3 proteins are naturally expressed, to understand the factors that regulate expression (transcriptionally and posttranscriptionally), to assign levels of expression of the different APOBEC3 proteins in different cell types and tissues, and to appreciate variations in expression levels both between individuals and within individuals at different points in time.

A number of analyses that address the expression and posttranslational regulation of APOBEC3 genes have already been reported. There is general agreement that several APOBEC3 genes are expressed in lymphocyte populations and that A3G and A3F are expressed in monocytes, dendritic cells (DCs), and hepatocytes (33, 60, 62, 74, 84). Furthermore, A3A is expressed in monocytes (60), and A3B and A3C are expressed in hepatocytes (12). As factors involved in resistance to infection, there is much interest in examining APOBEC3 protein expression in the context of the innate and inflammatory response to infection and, in particular, in response to type I interferon (IFN). Indeed, alpha IFN (IFN-α) was previously reported to induce A3G and A3A in monocytes (60, 61, 67, 74) and A3G, A3F, and A3B in hepatocytes (12, 67, 75), whereas the effect of IFN-α on A3G expression in CD4+ T cells is controversial, as induction has (14) or has not (65, 67, 74, 84) been observed. In fact, in vitro studies with panels of cytokines have revealed relatively little in terms of A3G regulation in T cells, with the exception of a modest induction with interleukin-2 (IL-2) or IL-15 over 5 days of treatment (74). It was also previously reported that the stimulation of the cell surface molecules CCR5 and CD40 can upregulate A3G expression in CD4+ T cells and DCs (62). More recently, a poly(I:C)-induced type I IFN response in DCs was reported to induce the expression of a lower-molecular-mass A3G protein without changes in A3G mRNA levels (77). The targeting of A3G to high-molecular-mass (HMM) ribonucleoprotein complexes, and, hence, the masking of catalytic and postentry antiviral activity, has also been associated with T-cell activation, which is indicative of further levels of functional regulation (16, 76).

In an effort to provide baseline information for future analyses addressing the effects of APOBEC3 expression phenotypes on HIV-1 pathogenesis, transmission, and sequence evolution, we have addressed the tissue and cell type expression profiles of human APOBEC3 genes, the variation in APOBEC3 mRNA expression levels between and within individuals, and alterations in expression in primary CD4+ T cells, macrophages, and DCs in response to extracellular stimuli, particularly IFN-α.

MATERIALS AND METHODS

Isolation and culture of primary cells.

Fresh peripheral blood was obtained by venipuncture, and peripheral blood mononuclear cells (PBMCs) were isolated using Lymphoprep (Axis-Shield, Oslo, Norway). Different PBMC subsets were separated using a fluorescence-activated cell sorter (FACS) (BD FACS Aria) with the following two antibody combinations (BD Pharmingen): combination 1 consisted of anti-CD3-phycoerythrin (PE), anti-CD19-fluorescein isothiocyanate (FITC), and anti-CD14-allophycocyanin to separate T cells (CD3+), B cells (CD19+), and monocytes (CD14+), and combination 2 consisted of anti-CD3-peridinin chlorophyll protein, anti-CD4-Alexa, anti-CD8-PBlue, anti-CD45RA-FITC, and anti-CD62L-PE to separate naïve CD4+ T cells (CD3+ CD4+ CD45RA+ CD62L+), naïve CD8+ T cells (CD3+ CD8+ CD45RA+ CD62L+), memory CD4+ T cells (CD3+ CD4+ CD45RA CD62L), and memory CD8+ T cells (CD3+ CD8+ CD45RA CD62L).

To assess A3G and/or A3F gene expression over time within donors, CD4+ T cells were isolated from three sequential 6-monthly cryopreserved PBMC samples from healthy participants from the Chicago component of the Multicenter AIDS Cohort Study using a human CD4 subset minicolumn kit (R&D Systems) according to the manufacturer's protocol.

