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IFN-α Treatment Inhibits Acute Friend
Retrovirus Replication Primarily through the
Antiviral Effector Molecule Apobec3
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of June 18, 2017.
Michael S. Harper, Bradley S. Barrett, Diana S. Smith, Sam
X. Li, Kathrin Gibbert, Ulf Dittmer, Kim J. Hasenkrug and
Mario L. Santiago
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J Immunol 2013; 190:1583-1590; Prepublished online 11
January 2013;
doi: 10.4049/jimmunol.1202920
http://www.jimmunol.org/content/190/4/1583
The Journal of Immunology
IFN-a Treatment Inhibits Acute Friend Retrovirus
Replication Primarily through the Antiviral Effector
Molecule Apobec3
Michael S. Harper,*,† Bradley S. Barrett,* Diana S. Smith,* Sam X. Li,*,‡
Kathrin Gibbert,x Ulf Dittmer,x Kim J. Hasenkrug,{ and Mario L. Santiago*,†,‡
T
he therapeutic potential of innate immune factors for
treating viral infections is highlighted by the use of IFN-a
in the clinic. IFN-a is routinely used for the treatment of
hepatitis B virus, hepatitis C virus, and Kaposi’s sarcoma (1). In
clinical trials, IFN-a administered to HIV-1–infected patients
significantly reduced plasma viral loads from 4- to 20-fold (2–5).
Administration of IFN-a in human T-lymphotropic virus type I
(HTLV-I)–infected patients also resulted in significant reductions
in proviral load and improved motor function (6, 7). These HIV-1
and HTLV-I clinical trials suggest the existence of IFN-a–induced
antiretroviral effector mechanisms that if understood in greater
*Department of Medicine, University of Colorado Denver, Aurora, CO 80045;
†
Integrated Department of Immunology, University of Colorado Denver, Aurora,
CO 80045; ‡Department of Microbiology, University of Colorado Denver, Aurora,
CO 80045; xInstitute for Virology, University of Duisburg-Essen, Essen 45122, Germany; and {Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, MT 59840
Received for publication October 19, 2012. Accepted for publication December 11,
2012.
This work was supported by National Institutes of Health (NIH) Grant R01 AI090795
(to M.L.S.), the University of Colorado Denver Early Career Scholar Program (to
M.L.S.), the Tim Gill Foundation (to M.S.H.), NIH/National Center for Research Resources Colorado Clinical and Translational Sciences Institute Grant TL1 TR000155
(to S.X.L.), and the Intramural Research Program at the National Institute of Allergy
and Infectious Diseases, NIH (to K.J.H.).
Address correspondence and reprint requests to Dr. Mario L. Santiago, Division of
Infectious Diseases, University of Colorado Denver, Mail Stop B-168, 12700 East
19th Avenue, Building RC2, Room 11420, Aurora, CO 80045. E-mail address: mario.
[email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: BM, bone marrow; DC, dendritic cell; dpi, day
postinfection; F-MuLV, Friend MLV helper virus; FV, Friend retrovirus; HTLV-I,
human T-lymphotropic virus type I; ISG, IFN-stimulated gene; KO, knockout;
LDV, lactate dehydrogenase–elevating virus; MLV, murine leukemia virus; poly(I:C),
polyinosinic:polycytidylic; WT, wild-type.
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202920
detail could result in more specific, and possibly more potent,
therapeutics.
IFN-a upregulates hundreds of IFN-stimulated genes (ISGs) that
could modulate diverse processes, including innate and adaptive
immunity (8, 9). IFN-a could indirectly inhibit virus replication
through its immunomodulatory properties involving MHC class I
upregulation and induction of NK and T cell responses (10). In
addition, IFN-a could upregulate ISGs that directly inhibit retroviral replication by physically interacting with virus components or
counteracting processes needed for replication (Supplemental Table I). These retroviral “restriction factors” have been the subject of
intense study in the last decade, especially because HIV-1 evolved
antagonists to counteract them (11). Restriction factors likely account for how IFN-a could inhibit acute retrovirus infection, before the peak of adaptive immune responses. However, efforts to
address this hypothesis in HIV-1 infection remain limited to correlative studies.