To assess the inducibility of APOBEC3 gene expression, naïve CD4+ T cells were isolated from PBMCs for cell culture by negative selection using the naïve CD4+ T-cell isolation kit (Miltenyi Biotec, Germany). Cells were subsequently cultured in RPMI 1640 medium (Gibco) supplemented with penicillin and streptomycin (Gibco) and 10% autologous serum in the presence or absence of 1,000 U/ml IFN-α (human leukocyte IFN; Sigma), 1,000 U/ml IFN-γ (R&D Systems), 20 U/ml recombinant IL-2 (R&D Systems), or 1 μg/ml plate-coated anti-CD3 (BD Pharmingen) with 5 μg/ml soluble anti-CD28 (BD Pharmingen). Cell aliquots were harvested up to 120 h after the start of culture and used for RNA isolation and/or immunoblotting.

To compare inductions of APOBEC3 mRNAs in naïve (CD45RO) or memory (CD45RO+) CD4+ T cells from the same donor, CD4+ T cells were isolated from PBMCs by negative selection using a CD4+ T-cell isolation kit (Miltenyi Biotec). CD4+ memory T cells were then positively selected using CD45RO MicroBeads (Miltenyi Biotec), and naïve CD4+ T cells were negatively selected by subsequently depleting any remaining CD45RO+ cells using triple the amount of CD45RO MicroBeads.

Monocytes were isolated from PBMCs using CD14 MicroBeads (Miltenyi Biotec). To obtain monocyte-derived macrophages (MDMs), CD14+ cells were allowed to adhere to plastic in RPMI 1640 medium supplemented with penicillin and streptomycin for 3 h and then cultured in the same medium containing 10% autologous serum and 100 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF; Miltenyi Biotec). After 5 days, adherent cells were cultured in the presence or absence of 1,000 U/ml IFN-α (Universal type I interferon; PBL InterferonSource).

To obtain immature DCs, CD14+ cells were cultured in RPMI 1640 medium supplemented with penicillin and streptomycin, 10% autologous serum, 10 ng/ml GM-CSF, and 100 ng/ml IL-4 (R&D Systems). Culture media and cytokines were refreshed every other day. After 5 days, immature DCs were cultured in the presence or absence of 1,000 U/ml IFN-α (PBL InterferonSource). Additionally, DC cultures were (or were not) stimulated with 100 ng/ml lipopolysaccharide (Sigma-Aldrich) for 24 h and stained with anti-HLA-DR-peridinin chlorophyll protein, anti-CD1a-allophycocyanin, anti-CD40-PE, and anti-CD83-FITC (BD Pharmingen) and analyzed by FACS (BD FACS Aria), confirming the immature phenotype of the unstimulated DCs (data not shown). From both MDM and DC cultures, cell aliquots were harvested up to 30 h after the addition of IFN-α and used for RNA isolation and/or immunoblotting.

RNA, cDNA, and quantitative real-time PCR.

RNA was isolated from harvested cells using the RNeasy kit including on-column DNase treatment (Qiagen). cDNA was made using the High Capacity cDNA archive kit (Applied Biosystems) and used for real-time PCR using TaqMan gene expression assays (Applied Biosystems) specific for human A3A (Hs00377444_m1), A3B (Hs00358981_m1), A3C (Hs00828074_m1), A3DE (Hs00537163_m1), A3F (Hs00736570_m1), A3G (Hs00222415_m1), A3H (Hs00419665_m1), CD3E (Hs00167894_m1), CD79a (Hs00233566_m1), colony-stimulating factor receptor (CSFR) (Hs00234617_m1), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Hs99999905_m1). Triplicate reactions were run according to the manufacturer's protocol using an ABI 7900HT sequence detection platform (Applied Biosystems), and SDS2.2 software (Applied Biosystems) was used for analysis. The gene specificity of each APOBEC3 TaqMan gene expression assay was confirmed by testing it against plasmid DNA containing a cDNA copy of A3A, A3B, A3C, A3D, A3F, A3G, or A3H (data not shown). Tissue-specific panels of pooled human cDNA were acquired from Clontech (human multiple tissue cDNA (MTC) panel I, human MTC panel II, and human immune system MTC panel). For relative quantification, samples were normalized for GAPDH mRNA content.

Absolute A3B, A3F, and A3G mRNA contents of CEM-SS and Jurkat cells were determined using standard dilutions of the respective APOBEC3 RNAs produced in vitro. Briefly, A3B, A3F, or A3G RNAs were produced using a MEGAscript T7 high-yield transcription kit (Ambion) and pcDNA3.1 vectors containing cloned cDNAs of the respective genes (7). Tenfold serial dilutions of APOBEC3 RNA were made in a background of RNA from a mouse cell line (NIH 3T3), keeping the total RNA concentration in all dilutions equal. CEM-SS and Jurkat cells were counted using a hemocytometer; 106 cells of each cell line were then used for RNA isolation, and cDNA was made using the High Capacity cDNA archive kit (Applied Biosystems). The absolute APOBEC3 mRNA contents of CEM-SS and Jurkat RNA were determined using an ABI 7900HT platform and SDS2.2 software (Applied Biosystems) in the absolute-quantification mode. When assuming equivalent GAPDH mRNA expression levels in different cell types, these absolute values can be used as the basis for estimating mRNA copy numbers for A3B, A3F, and A3G in primary samples.

Velocity sedimentation.

Velocity sedimentation was used to analyze A3G-containing ribonucleoprotein complexes (76). Specifically, CD4+ T cells were lysed in NP-40 buffer (0.62% NP-40, 10 mM Tris-acetate [pH 7.4], 50 mM potassium acetate, 100 mM NaCl, and 10 mM EDTA containing Roche EDTA-free protease inhibitor cocktail) by passage 20 times through a 20-gauge needle and cleared by centrifugation at 150 × g for 10 min, followed by centrifugation at 18,000 × g for 30 s. Subsequently, the lysate was treated with (or without) 250 μg/ml RNase A (Sigma) for 30 min at room temperature. One hundred microliters of cell lysate was layered onto a step gradient containing 200 μl each of 10%, 15%, 20%, 30%, and 50% sucrose in NP-40 buffer and centrifuged at 165,000 × g for 60 min at 4°C (TLA 100.2 rotor, Optima TLX ultracentrifuge; Beckman Coulter). Fractions of 80 μl were collected from the top of the gradient, precipitated with trichloroacetic acid, and analyzed by immunoblot analysis.

Immunoblot analysis.

Harvested cell aliquots were lysed in sample buffer containing 3% sodium dodecyl sulfate, 62.5 mM Tris-HCl, and 0.1 M dithiothreitol; resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and analyzed by immunoblotting using primary antibodies specific for HSP90α/β (H-114; Santa Cruz Biochemicals) and A3G and/or A3A (57), followed by secondary horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin antibodies (Pierce) and chemiluminescence (Pierce). The specificity of the A3G/A3A antibody was shown using immunoblot analysis of CEM-SS cells stably transduced to express A3A or A3G (data not shown).

RESULTS

APOBEC3 mRNA expression in PBMC subsets.

As discussed above, we sought to define the expression patterns of APOBEC3 genes in human tissues and cells and to address variations in natural expression levels between and within healthy blood donors. We first analyzed the relative mRNA expression levels of all APOBEC3 genes (A3A to A3H) in freshly isolated peripheral T cells, B cells, and monocytes using quantitative real-time PCR. For each gene, we determined the mRNA level in the respective FACS-sorted cell subsets relative to the level in whole PBMCs from the same individual. Figure Figure1A1A shows the median values for five healthy individuals. In general, the data show similar patterns for A3C, A3DE, A3F, and A3G, with expression being higher in B cells and T cells than in monocytes. Variations in this pattern were seen for A3A, which is expressed predominantly in monocytes; for A3B, which is highly expressed in B cells; and for A3H, where expression in T cells is higher than in B cells.

FIG. 1.
Expression of APOBEC3 mRNA in PBMC subsets. (A) Relative APOBEC3 mRNA expression levels in PBMC subsets. Median values for five healthy donors are depicted relative to mRNA levels in whole PBMCs from the same donor. (B) Relative mRNA expression in PBMCs ...

Analyses of this genre do not permit statements about absolute mRNA levels of individual APOBEC3 genes. In an endeavor to examine absolute expression for the APOBEC3 genes with the clearest HIV-1-inhibitory phenotypes, we first determined absolute mRNA copy numbers on a per-cell basis for A3G, A3F, and A3B in CEM-SS and Jurkat cells (Fig. 1B and C). These T-cell lines were then used as standards for analyses of primary cell samples. Results are normalized by GAPDH mRNA levels and displayed relative to T-cell line controls. Assuming that GAPDH mRNA levels are equivalent in different cell types, an approximation of actual expression is obtained by multiplying the absolute mRNA copy number for a given gene in the T-cell line by the relative mRNA expression in a given sample. This approach enables us to make statements about the relative expression of one APOBEC3 gene to another.