Based on transcriptional induction data, Tetherin/BST-2, APOBEC3G/F, ISG15, and OAS1 have each been implicated in the
clinical potency of IFN-a treatment against HIV-1 (4, 5). However,
it is currently unknown which antiretroviral effector molecules are
most relevant in reducing acute retrovirus replication in vivo.
Knocking down individual ISGs and evaluating retroviral control
after IFN-a treatment in vivo is not feasible in humans. Fortunately, the antiretroviral ISGs have direct evolutionary counterparts
in mice, likely reflecting the long history of genetic conflict between mammalian hosts and retroviruses (12) (Supplemental Table
I). In fact, even the completely unrelated proteins primate TRIM5a
and murine Fv1 appear to have undergone convergent evolution to
restrict a capsid-dependent postentry step in the retroviral life cycle
(13). Thus, assessing the therapeutic impact of IFN-a in mice
deficient in individual ISGs may provide basic insights on the
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Therapeutic administration of IFN-a in clinical trials significantly reduced HIV-1 plasma viral load and human T-lymphotropic
virus type I proviral load in infected patients. The mechanism may involve the concerted action of multiple antiretroviral effectors
collectively known as “restriction factors,” which could vary in relative importance according to the magnitude of transcriptional
induction. However, direct genetic approaches to identify the relevant IFN-a restriction factors will not be feasible in humans
in vivo. Meanwhile, mice encode an analogous set of restriction factor genes and could be used to obtain insights on how IFN-a
could inhibit retroviruses in vivo. As expected, IFN-a treatment of mice significantly upregulated the transcription of multiple
restriction factors including Tetherin/BST2, SAMHD1, Viperin, ISG15, OAS1, and IFITM3. However, a dominant antiretroviral
factor, Apobec3, was only minimally induced. To determine whether Apobec3 was necessary for direct IFN-a antiretroviral action
in vivo, wild-type and Apobec3-deficient mice were infected with Friend retrovirus, then treated with IFN-a. Treatment of infected
wild-type mice with IFN-a significantly reduced acute plasma viral load 28-fold, splenic proviral load 5-fold, bone marrow
proviral load 14-fold, and infected bone marrow cells 7-fold, but no inhibition was observed in Apobec3-deficient mice. These
findings reveal that IFN-a inhibits acute Friend retrovirus infection primarily through the antiviral effector Apobec3 in vivo,
demonstrate that transcriptional induction levels did not predict the mechanism of IFN-a–mediated control, and highlight the
potential of the human APOBEC3 proteins as therapeutic targets against pathogenic retrovirus infections. The Journal of
Immunology, 2013, 190: 1583–1590.
1584
commercially designed primers based on the Gene IDs in Supplemental
Table I and b-actin (Gene ID: 11461). Real-time PCR was performed in
a Bio-Rad CFX96 cycler using the following conditions: 95˚C for 10 min,
40 cycles of 95˚C for 15s, and 60˚C for 1 min. Melt curve analysis and
direct sequencing were performed to verify the purity and specificity of the
PCR amplicons, respectively. Threshold cycles were calculated automatically by CFX Manager Software (Bio-Rad), and fold induction was determined by the formula: Fold induction = 2^(2DDCt), where DDCt =
[Cttarget(IFN) 2 Ctactin(IFN)] 2 mean[Cttarget(PBS) 2 Ctactin(PBS)]. Fold
inductions were normalized against b-actin.
Plasma viral load quantification
FV copy numbers were measured by real-time PCR as previous described (31).