Figure Figure1B1B shows the expression of A3G, A3F, and A3B mRNAs in sorted T cells, B cells, and monocytes. In all cases, there was ~10-fold more A3G mRNA than A3F mRNA and comparatively little A3B mRNA. In addition, the expression of these three genes was clearly lower in monocytes than in lymphocytes. This highlights the value of examining actual expression levels; for instance, Fig. Fig.1B1B demonstrates that A3B mRNA is poorly expressed in PBMC subsets, including B cells, yet Fig. Fig.1A1A gives the misleading impression that B cells express substantial levels of A3B.

A3G and A3F mRNA expression patterns were next examined more closely in specific T-cell subsets, namely, sorted naïve (CD45RA+ CD62L+) and memory (CD45RA CD62L) CD4+ and CD8+ T cells (Fig. (Fig.1C).1C). No major differences in A3G or A3F mRNA expression levels were found between these cell populations. These results also show that A3G and A3F mRNA levels in lymphocytes may vary by about a log between healthy individuals.

In addition to variations in gene expression levels between individuals, it is also important to assess variation within individuals over time. To address this, we tested the A3G and/or A3F mRNA expression levels in CD4+ T cells of five healthy donors over three consecutive 6-monthly visits (Fig. (Fig.1D).1D). These data show relatively constant expression levels of A3G and A3F mRNA over a time span of 1 year for each of these donors and demonstrate that consistent variation in expression levels exists between individuals.

Tissue-specific expression of APOBEC3 mRNA.

Tissue gene expression patterns can give insight into protein function. To determine the tissue-specific expression patterns of the human APOBEC3 genes, we used quantitative PCR and commercially available panels of tissue-specific cDNA samples pooled from several donors. We determined the relative A3A, A3B, A3C, A3DE, A3F, and A3G mRNA contents in these panels in comparison to the vendor-supplied reference sample that was set to unity for all genes tested (Fig. (Fig.2A).2A). Because we had already found that T cells, B cells, and monocytes express APOBEC3 genes, we also determined the relative mRNA contents of the T-cell marker CD3, the B-cell marker CD79a, and the monocyte marker macrophage colony-stimulating factor receptor (M-CSFR) in each tissue-specific sample.

FIG. 2.
Relative quantities of A3A to A3G, CD3, CD79a, and M-CFSR mRNA in tissue samples. (A) Relative mRNA expression levels of A3A to A3G, CD3, CD79a, and the M-CFSR gene in a panel of tissue cDNA samples (Clontech). Results are plotted relative to an arbitrary ...

Inspection of the data immediately revealed a trend in that the relative expression profiles of the lymphocyte markers CD3 and CD79a resembled those of many of the APOBEC3 genes (Fig. (Fig.2A).2A). More specifically, a higher CD3 expression level frequently coincides with higher A3G and/or A3F expression levels, e.g., in spleen, lymph node, or lung. Accordingly, because A3G and A3F are expressed in lymphocytes, we interpret these findings as indicating that the lymphocyte content of a particular tissue is an important determinant of the A3G and/or A3F expression pattern. The clear exceptions to this trend are gonadal tissues (both testis and ovary), where comparatively high A3G and A3F contents were not matched by a high CD3 content. To address this more closely, we plotted A3G and A3F mRNA levels of each tissue sample tested (y axis) against the CD3 mRNA levels (x axis) (Fig. (Fig.2B).2B). The points corresponding to the ovary and testis samples are indicated by open symbols and are outliers in these plots. We interpret this as indicating that certain (nonlymphoid) cell types in both testis and ovary express high levels of A3G and A3F.

Induction of APOBEC3 expression in primary naïve CD4+ T cells.

As intrinsic viral resistance factors, APOBEC3 proteins have the capacity to inhibit HIV-1 replication at the onset of host infection or exposure. This may be during the first cycle of replication, potentially prior to proviral formation (16), or during subsequent viral production and spread. Therefore, it is attractive to reason that the expression of these proteins might be under the regulation of cytokines that play a role in early responses to virus infection, such as IFN-α or IFN-γ. Indeed, as discussed in the introduction, other studies previously investigated a possible effect of IFN-α on A3G expression. As CD4+ T cells are the major target cell for HIV-1 replication during natural infection, we first assessed the induction of APOBEC3 gene expression in naïve CD4+ T cells.