In brief, RNAwas extracted from 50 ml plasma using the RNAEasy kit (Qiagen),
then eluted in 100 ml RNAse-free water. RNA (10 ml) was added into a final
volume of 13 one-step TaqMan Reverse Transcriptase (RT)-PCR reaction
mixture (Applied Biosystems) that contained 10 pmol of the following primers:
FLV sense, 59-GGACAGAAACTACCGCCCTG-39; FLV antisense, 59-ACAACCTCAGACAACGAAGTAAGA-39; and FLV probe, FAM-TCGCCACCCAGCAGTTTCAGCAGC-TAMRA. Real-time PCR was performed in a BioRad CFX96 cycler using the following thermocycling conditions: 48˚C for 15
min, 95˚C for 10 min, 40 cycles of 95˚C for 10s, and 60˚C for 1 min. T7transcribed RNA standards were used to interpolate copy numbers.
Proviral load
FV copy numbers and cell equivalents were determined by qPCR. BM and
spleen cells from mice were obtained as described earlier, and DNA was
extracted using the DNAEasy Blood and Tissue kit (Qiagen). DNA (100 ng)
was added into a final volume of 13 TaqMan Universal Master Mix II
(Applied Biosystems) that contained 10 pmol of the FV primers and probe as
described earlier. FV DNA standards were used to interpolate copy numbers.
Cell equivalents were determined by genomic Apobec3 copy number, with 2
copies = 1 cell equivalent. DNA (100 ng) was added into a final volume of
13 TaqMan Universal Master Mix II that contained 10 pmol of the following
primers: mA3.sense, 59-ATCCTCTTCCTTGATAAGATTCGGTCCATG-39;
mA3.antisense, 59-GATCCCTGATTGCCACAGAGAACAC-39; mA3.probe,
FAM-ATCTACACCTCCCGCCTGTATTTCCACT-TAMRA. Both reactions were performed under the following thermocycling conditions: 95˚C
for 10 min, 40 cycles of 95˚C for 15 s, and 60˚C for 1 min. Plasmid
Apobec3 standards were used to interpolate copy numbers.
Infectious viremia
Materials and Methods
Mice
C57BL/6J (B6) mice were purchased from The Jackson Laboratory. Apobec3
knockout (KO) mice were derived from a gene-trap XN450 ES cell line
(BayGenomics) and backcrossed for nine generations into B6 (16). Although Fv2-resistant, older B6 mice are more susceptible to FV (28). B6
mice .6 mo old were therefore used in the FV infection studies. Mice were
handled according to Institutional Animal Care and Use Committee
guidelines at the University of Colorado Denver [Permit B-89709(10)1E].
FV infection and IFN-a treatment
The classical B-tropic FV stock containing Friend MLV helper virus (FMuLV), spleen focus-forming virus, and lactate dehydrogenase–elevating
virus (LDV) (29, 30) was prepared and titered in BALB/c mice. FV (104
spleen focus-forming units) were administered i.v. through the retro-orbital
sinus. At 3 d postinfection (dpi), 105 U Universal type I IFN (PBL IFNSource) or PBS was administered i.p. A lower IFN-a treatment dose (104
U) did not inhibit FV in B6 mice (data not shown). At 7 dpi, mice were
sacrificed and plasma, spleen, and bone marrow (BM) cells were obtained.
Infection studies were performed twice with a 2-mo gap, with four groups
(wild-type [WT] + PBS, WT + IFN-a, KO + PBS, and KO + IFN-a) of six
to eight mice each. Data from these two independent cohorts were combined. In other experiments, uninfected or 3 dpi B6 WT mice were treated
with PBS or 105 U IFN-a, and BM and spleen cells were analyzed after
6 h. CD11b+ cells were purified using magnetic beads (Miltenyi Biotec).
Infectious viremia was determined by incubating Mus dunni cells with plasma
from infected mice and determining the proviral copy numbers as described
earlier. Mus dunni cells were plated at a density of 4000–8000 cells/well in
a 48-well plate with 4 mg/ml polybrene (Sigma) and incubated for 24 h.
Plasma (5 ml) harvested from FV-infected mice was added to each well and
incubated for 48 h. DNA was extracted using the DNAEasy kit (Qiagen), and
quantitative PCR of FV DNA was performed on 100 ng input DNA.