Naïve CD4+ T cells were isolated from fresh PBMCs by negative selection and cultured in the presence or absence (mock) of IFN-α, IFN-γ, IL-2, or CD3/CD28 costimulation. Samples were taken at the start of culture (time point 0); after 3, 6, 18, or 30 h of culture, total RNA was then isolated and reverse transcribed, and relative mRNA induction was determined by quantitative PCR using GAPDH mRNA for normalization (Fig. (Fig.3).3). Importantly, the levels of GAPDH mRNA showed no significant variation over the duration of these experiments and remained constant relative to β-actin mRNA levels (data not shown). In initial experiments, we performed dose-response curves with the different culture supplements (IFN-α or IFN-γ used at 100, 500, 1,000, or 2,000 U/ml; IL-2 used at 5, 12.5, 25, or 50 U/ml; and anti-CD3 used at 0.1, 0.5, 1, or 2 μg/ml, with anti-CD28 held constant at 5 μg/ml) to determine the concentrations of cytokines and/or antibodies needed to stimulate the expression of known regulated genes. In particular, we monitored ISG15 (an IFN-stimulated gene), CD25 (the IL-2 receptor α-chain which is induced by IL-2), or CD69 (a T-cell activation marker which is induced by CD3/CD28 costimulation) mRNA expression levels and found that 1,000 U/ml IFN-α, 1,000 U/ml IFN-γ, 20 U/ml IL-2, or 1 μg/ml anti-CD3 yielded consistently good responses (data not shown).

FIG. 3.
Induction of APOBEC3 mRNA over time in primary naïve CD4+ T cells cultured in the presence of IFN-α (pink line), IFN-γ (yellow line), IL-2 (green line), or CD3/CD28 costimulation (purple line) or without an inducing agent ...

Figure Figure33 shows the mean relative inductions of A3A, A3B, A3C, A3DE, A3F, A3G, and A3H mRNAs over time in naïve CD4+ T cells of the five donors tested. The dark blue line represents the relative expression over time in the absence of stimulus (mock) and serves as a baseline against which levels of induction were determined. We found no significant induction of the various APOBEC3 genes after the addition of IFN-γ, IL-2, or anti-CD3/CD28. However, IFN-α potently induced A3A mRNA expression (>30-fold after 6 h of culture) and modestly induced A3G, A3F, and A3H mRNA levels (Fig. (Fig.3;3; see Table S1 in the supplemental material).

While it is both useful and commonplace to monitor mRNA levels as a measure of gene expression, protein expression levels are more relevant in most cases. Accordingly, we next used whole-cell lysates of parallel samples to assess A3G and A3A protein expression levels by immunoblotting. As would be expected from the mRNA analyses, the levels of the A3A protein were efficiently induced by IFN-α treatment, although detection was somewhat delayed relative to mRNA induction, with robust expression not seen until 18 h after the addition of IFN-α (Fig. (Fig.4).4). Analysis of A3G protein expression following IFN-α stimulation gave a very different result in that no significant or reproducible induction could be detected (including multiple independent experiments not shown here). We do not yet understand the basis for the discordance between A3G mRNA and protein expression levels, although one possibility may be that A3G mRNA is translationally repressed in response to IFN-α. In contrast, and consistent with measurements of mRNA levels, no clear induction of A3G protein levels could be seen following IFN-γ, IL-2, or anti-CD3/CD28 treatment. A minor increase in A3G levels following anti-CD3/CD28-mediated T-cell activation was noted (30- and 72-h time points), although the levels of the HSP90 loading control were also elevated in these samples.

FIG. 4.
Induction of the A3A and A3G proteins over time in primary naïve CD4+ T cells cultured in the presence of IFN-α, IFN-γ, IL-2, or CD3/CD28 costimulation. Naïve CD4+ T cells were harvested at different time ...

To test if memory CD4+ T cells would show similar results, we isolated both naïve (CD45RO) and memory (CD45RO+) CD4+ T cells from one donor. No differences were found between IFN-α induction of mRNAs of A3A to A3H in naïve and memory CD4+ T cells of this donor (data not shown).