Flow cytometry
BM cells were stained as previously described (32). In brief, 106 cells were
stained with mAb 720 for 30 min, washed 23 with PBS with 1% FBS,
then costained with Ter119-FITC (clone TER-119; BD Biosciences),
CD11c-PE-Cy7 (N418; eBioscience), and anti-mouse IgG1-allophycocyanin (Columbia Biosciences). Isotype controls and cells from uninfected
mice were used for gating. Cells were processed in an LSR-II cytometer
(BD Biosciences), collecting up to 250,000 events per sample. Datasets
were analyzed using FlowJo (TreeStar).
Statistical analysis
Median values were used to calculate fold-inhibition values if the data were
significantly skewed from a normal distribution using the Kolmogorov–
Smirnov test (p , 0.05). Otherwise, mean values were used. Data were
analyzed using a nonparametric two-tailed Mann–Whitney U test. Statistical analyses were performed using Prism 5 (GraphPad).
Quantitative PCR array for antiretroviral ISGs
Results
Relative expression of ISGs was measured by quantitative real-time PCR
using a custom RT2 Profiler Array (SABiosciences). RNA was extracted
from tissues using the RNAEasy kit (Qiagen), and cDNA was synthesized
using the RT2 First Strand kit (SABiosciences). cDNA was added to 13
RT2 SYBR qPCR Mastermix (SABiosciences) to a final concentration of
13 ng cDNA/25 ml reaction and applied to 96-well plates embedded with
IFN-a does not significantly induce Apobec3 relative to other
antiretroviral genes in vivo
IFN-a induces multiple antiretroviral ISGs, but a side-by-side
comparison of gene expression induction after in vivo IFN-a administration had not previously been performed. We therefore treated
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mechanism for how IFN-a could inhibit acute retroviral replication
in vivo. In particular, the concerted action of multiple effectors has
been suggested as necessary for IFN-a to directly inhibit acute
replication of different viruses (9, 14, 15). The magnitude of
transcriptional induction has also been used to tentatively gauge the
relative importance of a specific restriction factor in mediating
IFN-a–mediated virus control (4, 5). These assumptions on IFN-a
activity can be tested in a mouse model of retrovirus infection.
Using Apobec3-deficient mice, our group and others reported that
Apobec3 inhibited Friend retrovirus (FV) complex (16, 17), Moloney murine leukemia virus (MLV) (18), and mouse mammary tumor
virus during acute retrovirus infection (19). Pretreatment of mice
with LPS, a type I IFN inducer through TLR4, substantially inhibited
mouse mammary tumor virus 24 h posttreatment, but only in the
presence of Apobec3 (20). In contrast, another ISG, Tetherin, was
reported to be a key effector of polyinosinic:polycytidylic [poly(I:C)]
treatment against Moloney MLV (21). However, neither LPS nor
poly(I:C) are clinically relevant for antiviral therapy because they are
highly inflammatory in humans (22–25). In both studies, cell culture
experiments revealed that the impact of IFN-a on retrovirus replication are linked to Apobec3 (20) and Tetherin (21), respectively.
However, these studies did not provide data on inhibition of viremia after direct in vivo administration of IFN-a, the more clinically
relevant treatment regimen, into retrovirus-infected mice.
In this study, we used the FV model to obtain basic insights on
the direct effector mechanism of antiretroviral IFN-a therapy. FV
is a pathogenic retrovirus complex that causes severe splenomegaly
and erythroleukemia in adult immunocompetent mice. The host
genetics of FV infection has been extensively studied for .50 y
(26, 27) and is, therefore, an ideal system to unravel the complex
relationships of innate antiretroviral factors. Using the FV infection
model, we demonstrate for the first time, to our knowledge, that
IFN-a could act primarily through Apobec3 to inhibit acute infection of a pathogenic retrovirus in vivo.
IFN-a ANTIRETROVIRAL THERAPY AND Apobec3
The Journal of Immunology
1585
fection, we observed no to minimal induction of BM ISG by IFNa at 3 dpi (Fig. 2A). In contrast, FV/LDV induced multiple ISGs
in the spleen at 3 dpi, but not Apobec3 (Fig. 2B).
To determine whether antiretroviral genes could be induced by
IFN-a treatment in FV/LDV-infected mice, mice were treated with
IFN-a at 3 dpi, and ISG levels were measured 6 h later. In the BM,
Tetherin, ISG15, and IFITM3 were significantly induced (4- to 6fold; Fig. 2C), but the overall ISG induction levels after IFN-a
treatment of 3 dpi infected mice were lower compared with IFN-a
treatment of uninfected mice (Fig. 2C versus Fig. 1A). In contrast,
spleen ISG induction by IFN-a was similar between 3 dpi infected
and uninfected mice (Fig. 2D versus Fig. 1B). Thus, FV/LDV
infection suppressed the ability of administered IFN-a to induce
the transcription of antiretroviral ISGs at 3 dpi in the BM, but not
the spleen. Notably, Apobec3 was not induced by IFN-a in FVinfected mice in either compartment (Fig. 2C, 2D). Thus, IFN-a
is not a potent inducer of Apobec3 transcription in vivo in the
context of FV/LDV infection.
Compartmentalized induction of antiretroviral ISGs by IFN-a
in FV-infected mice
Apobec3 activity results in noninfectious particle release
during acute infection in B6 mice
We next evaluated ISG induction in FV-infected mice. Of note, the
FV stock used in this study and those that identified the classical Fv
restriction factors contained: 1) a replication-competent but nonpathogenic F-MuLV; 2) a replication-defective, but pathogenic
spleen focus-forming virus; and 3) LDV, an RNA virus endemic in
wild mouse populations (30). LDV induces a robust and systemic
IFN-a response that is rapidly downregulated (35, 36). Consistent
with the rapid downregulation of the IFN-a by FV/LDV coin-
The lack of Apobec3 induction and the existence of more highly
induced antiretroviral ISGs after IFN-a treatment suggested that
Apobec3 may not be a potent IFN-a effector. To directly test
whether Apobec3 was critical for the antiretroviral activity of IFN-a
in vivo, we administered IFN-a to FV-infected B6 WT and Apobec3
KO mice at 3 dpi and determined FV infection levels at 7 dpi (Fig.
3A). The 3 dpi time point was chosen for treating mice with IFN-a
because FV viremia and cellular infection levels remained low and
FIGURE 1. Induction of antiretroviral ISGs by IFN-a in vivo. (Left panels) Uninfected B6 mice (n = 4) were treated with 105 U IFN-a or PBS and after 6
h, BM and spleen mRNA were analyzed by quantitative PCR. Fold induction of ISGs post–IFN-a treatment (post-Rx) in (A) BM cells and (B) splenocytes.
(Right panels) CD11b+ myeloid cells were purified by magnetic bead separation from pooled BM or spleen from uninfected B6 mice (n = 2) treated for 6 h
with 105 U IFN-a or PBS. ISG fold induction is shown for (C) BM cells and (D) splenocytes. (A–D) (Left panels) Canonical ISGs Mx1 and ISG15 relative to
fold induction in b-actin, which was normalized to 1. (Right panels) Induction of antiretroviral ISGs, also normalized to b-actin (dashed lines). Error bars
correspond to SEM from triplicate measurements. Note that ISG15 was also reported as an antiretroviral gene (Supplementary Table I).
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B6 WT mice with either IFN-a or PBS and after 6 h evaluated
the relative mRNA expression of 10 antiretroviral genes in a
quantitative PCR array (Fig. 1; Supplemental Table I). We evaluated ISG induction relative to b-actin in the BM and spleen, the
major immune compartments targeted by FV (33, 34). As expected, IFN-a significantly induced (.15-fold) the expression of
canonical ISGs Mx1 and ISG15 in both the BM and the spleen
(Fig. 1A, 1B, left panels, respectively). Tetherin, Viperin, SAMHD1,
OAS1, IFITM3, PKR, and TRIM25 were also significantly upregulated (2- to 10-fold), whereas Fv1 was not induced (Fig. 1A, 1B,
right panels). Interestingly, Apobec3 was barely induced, with a
1.3-fold induction in BM (Fig. 1A) and no induction in spleen
(Fig. 1B). Even in purified CD11b+ myeloid cells, Apobec3 was
induced no more than 2-fold in the BM (Fig. 1C) and not induced
in the spleen (Fig. 1D). Apobec3 was also not induced at 24 h
post–IFN-a treatment in either compartment (data not shown).