Thus, although we were able to detect a modest induction of A3G mRNA levels in response to IFN-α in resting CD4+ T cells, this response was not mirrored by increased A3G protein levels.

Detection of A3G in HMM and LMM complexes.

A3G can be found in HMM ribonucleoprotein complexes that mask its catalytic activity or in a catalytically active form in low-molecular-mass (LMM) complexes (16, 76), implying that A3G activity can be regulated through associations with cellular factors. To assess whether different stimuli can influence the segregation of A3G between such complexes, we used velocity sedimentation to separate LMM and HMM A3G complexes from lysates of CD4+ T cells after incubation with IFN-α, IFN-γ, or IL-2 or following anti-CD3/CD28 costimulation. Gradient fractions were precipitated and analyzed by immunoblotting (Fig. (Fig.55).

FIG. 5.
Effects on A3G protein complex mass in primary naïve CD4+ T cells cultured in the presence of IFN-α (1,000 U/ml), IFN-γ (1,000 U/ml), IL-2 (20 U/ml), or CD3 and CD28 costimulation (1 μg/ml and 5 μg/ml, respectively). ...

As previously shown (16, 74), A3G accumulates in LMM complexes in resting CD4+ T cells (i.e., A3G is found toward the top of the gradient [Fig. [Fig.5,5, top]). After RNase treatment and, hence, the disruption of ribonucleoprotein complexes, a slight shift toward even-lower-molecular-mass complexes was observed (Fig. (Fig.5,5, second panel), indicating that some A3G is associated with cellular RNA and RNA binding proteins in resting CD4+ T cells. Treatment with 1,000 U/ml IFN-α or IFN-γ for 30 h did not cause a change in the sedimentation of A3G-containing complexes, while anti-CD3/CD28 costimulation clearly induced the formation of HMM A3G complexes, as shown by the more rapid sedimentation of A3G (Fig. (Fig.5,5, bottom). Others previously reported the recruitment of A3G into HMM complexes in CD4+ T cells after 5 to 7 days of culture in the presence of 50 ng/ml IL-2 (74). However, we found that treatment with 20 U/ml IL-2 for 3 days induced HMM A3G complexes to only a very minor degree (Fig. (Fig.5,5, lane 11, fifth panel).

In conclusion, the treatment of primary CD4+ T cells with IFN-α, IFN-γ, or IL-2 had no significant effect on the distribution of A3G between ribonucleoprotein complexes with differing sedimentation characteristics. Thus, we have not obtained persuasive evidence for A3G protein expression being regulated by IFN or IL-2 in primary CD4+ T cells in the context of a potentially rapid response to infection.

Induction of APOBEC3 expression in primary macrophages and DCs.

While CD4+ T cells are the major target for HIV-1 replication, other cell types including macrophages and DCs can also be infected. These cell types are crucial for generating antiviral immune responses but are also thought to play an important role in establishing systemic HIV-1 infection (reviewed in reference 28). We therefore sought to assess the regulation of APOBEC3 genes in MDMs and monocyte-derived immature DCs. CD14+ monocytes were isolated from fresh PBMCs by positive selection and either allowed to adhere to plastic and cultured in the presence of GM-CSF to generate MDMs or cultured in the presence of IL-4 and GM-CSF to obtain DCs. Differentiated macrophages were identified by morphology, and the phenotype of differentiated DCs was confirmed both by morphology and by FACS staining before and after lipopolysaccharide stimulation (see Materials and Methods) (data not shown). Both MDMs and DCs were cultured in the presence or absence (mock) of IFN-α, and samples were collected for RNA and protein analyses as described above.

In both MDMs and immature DCs, we found potent inductions of A3A, A3F, and A3G mRNAs (Fig. 6A and B; see Tables S2 and S3 in the supplemental material). The induction of A3A and A3G mRNAs in response to IFN-α was mirrored by increased A3A and A3G protein levels in MDMs and DCs after IFN-α exposure (Fig. (Fig.6C).6C). Although this effect was previously shown for MDMs (61, 74, 84), this is the first demonstration of IFN-α-induced expression of A3G and A3A in DCs. A3DE and A3H mRNA levels were also substantially induced in MDMs, albeit with slower kinetics (Fig. (Fig.6A),6A), but only modestly induced in DCs in response to IFN-α. The potent induction of A3G and A3F and other APOBEC3 genes in these cell types by IFN-α implies that APOBEC3 protein activity is integrated with early antiviral innate immune and inflammatory responses.