These results reveal that IFN-a is not a potent inducer of Apobec3
transcription in the BM and spleen in vivo.
1586
IFN-a ANTIRETROVIRAL THERAPY AND Apobec3
were not significantly different between mice that express or do not
express the B6 Apobec3 gene (data not shown).
First, we tested for the signature Apobec3 phenotype of noninfectious particle release. In cell culture, Apobec3 is packaged into
retrovirus particles and inhibits reverse transcription and/or induces
lethal G-to-A hypermutation in the next target cell (37). Thus,
whereas virion output is not decreased by Apobec3 activity, the
infectivity of the resulting virions is significantly inhibited. This
phenotype was observed in 7 dpi plasma from FV-infected (B6 3
BALB/c)F1 and (B6 3 A.BY)F1 mice (38). We extend this observation to B6 mice. Plasma viral RNA loads of PBS-treated B6 WT
and Apobec3 KO mice were not significantly different (Fig. 3B),
suggesting similar physical numbers of retrovirus particles. In contrast, there was a 30-fold lower infectious virus titer in B6 WT
versus Apobec3 KO mice (Fig. 3C). Thus, the majority of FV virions
circulating in 7 dpi WT mice plasma were noninfectious, demonstrating that Apobec3 was functioning as expected in this study.
Inhibition in plasma viremia and cellular proviral load by
IFN-a requires Apobec3
We next evaluated the therapeutic impact of IFN-a on plasma viral
load and infectious viremia. IFN-a treatment resulted in a 28-fold
reduction in plasma viral load in B6 WT mice, whereas no significant reduction was observed with Apobec3 KO mice (Fig. 3B).
Although not statistically significant, IFN-a treatment resulted in
a 73-fold reduction in infectious viremia in WT mice, but only a 2fold reduction in Apobec3 KO mice (Fig. 3C). Thus, the therapeutic
impact of IFN-a on acute FV plasma viremia could primarily be
attributed to Apobec3.
To evaluate whether Apobec3 was critical for IFN-a–mediated
inhibition of cellular FV infection, we measured proviral DNA levels
at 7 dpi in the BM and spleen by quantitative PCR (Fig. 3D, 3E).
Proviral loads of PBS-treated B6 WT and Apobec3 KO mice were not
significantly different from each other, suggesting that next-round
inhibition of Apobec3 was not yet apparent at 7 dpi in the B6 genetic background. Importantly, after IFN-a treatment, we observed
a 5-fold and 14-fold reduction in proviral load in B6 WT mice in the
spleen (Fig. 3D) and BM (Fig. 3E), respectively. However, we did not
observe statistically significant reductions in B6 Apobec3 KO mice
(Fig. 3D, 3E). Thus, the therapeutic effect of IFN-a on cellular FV
infection in BM and spleen was mediated primarily through Apobec3.
Notably, we observed a wide range in infection levels in IFN-a–
treated mice. For example, nearly half of IFN-a–treated WT mice
showed undetectable infectious titers, whereas some showed infectious titers similar to untreated mice (Fig. 3C). In contrast with
antiretroviral compounds that directly target specific viral proteins, the antiviral activity of IFN-a is indirect, requiring signaling
pathways to activate downstream effectors. Immune responses,
including innate responses in inbred mice, are often affected by
stochastic events, so it is not too surprising that immunomodulatory agents would also be subject to individual differences. Interestingly, a wide range in IFN-a efficacy was also observed in human
IFN-a trials against HIV-1 (2–5).