FIG. 6.
APOBEC3 mRNA expression in macrophages and DCs. Shown are data for the induction of APOBEC3 mRNA over time in MDMs (A) and DCs (B) cultured in the presence (pink line) or absence (mock) (blue line) of IFN-α. The induction of APOBEC3 mRNA expression ...

DISCUSSION

In this report, we present detailed analyses of “basal” levels of APOBEC3 gene expression in human hematopoietic cell populations (Fig. (Fig.1)1) and a panel of human tissue samples (Fig. (Fig.2).2). We extend these studies to include an evaluation of both mRNA and, in the cases of A3G and A3A, protein expression in response to innate immune and inflammatory mediators, particularly IFN-α (Fig. (Fig.33 to to6).6). Knowledge of cell type-specific expression and its variation is an important foundation for investigating the relationships between APOBEC3 expression and HIV-1 pathogenesis in humans.

Analysis of T cells, B cells, and monocytes purified from peripheral blood revealed a general trend in that most APOBEC3 family members are expressed at higher levels in both T cells and B cells than in monocytes (Fig. (Fig.1A).1A). The obvious outliers to this trend are A3A and A3B, which are expressed predominantly in monocytes and B cells, respectively. In vitro experiments have revealed many viral and transposon substrates for APOBEC3 protein-mediated inhibition. Which of these will ultimately be assigned as physiological targets of the respective APOBEC3 proteins remains a matter of conjecture, although the susceptibility of HIV-1 to A3G and A3F is well accepted (see below). Accordingly, any biological significance of the skewed expression patterns for A3A and A3B awaits a clearer understanding of the in vivo functions of these proteins (discussed further below).

Focusing further on A3G, A3F, and A3B, three family members with HIV-1-inhibitory function (6, 43, 69, 82, 87), we used measurements of absolute mRNA expression levels of each gene in cell line standards to address levels of expression relative to each other (Fig. 1B and C). This approach reveals that A3G mRNA is ~10-fold more abundant than A3F mRNA in T cells, B cells, and monocytes and ~100- to ~1,000-fold more abundant than A3B mRNA. These data support the view that A3G is the principal APOBEC3 antagonist of HIV-1 replication in T cells and that A3F, but not A3B, also contributes to antiviral action. The fact that HIV-1 Vif inhibits both A3G and A3F, but not A3B (6, 20, 23, 64), and the noted bias for G-to-A hypermutations occurring at A3G consensus target sites during natural HIV-1 infection of humans (24, 32, 37, 38, 41, 59, 81) are each in accordance with this observation.

In addition to cross-sectional analyses of expression in multiple individuals, we also examined the relative expression levels of A3G and A3F in five individuals over the course of a year (Fig. (Fig.1D).1D). Variations in expression levels between individuals were seen, and these appeared to be maintained over time. Thus, some individuals consistently express higher levels of A3G and/or A3F. As outlined above, some previous studies focused on whether this can impact the course of natural HIV-1 infection, viral sequence evolution, and/or viral transmission, but more future work is needed to clarify these issues.

We next examined the expression patterns of APOBEC3 family members using a panel of human tissue samples (Fig. (Fig.2).2). We highlight the results for A3G and A3F because their expression in T cells and B cells is established. Notably, the expression of both A3G and A3F generally correlates well with the mRNA levels of the lymphocyte markers CD3 and CD79a (Fig. (Fig.2),2), leading us to propose that A3G and A3F are preferentially expressed in T cells and B cells and that the residence of these cell types in different tissues contributes substantially to tissue expression profiles (Fig. (Fig.2B).2B). In contrast, A3B, which is expressed at comparatively low levels in lymphocytes (Fig. (Fig.1B)1B) yet at similar levels in many tissues (Fig. (Fig.2A),2A), is presumably poorly expressed in many cell types. To enable future analyses of the tissue- and/or cell type-specific expression of individual APOBEC3 family members, it will be necessary either to isolate “pure” cell populations from tissues or to develop gene-specific probes for in situ hybridization and specific antibodies for immunocytochemistry.

The two tissues displaying clear variance between A3G and/or A3F expression and T- and B-cell marker content are testis and ovary, where the level of gene expression is comparatively high (Fig. (Fig.2B)2B) (see also reference 33). One potential biological explanation for this might be that APOBEC3 proteins contribute to the preservation of the germ line by providing protection against the detrimental effects of mobile element transposition, and this activity would be favored by higher levels of expression.

Since virus restriction factors contribute to the initial defense against viral infection, we characterized APOBEC3 gene expression responses in natural target cells of HIV-1 to a number of innate immune and inflammatory mediators, particularly type I IFN (Fig. (Fig.3,3, ,4,4, and and6).6). Indeed, the expression of other restriction factors such as TRIM5α and tetherin/CD317/BST2 has been shown to be IFN-α inducible (56, 63, 66). We found that A3G, examined here at both the mRNA and protein levels, is constitutively expressed in naïve and memory CD4+ T cells (Fig. (Fig.11 and and4),4), MDMs, and DCs (Fig. (Fig.6).6). Following treatment with IFN-α, levels of A3G mRNA increased modestly in T cells but more substantially in MDMs and DCs, and A3F displayed similar phenotypes. In contrast, the A3G protein was not induced in T cells, but clear increases in expression were seen in MDMs and DCs (Fig. (Fig.44 and and6C).6C). Thus, A3G is expressed constitutively and broadly in hematopoietic cells and is induced by IFN-α in a more limited subset of cell types, findings that are in keeping with those described previously by a number of groups (12, 61, 67, 74, 75, 84). Determining the precise significance of IFN-α induction of A3G and/or A3F as well as the causative variations in signaling that evoke the differences between cell types is a subject for future investigation. It is also possible that the A3G expression level in T cells is relatively efficient such that there is no potential for further induction. Future analyses of the regulatory elements that govern A3G expression will help answer such questions.

While A3DE and A3H show some responsiveness to IFN-α in terms of mRNA levels, the most striking effects were seen for A3A. In all cell types tested, A3A mRNA and protein levels are potently induced by the addition of IFN-α (Fig. (Fig.3,3, ,4,4, and and6)6) (61). In fact, we find that A3A is one of the most IFN-α-responsive human genes that we have encountered. These observations implicate A3A as an important effector during innate and inflammatory responses. It will be interesting to define the relevant IFN-α-inducible functions and targets of A3A. These likely do not include the inhibition of HIV-1 (6; F. A. Koning, unpublished results) but could relate to its effects on endogenous retroelements (10, 11, 13, 39, 53), adeno-associated virus (13, 55), or human papillomavirus (80). We also note that a recent study reported the IFN-α-mediated induction of ~20-kDa A3G-related peptides in DCs in the absence of mRNA induction (77). Since anti-A3G sera raised against the C terminus of A3G readily detected the A3A protein (Fig. (Fig.44 and and6C),6C), we suggest that the peptides described previously by Trapp and colleagues represent A3A expressed from induced A3A mRNA rather than small A3G proteins.

In sum, this study discusses a detailed analysis of APOBEC3 mRNA and, where technically possible, protein expression in human hematopoietic cells as well as tissues. With respect to the significance for HIV-1, we report that A3G and A3F are widely expressed in cell types that are susceptible to infection, that levels of A3G expression are ~10-fold higher than those of A3F, that expression levels vary in a sustained fashion among individuals, and that the A3G protein can be significantly induced in response to IFN-α in myeloid cell types.

Supplementary Material

[Supplemental material]

Acknowledgments

This work was supported by the National Institutes of Health (grant AI070072), the UK Medical Research Council, the Guy's and St. Thomas' Charity, and the Department of Health via a National Institute for Health Research comprehensive Biomedical Research Centre award to Guy's and St. Thomas' NHS Foundation Trust in partnership with King's College London and King's College Hospital NHS Foundation Trust. F.A.K. is a fellow of the European Molecular Biology Organization, and M.H.M. is an Elizabeth Glaser Scientist.

Footnotes

[down-pointing small open triangle]Published ahead of print on 8 July 2009.

Supplemental material for this article may be found at http://jvi.asm.org/.

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