Apobec3 is critical for the antiretroviral impact of IFN-a in
multiple BM cell subpopulations
We next performed flow cytometry to investigate the role of
Apobec3 in IFN-a antiretroviral activity in the major target cells
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FIGURE 2. Compartmentalized induction of antiretroviral ISGs in the context of FV infection. (A and B) B6 mice (n = 4) were inoculated with 104 SFFU
of FV complex or PBS. At 3 dpi, BM or spleen mRNA were analyzed by quantitative PCR relative to PBS-treated mice. Fold induction of ISGs after FV
infection in (A) BM cells and (B) splenocytes. Bar graph scales were drawn to allow for comparison of fold-induction levels in BM versus spleen. (C and D)
B6 mice (n = 4) were treated with IFN-a at 3 dpi and fold induction of ISGs after 6 h was measured in (C) BM cells and (D) splenocytes. (A–D) (Left
panels) Canonical ISGs Mx1 and ISG15 relative to b-actin fold induction, which was normalized to 1. (Right panels) Induction of antiretroviral ISGs. Error
bars correspond to SEM from triplicate measurements. Dashed lines corresponding to no change in expression relative to b-actin are shown.
The Journal of Immunology
of FV (33, 34) that include CD11c+ dendritic cells (DCs), Ter119+
erythroblasts, and CD19+ B cells (Fig. 4A). Infected cells were
detected with a FV Env–specific mAb 720. We focused our
analyses in the BM due to a more pronounced IFN-a treatment
effect on FV proviral load in the BM (Fig. 3E) compared with
spleen (Fig. 3D). Moreover, the median percentage of FV+ cells in
the spleen of each cohort was very low (5–7%) relative to the
proviral load data (Fig. 3D), suggesting that FV Env was downregulated in infected splenocytes, possibly by an emerging FV Ab
response.
Consistent with the BM proviral load data (Fig. 3E), IFN-a
treatment reduced FV infection in total BM cells by 7.3-fold in
WT mice, but no significant inhibition was achieved in Apobec3
KO mice (Fig. 4B). Interestingly, IFN-a treatment also significantly reduced FV infection in CD11c+ (1.6-fold) and Ter119+
cells (1.2-fold) of B6 Apobec3 KO mice (Fig. 4C, 4D). However,
the fold-inhibition values were lower compared with WT mice,
where IFN-a treatment reduced FV infection 7.0-fold and 6.0-fold
in CD11c+ and Ter119+ cells, respectively (Fig. 4C, 4D). Thus,
∼80% of the antiretroviral activity of IFN-a in BM DCs and
FIGURE 4. Impact of IFN-a treatment on BM cell subpopulations. (A)
Gating strategy for total BM cells. (Right panels) Representative histograms showing reduction of FV infection by IFN-a treatment (Rx) in B6
WT but not B6 Apobec3 KO mice. Flow cytometry was performed with
a mAb specific to FV Envelope gp70, mAb 720. FV-infected cells were
quantified for gated (B) total BM, (C) CD11c+ DCs, (D) Ter119+ erythroblasts, and (E) CD19+ B cells. Median values are indicated by solid lines.
Each dot in (B)–(E) corresponds to an infected mouse. Statistical difference
was evaluated using a two-tailed Mann–Whitney U test, with exact p
values shown. Fold differences of non–log-transformed values between
PBS- and IFN-a–treated mice are indicated for differences that were statistically significant (p , 0.05).
erythroblasts could be attributed to Apobec3. In contrast, IFN-a
did not significantly inhibit FV infection in B cells of B6 Apobec3
KO mice (Fig. 4E), suggesting that Apobec3 accounted for the
antiretroviral activity of IFN-a in B cells. Thus, the direct antiretroviral impact of IFN-a therapy in the major FV target cells in
the BM can primarily be attributed to Apobec3.
Discussion
Unraveling the direct effector mechanism(s) that confers antiretroviral activity of IFN-a in vivo is critical for improving the
therapeutic efficacy of this cytokine. In this study, we report in a
mouse model of pathogenic retrovirus infection that IFN-a therapy could act primarily through the antiviral gene, Apobec3. This
key finding was unexpected because: 1) Apobec3 mRNA was not
significantly induced in the major compartments that support FV
replication, the BM and spleen, respectively; and 2) ISGs that were
highly induced by IFN-a in both compartments were known to
exhibit potent antiretroviral activity in vitro.
We provide three possible explanations for how Apobec3
trumped more highly induced antiretroviral genes. First, it may be
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FIGURE 3. Apobec3 accounts for IFN-a–mediated control of plasma
viremia and proviral load. (A) Infection and treatment timeline. B6 WT or
Apobec3 KO mice were infected with 10,000 SFFU of FV at day 0, then
treated with 105 U IFN-a at day 3. Biological samples were analyzed at 7
dpi. Data from two independent cohorts were combined. (B) Inhibition of
plasma viral load. Viral RNA copies per milliliter plasma at 7 dpi were
measured by quantitative PCR. (C) Infectious viremia. Plasma (5 ml) was
incubated with Mus dunni cells, and viral DNA levels were measured after
2 d. Median values are indicated. (D) Spleen and (E) BM proviral load
were measured by quantitative PCR. Statistical difference between the
datasets was evaluated using a two-tailed Mann–Whitney U test, with
exact p values shown. For all panels except (B) that showed a non-Gaussian
distribution, mean values were indicated and used to calculate fold differences.
1587
1588
by IFN-a treatment. Interestingly, discrepancies in the transcriptional regulation of the human homolog, APOBEC3G, was also
observed between the major target cells of HIV-1, CD4+ T cells
versus macrophages, as described later.
Mice encode a single Apobec3 gene that expanded into seven
APOBEC3 members in primates, APOBEC3A to H (48, 49).
Human APOBEC3A expression is the most highly inducible by
IFN-a, but APOBEC3A does not incorporate into virions, and
thus could not reduce virion infectivity (50, 51). In contrast, human APOBEC3D, F, G, and H could significantly inhibit HIV-1
in vitro (52, 53). Of these seven human APOBEC3 members,
APOBEC3G is the most highly expressed in HIV target cells and
is considered the most potent APOBEC3 member against HIV-1
and HTLV-I (54–56). Thus, APOBEC3G is considered a prime
candidate for the direct antiretroviral effector of IFN-a.
Similar to the lack of mouse Apobec3 induction by IFN-a
in vivo in this study, IFN-a does not significantly induce APOBEC3G expression in activated CD4+ T cells (57–61). In contrast,
APOBEC3G could be upregulated in monocytes, macrophages,
and DCs (50, 51, 57–59, 62–64). Thus, studies that showed an
increase in APOBEC3G mRNA expression in bulk PBMCs after
IFN-a treatment (5, 58) were most likely due to an effect on
myeloid cells. However, activated CD4+ T cells produce 99% of
HIV-1 in plasma (65), whereas restriction blocks because of another factor, SAMHD1, limit HIV-1 replication in myeloid cells
(66, 67). Thus, the notion that IFN-a upregulates APOBEC3G
transcription to reduce HIV-1 viremia in vivo remains a matter of
debate. In fact, if the magnitude of transcriptional induction is
used as a sole basis for how IFN-a could counteract HIV-1, then
APOBEC3G may not be the best candidate for the direct IFN-a
effector. Tetherin mRNA induction, but not APOBEC3G mRNA
induction, significantly correlated with HIV-1 plasma viral load
reduction after IFN-a treatment (5). We also observed that Tetherin was among the most highly induced ISGs in mice after IFN-a
treatment. However, our results revealed that although Apobec3
was not induced as the other ISGs, it was critical for the antiretroviral efficacy of IFN-a. Thus, we conclude that at least in the
FV model, ISG mRNA induction was a poor predictor of critical
IFN-a effector mechanisms in vivo. The dominance of Apobec3
over other ISGs as an antiretroviral IFN-a effector suggests that
therapeutic strategies to augment APOBEC3 activity are relevant
for counteracting pathogenic human retrovirus infections.
Acknowledgments
We thank Charles Dinarello for critically reviewing the manuscript and
Thomas Campbell for insights on IFN-a therapy.
Disclosures
The authors have no financial conflicts of interest.
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